PURIFICATION AND CHARACTERIZATION OF PROTEINASE INHIBITOR FROM GARLIC (ALLIUM SATIVUM)

SUBMITTED FOR THE AWARD OF THE DEGREE OF

Doctor of Philosophy

IN BIOCHEMISTRY

BY

MOHD FAIZAN SIDDIQUI

UNDER THE SUPERVISION OF

Maulana Azad Library, Aligarh Muslim University PROFESSOR BILQEES BANO

DEPARTMENT OF BIOCHEMISTRY FACULTY OF LIFE SCIENCES ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2019

Certificate

This is to certify that the work presented in this thesis entitled

“Purification and characterization of cysteine proteinase inhibitor from garlic (Allium sativum)” is an original work done by Mr. Mohd

Faizan Siddiqui under my supervision and is suitable for submission for the award of Ph.D. degree in Biochemistry.

Professor Bilqees Bano

Maulana Azad Library, Aligarh Muslim University ANNEXURE-I

CANDIDATES DECLARATION

I, MOHD FAIZAN SIDDIQUI, Department of Biochemistry, Faculty of Life Sciences, certify that the work embodied in this Ph.D. thesis is my own bonafide work carried out by me under the supervision of PROF. BILQEES BANO at Aligarh Muslim University, Aligarh. The matter embodied in this Ph.D. thesis has not been submitted for the award of any other degree.

I declare that I have faithfully acknowledged, given credit to and referred to the research workers wherever their works have been cited in the text and the body of the thesis. I further certify that I have not wilfully lifted up some other’s work, para, text, data, result, etc. Reported in the journals, books, magazines, reports, dissertations, theses, etc., or available at web-sites and included them in this Ph.D. thesis and cited as my own work.

Date: ...... (Signature of the candidate)

MOHD FAIZAN SIDDIQUI

......

Certificate from the Supervisor

This is to certify that the above statement made by the candidate is correct to the best of my knowledge.

Signature of the Supervisor: Maulana Azad Library, Aligarh Muslim University Name & Designation: Dr. BILQEES BANO, PROFESSOR Department: Department of Biochemistry, F/o Life Sciences, AMU, Aligarh

(Signature of the Chairman of the Department with seal)

ANNEXURE – II

COURSE/COMPREHENSIVE EXAMINATION/PRE-SUBMISSION

SEMINAR COMPLETION CERTIFICATE

This is to certify that Mr. MOHD FAIZAN SIDDIQUI, Department of Biochemistry, Faculty of Life Sciences, AMU, has satisfactorily completed the Course work/ comprehensive examination and pre-submission seminar requirement which is part of his/her Ph.D. programme.

Date: ...... (Signature of the Chairman of the Department)

Maulana Azad Library, Aligarh Muslim University

ANNEXURE - III

COPYRIGHT TRANSFER CERTIFICATE

Title of the Thesis: PURIFICATION AND CHARACTERIZATION OF CYSTEINE PROTEINASE INHIBITOR FROM GARLIC (ALLIUM SATIVUM)

Candidate’s Name: MOHD FAIZAN SIDDIQUI

Copyright Transfer

The undersigned hereby assigns to the Aligarh Muslim University, Aligarh copyright that may exist in and for the above thesis submitted for the award of the Ph.D. degree.

Signature of the candidate

Note: However, the author may reproduce or authorize others to reproduce material extracted verbatim from the thesis or derivative of the thesis for the author’s personal use provide that the source and the University’s copyright notice are indicated.

Maulana Azad Library, Aligarh Muslim University

Acknowledgment

I begin my gratitude with Allah, The most Beneficial and Merciful. Oh Allah! indeed you are Ar-Razzaaq (The provider) and Al-Haadi (The Guide). I bow my head to you for being with me and helping me throughout my existence.

I am highly grateful to my parents and my siblings for their endless love and support. They were the ones who kept me going when I wanted to give up. Their ‘with-me’ attitude strengthened me and put me all together whenever I was fragile and broken…………. especially you AMMA.

With sincere feelings, I thank my esteemed supervisor Prof. Bilqees Bano, for her expert guidance, and invaluable advice; and for nurturing in me the ability for work, observation and study with her scientific acumen. Her critical commentary on my work has played a major role in both the content and presentation of my discussion and arguments. I am highly grateful to her for guidance and support in carrying out this research work.

I thank Prof. Mohd. Tabish, (Chairman, Department of Biochemistry), and Prof. Riaz Mahmood, for providing all the necessary facilities required to carryout this work. I extend my gratitude to all the teachers of Department of Biochemistry, Prof. S.M. Hadi, Prof. Masood Ahmad, Prof. Qayyum Husain, Prof. Imrana Naseem, Prof. Saleem Javed, Dr. Fahim Halim Khan, Dr. Farah Khan, Dr. Aabgeena Naeem, Dr. Shamila Fatima and Dr. Samreen Amani.

I am highly grateful to Dr. Azad Alam Siddiqui who helped me a lot during my experimentsMaulana since myAzad master’s Library, dissertation Aligarh on . Muslim I am University also thankful to all of my seniors for their help and constructive suggestions especially Dr. Aamir Sohail, Dr. Asim Rizvi, Dr. Azaj Ahmed, Dr. Anas Shamsi, Dr. Zeba Farooqui, Dr. Naureen Fatima, Dr. Shariq Qayyum, Dr. Nazim Husain. I extend my gratitude to all the other members of the lab for their cooperation and help.

I am highly blessed for having few gems in my life whom I call my friends my buddies and to whom a word “thanks” is not enough- Saniyya Khan, Yusra Rahman (Sci.), Samra Hasan (CB), Faizan Ahmed (Rockstar et al., 2009), Saman Khan, Nazia Parveen, Anzar Abdul Mujeeb, Saquib Khan, Arbab Husain, Zia ul Hasan. I thank you all for being there whenever I needed and for teaching me other important lessons of life (B.Sc 2009-12).

I thank my colleague Faisal Asar, Shahbaz, Kashan and Moasfer and juniors Sharmin, Samreen, Azra, Amin and Amir, for encouraging me to achieve my goal through healthy interactions in the department. I also thankful to my childhood friends Suhail, Ahmad, Rifat, Muratza, Danish, and Qasim for their continuous support. I also thank to someone special in my life. Thank You

I am thankful to all the non-teaching staff for their supportive and helping attitude. I would also like to thank whole staff of USIF for proving their facility and for their friendly and cordial attitude. Thanks are due to the UGC MANF SRF Fellowship, New Delhi, India for providing JRF/SRF fellowship [No. F1-17.1/2016-17/MANF-2015- 17-UTT-55522] during the tenure of my Ph.D.

(Mohammad Faizan Siddiqui)

Maulana Azad Library, Aligarh Muslim University

Abstract

ABSTRACT

Proteolytic or proteinases are the peptide bond hydrolyzing enzymes. Proteinases with a cysteine residue at the are called as cysteine proteinases. These endogenous thiol proteinases are widely distributed in the living system and are involved intracellular of protein as well as peptide catabolism, cell invasion, and processing of surface proteins to the development and function of the . Since they play a vital role in the prevention of unwanted proteolysis, therefore, any alteration in their activity can result in devastating tissue and proteolytic cellular damage. Therefore, function of proteinases should be regulated strictly. One of the mechanisms to regulate the function of thiol proteinases is by their interaction with cysteine proteinase inhibitors usually known as . The cystatins specifically inhibit the activity of like cysteine proteinases. They bind to the cysteine proteinases and hamper their activity. Thereby maintaining the balance between cellular proteinase and anti-proteinase activity.

Cystatins are the natural regulators of cysteine proteinases. They are ubiquitously present in microorganisms, plants, and animals. Cystatins have been found to be evolutionary, structurally, and functionally related to each other forming cystatin superfamily. They are classified on the basis of molecular weight, structure, and complexity into the following three families, namely: Family I, II, and III. They are reversible, competitive, and non-covalent binding inhibitors. Family I, also called as stefins include members of low molecular weight proteins (~11 kDa) which lack disulfide bonds and carbohydrate content. This family includes Stefin A, B, and C. Family II is known as cystatins which represent inhibitors of a bit higher molecular weightMaulana proteins (~ 13Azad kDa) Library,possessing Aligarhdisulfide bonds Muslim towards University carboxy-terminal. This family comprises of cystatin C, D, E/M, and F. Family III, also known as kininogens are high molecular weight – glycosylated inhibitors containing multiple disulfide linkages. They are found in blood plasma. Another family has been added in the cystatin superfamily after the identification of cysteine proteinase inhibitors in plants and is called as phytocystatin. Thus, phytocystatin is the fourth family of cystatin superfamily and is the counterpart of animal cystatin which inhibits cysteine proteinases in plants. This family has been classified into three distinct groups, namely group I, group II, and group III phytocystatin based on the molecular mass

1

Abstract and conserved domains. Group I contains a single cystatin domain and has a molecular weight of 12 – 16 kDa. Group II contains a highly conserved cystatin domain at N- terminal and an additional extension at C- terminal of 10 kDa along with a conserved SNSL motif. The group II phytocystatin members have a molecular weight of around 23 kDa. Group III phytocystatins contain multi-domain and are of high molecular weight of around 85 kDa. Phytocystatins have well-conserved motifs of cystatin superfamily as Gln-Xaa-Val-Xaa-Gly motif at the central region of the polypeptide chain (Xaa is any amino acid), along with a Proline-Tryptophan dipeptide motif at the C-terminal region, and a conserved Gly residue at the N-terminal region. Furthermore, they also contain an N-terminal conserved motif [LVI]-[AGT]-[RKE]- [FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-N, known as LARFAV- like motif which constitutes the alpha-helix in the cystatin structure. Phytocystatins are responsible for regulating protein turnover in developing and germinating seeds. They are also involved in numerous other physiological processes like controlling intracellular proteolysis as well as extracellular proteolysis of cysteine proteinases of pathogenic nematodes and arthropods. They also act as a potent inhibitor of gut proteinases leading to the plant defense against insects and bacterial phytopathogens.

Garlic (Allium sativum) is a perennial plant of the genus Allium and family amaryllis (Amaryllidaceae). It has been used as a food flavoring agent as well as traditional medicine. Allicin is the principal bioactive compound present in the aqueous extract of garlic. The fresh garlic cloves contain different organosulfur compounds, phenolic and steroidal compounds such as diallyl disulfide (DADS) and diallyl trisulfide (DATS), and g-glutamyl S- allyl cysteine. Garlic is involved in the treatment of different ailments, such as blood pressure, cardiovascular, diabetes, ,Maulana and Azad cancer. Library, Phytocystatin Aligarhs ha Muslimve been previouslyUniversity isolated from different plant sources such as tomato, maize, black gram, almond, chickpea, and mustard seeds. However, no report was available in the literature for garlic phytocystatin. Hence, the thesis describes the isolation and characterization of phytocystatin from garlic cloves.

The first chapter of the thesis deals with the isolation and characterization of phytocystatin from garlic cloves. Garlic phytocystatin (GPC) after preliminary treatment was purified using ammonium sulfate fractionation (30-60%) and gel filtration chromatography on Sephacryl S-100HR with a fold purification of 152.6 and

2

Abstract yield of 48.9%. The molecular mass was calculated as 12.5 kDa using SDS-PAGE technique and 12.0 kDa using gel filtration chromatography. The SDS-PAGE also indicated that GPC is a single polypeptide chain with no subunit structure. It was also found to be stable under a broad range of pH (6–8) and temperature (30 ◦C–60 ◦C). GPC was found to be devoid of carbohydrate and thiol content. Its Stokes radius and the diffusion coefficient were 17.8 Å and 12.4 x 10-7 m2 sec-1, respectively indicating globular nature of the inhibitor. Kinetic studies suggested non-competitive inhibition -8 for GPC, with the maximum inhibition towards papain (Ki = 8.5x10 M) followed by -7 -7 ficin (Ki = 1.2x10 M) and (Ki = 1.38x10 M). Ultra-violet absorption spectroscopy and fluorescence spectroscopy revealed significant conformational change within GPC upon GPC-papain complex formation. The secondary structure analysis showed that GPC possessed 33.9% alpha-helical content as assessed by circular dichroism spectroscopy. Hence, the low molecular weight and observed parameters suggest that the newly purified phytocystatin from garlic cloves belongs to group I phytocystatin.

In the second chapter, we have investigated the denaturation effect of urea and guanidine hydrochloride (GdnHCl) on the purified GPC. Urea and GdnHCl are the two chemical denaturants used in labs to decipher the folding – unfolding pattern of proteins. Phytocystatins are crucial proteins and play an essential role in maintaining the proper functioning of all living cells by virtue of their thiol regulatory properties within plants. Hence, malfunctioning of this protein could result in the disruption of physiological activities and may result in poor growth and development of plants. The present chapter deals with the chemical denaturation of GPC with the aid of urea and guanidine hydrochloride to characterize the unfolded and denaturedMaulana state. Azad The cysteine Library, proteinase Aligarh inhibitory Muslim activity University of GPC decreases with the increasing concentration of urea and GdnHCl. GPC loses its 50% cysteine proteinase inhibitory activity at 3 M urea and 2 M GdnHCl. Increased fluorescence intensity along with red shift of 13 nm and 8 nm reflected the unfolding of GPC at higher concentration of urea and GdnHCl, respectively. GdnHCl induced unfolding showed the presence of indiscernible intermediate as evident by ANS binding studies. However, denaturation by urea did not show any such intermediates. Urea and GdnHCl induced denaturation of GPC showed a decline in the ANS fluorescence intensity along with the red shift of 10 nm and 5 nm, respectively. Mid-point

3

Abstract transition was observed at 4.7 ± 0.1 M urea and 2.32 ± 0.1 M GdnHCl. Circular dichroism and FTIR results indicated 50% loss of secondary structure at 5 M urea and 2.5 M GdnHCl. This study provided an intriguing insight into the possible alteration in structure, stability, and function of GPC induced by urea and GdnHCl.

Pesticides are creating havoc in the health and safety of humans as well as animals. Their excessive usage has polluted the environment, and biomagnification has severely affected the biosystems and ecosystems, rendering them unfit for flora and fauna. Though pesticides aid in crop yield and food production, still the level of environmental destruction and degradation has made it quite essential to check their usage on a global level. The third chapter reports the effect of carbendazim (fungicide) and oxyfluorfen (herbicide) on GPC. Carbendazim is a broad-spectrum benzimidazole fungicide which is used to ensure plants' protection from pest and pathogens' invasion. Functional activity of GPC was monitored by the cysteine proteinase inhibitory assay, which suggested that incubation of GPC with the higher concentration of carbendazim disrupts the inhibitory function of GPC. UV spectroscopy confirmed the formation of GPC-carbendazim complex. Intrinsic fluorescence studies suggest binding of carbendazim to GPC and reflect changes in the microenvironment around tryptophan residues of GPC. Isothermal titration calorimetry suggests that interaction of carbendazim to GPC is an exothermic reaction. Secondary structure analysis confirmed that the binding of carbendazim decreases the alpha-helical content of GPC. Collectively, these results demonstrated that GPC exhibited significant structural and functional alteration upon interaction with carbendazim.

Oxyfluorfen is a broad range nitrophenyl ether herbicide used to combat Maulana Azad Library, Aligarh Muslim University different kinds of harmful pest. The cysteine proteinase inhibitory assay was done to assess the inhibitory action of GPC in the presence of oxyfluorfen. The cysteine proteinase inhibitory activity of GPC declines with increasing concentration of oxyfluorfen. The complex formation of GPC-oxyfluorfen was shown by UV absorption spectroscopy. The intrinsic fluorescence experiment affirmed the quenching of GPC in the presence of oxyfluorfen. Synchronous fluorescence study showed the alteration in the microenvironment around aromatic residues of oxyfluorfen. The isothermal titration experiment suggests that the interaction of oxyfluorfen with GPC is an exothermic and thermodynamically favorable reaction.

4

Abstract

Secondary structure analysis by circular dichroism revealed alteration in the secondary structure of GPC in the presence of an increasing concentration of oxyfluorfen. The circular dichroism result showed reduction in the alpha-helical content of GPC upon interaction with oxyfluorfen. Consequently, all these outcomes affirmed the formation of GPC–oxyfluorfen complex along with the structural and functional alteration within GPC. Since GPC is involved in various regulatory processes, therefore, its structural or functional alteration may lead to disruption of physiological and biological balance within the plant. Hence, the study signifies that exposure of carbendazim and oxyfluorfen to plant exerts physicochemical changes and induces stress within the plant system.

Intrinsic and extrinsic factors are responsible for the transition of soluble proteins into aggregated form. Trifluoroethanol is among such potent extrinsic factor which facilitates the formation of aggregated structure. It disrupts the interactive forces and destabilizes the native structure of the protein. The fourth chapter investigates the effect of trifluoroethanol (TFE) on garlic phytocystatin. GPC was incubated with increasing concentration of TFE (0–90% v/v) for 4 h. Incubation of GPC with TFE induced structural changes, thereby resulting in the formation of aggregates. Inactivation of GPC was confirmed by cysteine proteinase inhibitory activity. GPC incubated with 30% TFE exhibited native-like secondary structure and high ANS fluorescence, suggesting the presence of molten globule state. Circular dichroism study confirmed the transition of the native alpha-helical structure to the beta-sheet structure at 60% TFE. Furthermore, increased Thioflavin-T fluorescence and redshift in Congo red absorbance assay confirmed the presence of aggregates. Rayleigh and turbidity assay were also performed to validate the aggregation results. Finally,Maulana the scanning Azad electron Library, microscopy Aligarh was followed Muslim to analyzeUniversity the morphological changes which confirmed the presence of sheath-like structure at 60% TFE. The study highlights the conformational behavior of a plant protein under extrinsic stress conditions.

Heavy metal-induced abiotic stress is associated with retarded growth and development of plants. Therefore, it is essential to have an insight into the potential toxic effects of heavy metals on crucial plant regulatory proteins like phytocystatins. Thus, the final chapter of the thesis deals with the effect of two heavy metals viz. zinc (Zn+2) and cadmium (Cd+2) on the structure and function of GPC. Binding of

5

Abstract zinc (Zn+2) and cadmium (Cd+2) with GPC resulted in the reduced inhibitory activity of GPC as evaluated by cysteine proteinase inhibitory assay. UV-vis absorption spectroscopy revealed the complex formation of zinc and cadmium with GPC. Further, fluorescence quenching experiment confirmed the quenching of fluorophores upon their binding. In addition, synchronous and three-dimensional fluorescence spectroscopy suggested the alteration in the microenvironment around aromatic residues of GPC upon their binding. Reduction in alpha-helical content of native GPC was also observed as evident by secondary structure analysis by circular dichroism. The possibility of formation of GPC aggregates was checked by aggregation specific assays. The Thioflavin-T and Congo red assays showed increased fluorescence intensity and absorbance, respectively in the presence of zinc (Zn+2) and cadmium (Cd+2) as compared to native GPC, which is indicative of the formation of aggregates. Scanning electron micrographs showed the morphological changes in the native GPC upon addition of zinc (Zn+2) and cadmium (Cd+2). The observations confirmed the alteration in function and conformation of GPC upon interaction with zinc and cadmium. Hence, it can be evidently concluded that high concentration of zinc (Zn+2) and cadmium (Cd+2) might alter the functioning of cysteine proteinase inhibitor present in garlic and affects the growth and development of garlic crops.

Maulana Azad Library, Aligarh Muslim University

6

CONTENTS Page No. LIST OF ABBREVIATIONS i-ii LIST OF FIGURES iii-vii LIST OF TABLES viii ABSTRACT ix-xiv

INTRODUCTION 1-71 1. Proteinases: General 1-18 1.1 Serine proteinases 1.2 Aspartate proteinases 1.3 Metalloproteinases 1.4 Threonine proteinases 1.5 Glutamic proteinases 1.6 peptide 1.7 Cysteine proteinases 1.8 Mammalian cysteine proteinases 1.9 Plant cysteine proteinases 1.10 Bacterial and viral cysteine proteinases 1.11 Mechanism of action of proteinases 1.12 Regulation of cysteine proteinases 2. Mammalian proteinase inhibitors 19 3. Plant proteinase inhibitors 20-21 4. Specific inhibitor of cysteine proteinases: Cystatins 21-36 4.1 Classification of the cystatin superfamily 4.2 Family I: Type I Cystatin: The Stefins 4.3 Family II: Type II Cystatin: The Cystatins 4.4 Family III: Type III Cystatin: The Kininogens Maulana4.5 Family Azad IV: TheLibrary, Phytocystatins Aligarh Muslim University 5. Characteristic features of phytocystatins 36-43 6. Evolution of phytocystatins as separate superfamily 43-44 7. Garlic plant 44-50 7.1 Medical significance of garlic 8. Importance of phytocystatins 50-67 9. Scope of the thesis 68-71

MATERIALS AND METHODS A. MATERIALS 72 B. METHODS 73-98 I. PURIFICATION AND CHARACTERIZATION OF GARLIC PHYTOCYSTATIN 73-84 1. Homogenate preparation 73 2. Ammonium sulfate fractionation 73 3. Gel-filtration chromatography 73 4. Colorimetric analysis 74 4.1 Determination of protein concentration 4.2 Carbohydrate estimation 4.3 Thiol group estimation 4.4 Cysteine proteinase inhibitory activity assay 5. Gel electrophoresis 76 5.1 Polyacrylamide gel electrophoresis (PAGE) 5.2 SDS Polyacrylamide gel electrophoresis (SDS-PAGE) 5.3 Staining of the gel: Coomassie Brilliant Blue staining 6. Molecular mass determination 77 6.1 Molecular mass determination by SDS-PAGE 6.2 Molecular mass determination under native conditions by gel filtration chromatography 7. Determination of hydrodynamic properties 78 8. pH stability 79 9. Thermal stability 79 10. Inhibition kinetics 79

10.1 Determination of Michaelis constant (Km)

10.2 Determination of inhibition constant (Ki)

10.3 Determination of dissociation rate constant (K-1) 10.4 Determination of association rate constant (K ) Maulana Azad Library, Aligarh Muslim+1 University 10.5 Half-life of complex

10.6 IC50 value 11. Immunological studies 81 11.1 Production of antisera 11.2 Immunodiffusion 11.3 Direct binding -linked immunosorbent assay (ELISA) 12. Spectroscopic analysis 82 12.1 Ultraviolet absorption spectroscopy 12.2 Fluorescence spectroscopy 12.3 Circular dichroism spectroscopy 12.4 Fourier transform infrared spectroscopy

II. UNFOLDING STUDIES OF GARLIC PHYTOCYSTATIN IN PRESENCE OF DENATURANTS 84-88 13. Unfolding of GPC in the presence of urea and guanidine 84 hydrochloride 14. Cysteine proteinase inhibitory assay of GPC in the presence urea 84 and GdnHCl 15. Intrinsic fluorescence spectroscopy 85 16. ANS fluorescence analysis 85 17. Acrylamide quenching experiment 86 18. Circular dichroism (CD) analysis 86 19. Fourier transform infrared (FTIR) spectroscopy 87 20. Equilibrium denaturation experiment of GPC 87

III. INTERACTION OF PESTICIDES (CARBENDAZIM AND OXYFLUORFEN) WITH GARLIC PHYTOCYSTATIN 88-91 21. Sample preparation 88 22. Cysteine proteinase inhibitory assay of GPC in the presence of carbendazim and oxyfluorfen 88 23. Ultraviolet absorption spectroscopy 89 24. Intrinsic fluorescence spectroscopy 89 25. Synchronous fluorescence spectroscopy 90 26. Isothermal titration spectroscopy 90 27. Circular dichroism (CD) analysis 91

IV. AGGREGATION STUDY OF GARLIC PHYTOCYSTATIN ASSISTED BY TRIFLUOROETHANOL (TFE) 91-94 28. Cysteine proteinase inhibitory assay of GPC in the presence of TFE 91 29. Intrinsic fluorescence spectroscopy 92 30. ANS fluorescence spectroscopy 92 31. CircularMaulana dichroism Azad (CD) Library, analysis Aligarh Muslim University 92 32. Turbidity measurement 93 33. Rayleigh scattering measurement 93 34. Thioflavin- T (ThT) assay 93 35. Congo red assay 94 36. Scanning electron microscopy 94

V. EFFECT OF HEAVY METALS (Zn+2 AND Cd+2) ON GARLIC PHYTOCYSTATIN 94-98 37. Cysteine proteinase inhibitory assay of GPC in the presence of zinc and cadmium 94 38. Ultraviolet absorption spectroscopy 95 39. Intrinsic fluorescence spectroscopy 95 40. Synchronous fluorescence spectroscopy 96 41. Three-dimensional spectroscopy 96 42. Circular dichroism (CD) analysis 96 43. Thioflavin-T (ThT) assay 97 44. Congo red assay 97 45. Scanning electron microscopy 98 46. Statistical analysis 98

RESULTS AND DISCUSSION RESULTS CHAPTER 1: PURIFICATION AND CHARACTERIZATION OF GARLIC PHYTOCYSTATIN (GPC) 99-135 1. Purification of garlic phytocystatin (GPC) 99 2. Gel filtration chromatography 99 3. Homogeneity of the purified GPC 102 4. Reducing (SDS + βME) and non-reducing SDS-PAGE 102 5. Properties of the purified GPC 102 5.1 Molecular mass determination 5.2 Stokes radius 5.3 Diffusion coefficient 5.4 Carbohydrate estimation 5.5 Thiol group estimation 5.6 Effect of pH on the activity of GPC 5.7 Effect of temperature on the activity of GPC 6. Immunological properties 109 6.1 Antibody titer 6.2 Cross reactivity 7. Kinetic propertiesMaulana of GPC Azad Library, Aligarh Muslim University113 7.1 Stoichiometry of inhibition 7.2 Inhibition of different proteinases by GPC

7.3 Determination of inhibition constant (Ki)

7.4 Determination of dissociation rate constant (K-1)

7.5 Determination of association rate constant (K+1) 8. Spectroscopic analysis of GPC 125 8.1 Ultraviolet absorption spectroscopy 8.2 Fluorescence spectroscopy 8.3 Circular dichroism (CD) spectroscopy 8.4 Fourier transform infrared spectroscopy (FTIR) DISCUSSION 131

CHAPTER 2: UNFOLDING STUDIES OF GARLIC PHYTOCYSTATIN (GPC) IN THE PRESENCE OF UREA AND GUANIDINE HYDROCHLORIDE 136-168 1. Functional study 136 1.1 Effect of urea on the cysteine proteinase inhibitory activity of GPC 1.2 Effect of guanidine hydrochloride on the cysteine proteinase inhibitory activity of GPC 2. Structural studies 139 2.1 Effect of urea on the intrinsic fluorescence of GPC 2.2 Effect of GdnHCl on the intrinsic fluorescence of GPC 3. ANS fluorescence studies of GPC in the presence of denaturants 141 3.1 Effect of urea on the ANS fluorescence of GPC 3.2 Effect of GdnHCl on the ANS fluorescence of GPC 4. Acrylamide quenching studies of GPC in the presence of denaturants 145 4.1 Acrylamide quenching analysis of GPC in the presence of urea 4.2 Acrylamide quenching analysis of GPC in the presence of GdnHCl 5. Secondary structure analysis of GPC in the presence of urea 146 and guanidine hydrochloride 5.1 Effect of urea on the secondary structure of GPC 5.2 Effect of GdnHCl on the secondary structure of GPC 6. Fourier transform infrared measurements of GPC in the presence 154 of denaturants 6.1 Fourier transform infrared analysis of GPC in the presence of urea 6.2 Fourier transform infrared analysis of GPC in the presence of GdnHCl 7. Equilibrium denaturation study of GPC in the presence of denaturants 157 7.1 Equilibrium denaturation study of GPC in the presence of urea 7.2 Equilibrium denaturation study of GPC in the presence of Maulana GdnHCl Azad Library, Aligarh Muslim University DISCUSSION 163

CHAPTER 3 A: INTERACTION OF CARBENDAZIM (FUNGICIDE) WITH GARLIC PHYTOCYSTATIN 169-191 1. Functional study 169 1.1 Effect of carbendazim on the cysteine proteinase inhibitory activity of GPC 2. Structural study 169 2.1 Ultra-violet absorption study of GPC in the presence of carbendazim 2.2 Intrinsic fluorescence of GPC in the presence of carbendazim 3. Synchronous fluorescence of GPC in the presence of carbendazim 176 3.1 Effect of carbendazim on the synchronous fluorescence of GPC 4. Isothermal titration calorimetric study of GPC in the presence of carbendazim 180 5. Secondary structure analysis of GPC in the presence of carbendazim 184 5.1 CD analysis DISCUSSION 187 CHAPTER 3 B: INTERACTION OF OXYFLUORFEN (HERBICIDE) WITH GARLIC PHYTOCYSTATIN 192-215 1. Functional study 192 1.1 Effect of oxyfluorfen on the cysteine proteinase inhibitory activity of GPC 2. Structural study 194 2.1 Ultra-violet absorption study of GPC in the presence of oxyfluorfen 2.2 Intrinsic fluorescence study of GPC in the presence of oxyfluorfen 3. Synchronous fluorescence study of GPC in the presence of Oxyfluorfen 203 3.1 Effect of oxyfluorfen on the synchronous fluorescence of GPC 4. Isothermal titration calorimetric study of GPC in the presence of oxyfluorfen 206 5. Secondary structure analysis of GPC in the presence of Oxyfluorfen 206 5.1 CD analysis DISCUSSION 211 CHAPTER 4: AGGREGATION STUDY OF GARLIC PHYTOCYSTATIN ASSISTED BY TRIFLUOROETHANOL 216-237 1. Functional study 216 1.1 Effect of TFE on the cysteine proteinase inhibitory activity of GPC 2. Structural study 216 2.1 Effect of TFE on the intrinsic fluorescence of GPC 3. ANS fluorescence studies of GPC 218 3.1 Effect of TFE on the ANS fluorescence of GPC in the presence of TFE Maulana Azad Library, Aligarh Muslim University 4. Secondary structure analysis of GPC in the presence of TFE 221 4.1 Circular dichroism measurements of GPC in the presence of TFE 5. Aggregation specific assays of GPC in the presence of TFE 224 5.1 Turbidity assay of GPC in the presence of TFE 5.2 Rayleigh scattering assay of GPC in the presence of TFE 5.3 Thioflavin-T fluorescence measurements of GPC in the presence of TFE 5.4 Congo red assay of GPC in the presence of TFE 6. Scanning electron microscopy of GPC in the presence of TFE 230 DISCUSSION 234

CHAPTER 5: EFFECT OF HEAVY METALS (Zn+2 & Cd+2) ON STRUCTURE AND FUNCTION OF GARLIC PHYTOCYSTATIN 238-275 1. Functional study 238 1.1 Effect of heavy metals (Zn+2 and Cd+2) on the cysteine proteinase inhibitory activity of GPC 2. Structural study 238 2.1 Ultra-violet absorption study of GPC in the presence of heavy metals (Zn+2 and Cd+2) 2.2 Intrinsic fluorescence study of GPC in the presence of heavy metals (Zn+2 and Cd+2) 2.3 Stern-Volmer quenching analysis of GPC in the presence of heavy metals (Zn+2 and Cd+2) 3. Synchronous fluorescence study of GPC in the presence of heavy metals (Zn+2 and Cd+2) 246 4. Three-dimensional fluorescence study of GPC in the presence of heavy metals (Zn+2 and Cd+2) 252 5. Secondary structure analysis of GPC in the presence of heavy metals (Zn+2 and Cd+2) 260 5.1 CD analysis 6. Aggregation specific assay of GPC in the presence of heavy metals 262 (Zn+2 and Cd+2) 6.1 Thioflavin-T fluorescence of GPC in the presence of Zn+2 and Cd+2 6.2 Congo red measurements of GPC in the presence of Zn+2 and Cd+2 7. Scanning electron microscopy of GPC in the presence of heavy metals (Zn+2 and Cd+2) 264 DISCUSSION 269

CONCLUSION 276-279

BIBLIOGRAPHYMaulana Azad Library, Aligarh Muslim University 280-330

BIOGRAPHY

LIST OF ABBREVIATIONS

ANS 8-Anilino-1-naphthalenesulfonic acid

APS Ammonium persulfate

CD Circular dichroism

DTNB Dithionitrobenzoic acid

EDTA Ethylene diamine tetra acetic acid

ELISA Enzyme linked immunosorbent assay

FTIR Fourier transform infrared spectroscopy

g Gram

GPC Garlic phytocystatin

H Hours

kDa Kilo Dalton

mdeg Millidegree

M Molar

Min Minute

Maulanamg Azad Library,Milligram Aligarh Muslim University

ml Millilitre

mM Millimolar

MRE Mean residual ellipticity

PAGE Polyacrylamide gel electrophoresis

SDS Sodium dodecyl sulphate

i

SEM Scanning electron microscopy

TCA Trichloracetic acid

TEMED Tetraethyl methyl ethylene diamine

ThT Thioflavin T v/v Volume by volume

βME Beta mercapto ethanol

μg Microgram

μl Micro litre

μM Micro molar

Maulana Azad Library, Aligarh Muslim University

ii

LIST OF FIGURES

Figure No. Page No. INTRODUCTION

1 Mechanism of action of proteinases 8 2 Schematic representation of the catalytic mechanism involved in 17 various type of 3 Representation of the chain structure of cysteine proteinase inhibitors 25 4 Structure of human cystatin F 26 5 Representation of the proposed “Trunk model” 31 6 Structure of phytocystatin purified from sesame indicum 39 7 Scheme representing the classification of cystatin superfamily 40 8 Alignment of known phytocystatin representing the conserved 42 regions CHAPTER – 1

9 Elution profile of GPC on a Sephacryl S-100 HR column. 101 10 Gel electrophoresis of purified garlic phytocystatin. 103 11 SDS-PAGE of garlic phytocystatin under non-reducing and reducing 104 conditions. 12 Molecular mass determination of purified GPC using Sephacryl S- 106 100 HR gel filtration chromatography. 13 Log M (molecular weight) of marker proteins against relative 107 mobility (Rm) of molecular weight markers for determination of molecular mass. Maulana Azad Library, Aligarh Muslim University 14 Determination of Stokes radius of GPC 108 15 pH stability profile of GPC 110 16 Effect of temperature on the inhibitory activity of GPC. 111 17 Direct binding ELISA plot 112 18 Ouchterlony immunodiffusion of GPC. 114 19 Inhibitory activity of GPC against different proteinases. 115 20 Lineweaver-Burk plot representing the inhibitory effect of GPC on 117 papain.

iii

21 Dixon plot for determination of apparent Ki of GPC for papain. 118 22 Lineweaver-Burk plot representing the inhibitory effect of GPC on 119 bromelain.

23 Dixon plot for determination of apparent Ki of GPC for bromelain. 120 24 Lineweaver-Burk plot representing the inhibitory effect of GPC on 121 ficin.

25 Dixon plot for determination of apparent Ki of GPC for ficin. 122 26 UV-visible absorption spectra of GPC and GPC-papain complex. 126 27 Fluorescence emission spectra of GPC and GPC-papain complex. 127 28 Far-UV CD spectra of GPC and GPC - papain complex. 128 29 FTIR spectra of GPC and GPC - papain complex. 130 CHAPTER – 2

30 Cysteine proteinase inhibitory assay of GPC in the presence of urea 137 31 Cysteine proteinase inhibitory assay of GPC in the presence of 138 GdnHCl 32 Intrinsic fluorescence analysis of GPC in the presence of urea 140 33 Intrinsic fluorescence analysis of GPC in the presence of GdnHCl 142 34 ANS fluorescence analysis of GPC in the presence of urea 143 35 ANS fluorescence analysis of GPC in the presence of GdnHCl 144 36 Acrylamide quenching analysis of GPC in the presence of urea 147 37 Acrylamide quenching analysis of GPC in the presence of GdnHCl 148 38 Circular dichroism analysis of GPC in the presence of urea 151 39 Circular dichroism analysis of GPC in the presence of GdnHCl 152 40 FTIR analysis of GPC in the presence of urea 155 41 FTIRMaulana analysis of GPCAzad in theLibrary, presence Aligarh of GdnHCl Muslim University156 42 Unfolding of GPC in the presence of urea 158 43 Conformational Stability plot of GPC in the presence of urea 159 44 Unfolding of GPC in the presence of GdnHCl 160 45 Conformational stability plot of GPC in the presence of GdnHCl 161

iv

CHAPTER – 3 A

46 Cysteine proteinase inhibitory assay of GPC in the presence of 170 carbendazim 47 A UV-absorption analysis of GPC in the presence of carbendazim 172 (CAR) 47 B Relative absorption plot of GPC in the presence of carbendazim 173 48 A Intrinsic fluorescence spectra of GPC in the presence of carbendazim 174 (CAR) 48 B Relative fluorescence plot of GPC in the presence of carbendazim. 175 49 A Stern-Volmer analysis of GPC in the presence of carbendazim 177 49 B Modified Stern-Volmer analysis of GPC in the presence of 178 carbendazim 50 A Synchronous fluorescence spectra of GPC in the presence of 181 carbendazim (CAR) 50 B Synchronous fluorescence spectra of GPC in the presence of 182 carbendazim (CAR) 51 Isothermal titration calorimetry plot of GPC in the presence of 183 carbendazim 52 Circular dichroism analysis of GPC in the presence of carbendazim 185 (CAR) CHAPTER – 3 B

53 Cysteine proteinase inhibitory assay of GPC in the presence of 193 oxyfluorfen 54 A MaulanaUV-absorption Azad analysis Library, of GPC Aligarh in the presence Muslim of oxyfluorfen University 195 54 B Relative absorption plot of GPC in the presence of oxyfluorfen 196 55 A Intrinsic fluorescence spectra of GPC in the presence of oxyfluorfe 198 55 B Relative fluorescence plot of GPC in the presence of oxyfluorfen 199 56 A Stern-Volmer analysis of GPC in the presence of oxyfluorfen 200 56 B Modified Stern-Volmer analysis of GPC in the presence of 201 oxyfluorfen 57 A. Synchronous fluorescence spectra of GPC in the presence of 204 oxyfluorfen

v

57 B. Synchronous fluorescence spectra of GPC in the presence of 205 oxyfluorfen 58 Isothermal titration calorimetry plot of GPC in the presence of 207 oxyfluorfen 59 Circular dichroism analysis of GPC in the presence of oxyfluorfen 209 CHAPTER – 4

60. Cysteine proteinase inhibitory assay of GPC in the presence of TFE 217 61 A. Intrinsic fluorescence emission spectra of GPC in the absence and 219 presence of TFE 61 B. Relative fluorescence plot of GPC in the presence of TFE 220 62 A. ANS fluorescence emission spectra of GPC in the presence of TFE 222 62 B. Relative ANS fluorescence plot of GPC in the presence of TFE 223 63. Circular dichroism spectra of GPC in the absence and presence of 225 TFE 64. Turbidity assay of GPC in the presence of TFE 226 65. Rayleigh scattering measurement of GPC in the presence of TFE 228 66. ThT fluorescence spectra of GPC in the presence of TFE 229 67. Congo red absorbance spectra of GPC in the presence of TFE 231 68. Scanning electron microscopy of GPC in the presence of TFE 232 CHAPTER – 5

69. Cysteine proteinase inhibitory assay of GPC in the presence of heavy 239 metals (Zn+2 and Cd+2) 70 A. UV-absorption spectra of GPC treated with zinc 241 70 B. UV-absorptionMaulana spectra Azad of Library,GPC treated Aligarh with cadmium Muslim University242 71 A. Intrinsic fluorescence spectra of GPC treated with zinc 244 71 B. Intrinsic fluorescence spectra of GPC treated with cadmium 245 72 A. Stern-Volmer analysis of GPC in the presence of zinc 247 72 B. Stern-Volmer analysis of GPC in the presence of cadmium 248 72 C. Modified Stern-Volmer analysis of GPC in the presence of zinc 249 72 D. Modified Stern-Volmer analysis of GPC in the presence of cadmium 250 73 A. Synchronous fluorescence spectra of GPC in the presence of zinc 253 73 B. Synchronous fluorescence spectra of GPC in the presence of zinc 254

vi

73 C. Synchronous fluorescence spectra of GPC in the presence of 255 cadmium 73 D. Synchronous fluorescence spectra of GPC in the presence of 256 cadmium 74. Three-dimensional fluorescence spectra of GPC in the presence of 258 zinc 75. Three-dimensional fluorescence spectra of GPC in the presence of 259 cadmium 76. Circular dichroism analysis of GPC in the presence of Zn+2 and Cd+2 261 77. ThT fluorescence spectra of GPC in the presence of Zn+2 and Cd+2 263 78. Congo red absorbance of GPC in the presence of Zn+2 and Cd+2 265 79. Scanning electron microscopy of GPC in the absence and presence of 266 Zn+2 and Cd+2

Maulana Azad Library, Aligarh Muslim University

vii

LIST OF TABLES Table No. Page No. INTRODUCTION Table 1 Classification of the proteinases on the basis of their catalytic 5 mechanism, amino acid residue presents at the active sites and their specific inhibitor. Table 2 Molecular weight and number of amino acid residues present in 41 different phytocystatins. CHAPTER – 1 Table 3 Purification table of phytocystatin from garlic (Allium sativum). 100 Table 4 Kinetic parameters obtained upon interaction of GPC with 124 different proteinases– papain, ficin, and bromelain. CHAPTER – 2 Table 5 Stern-Volmer quenching constant (Ksv) of GPC in the presence 149 of an increasing concentration of urea and guanidine hydrochloride Table 6 Secondary structure analysis of GPC under denaturing conditions 153 using urea and guanidine hydrochloride. Table 7 Urea and guanidine hydrochloride induced unfolding parameters 162 of GPC CHAPTER – 3 A Table 8 Stern-Volmer quenching constant (Ksv) and binding constant (Kb) 179 for the interaction of GPC with carbendazim. Table 9 Secondary structure analysis of GPC incubated with carbendazim 186 Maulana Azad Library,CHAPTER Aligarh – Muslim 3 B University Table 10 Stern-Volmer quenching constant (Ksv) and binding constant (Kb) 202 for the interaction of GPC with oxyfluorfen. Table 11 Secondary structure analysis of GPC after 4 hours incubation 210 with oxyfluorfen. CHAPTER – 5 Table 12 Stern-Volmer quenching constant (Ksv) and binding constant 251 +2 +2 (Kb) for the interaction of GPC with Zn and Cd .

viii

Introduction

Maulana Azad Library, Aligarh Muslim University

Introduction

INTRODUCTION

1. PROTEINASES: GENERAL

Proteinases are the enzymes which catabolize proteins by hydrolysis of peptide bonds. They are ubiquitously found in animals, plants, bacteria, archaea, and viruses (Puente et al., 2003). Proteinases were previously considered mainly as a degradative enzyme whose primary function is the breakdown of proteins into amino acid residues and turnover of proteins. However, the concept of limited cleavage of proteins distinguishes proteinases from the simple non- specific degradation of proteins to specific cleavage of peptide bonds (Neurath, 1999). The specificity of proteinases have a vital role in the regulation of signaling pathways and therefore are involved in different processes viz. spore germination, blood coagulation, blood pressure control, proenzyme activation, processes and intestinal cell function (Antalis et al., 2007; Johnson and Pellecchia, 2006; Neurath, 1984). The signaling pathways are highly regulated, and any imbalance in the proteinase action may be responsible for various pathologies such as cancer, inflammatory diseases, osteoporosis, neurological and cardiovascular diseases (Turk, 2006). The proteinases are also involved in controlling different stages of growth and development, bone formation, ovulation, neuronal outgrowth, antigen presentation, cell-cycle regulation, wound healing, immune and inflammatory cell migrationMaulana as Azadwell as Library, activation, Aligarh angiogenesis Muslim, and apoptosisUniversity (Barrett et al., 1998; Kepler, 2002). Proteinase genes share about 2% of the mammalian genome whose expression results are enzymes which are important for biological processes such as development, cell death, coagulation, immunity and inflammation (Turk, 2006). It is estimated that approximately 10% of the enzymes are included in the enzyme classification list (Barrett et al., 2001; Rawlings et al., 2004). Proteinases are also known as peptidases which comprise one of the largest functional groups of proteins with more than 560 members (Barrett et al., 1998). Peptidases are broadly classified on the basis of 1 | P a g e

Introduction site of action. Exopeptidases cleave N- or C- terminal peptide bonds in proteins while endopeptidases act on the internal peptide bonds. The exopeptidases comprise of aminopeptidases and carboxypeptidases (Rao et al., 1998). Aminopeptidases cleave peptide bond from the N-terminal of the protein chain and generate either single amino acid or dipeptide. On the other hand, carboxypeptidases act on the C-terminal of the protein chain and generate single amino acids or dipeptides. They are also classified on the basis of pH range (acid, neutral or alkaline) and specificity (keratinase, elastase and collagenase). The proteinases have been classified into four major classes viz. serine proteinases, aspartic proteinases, cysteine proteinases, and metalloproteinases on the basis of their catalytic mechanism and affinity to respective inhibitors (Table 1) (Hartley, 1960). However recently, the proteinases have been classified into seven families according to the key catalytic group such as serine (Ser), threonine (Thr), cysteine (Cys), aspartic (Asp), asparagine (Asn), glutamate (Glu), or metal ion present at their active site (Oda, 2012). Glu, Asp and metalloproteinases activate a water molecule which further acts as a nucleophile while Cys, Ser, and Thr directly act as a nucleophile and attack amide carbonyl.

1.1 SERINE PROTEINASES (EC 3.4.21)

Serine proteinases have emerged as the most abundant and functionally diverse proteinase group (M. J. Page and Di Cera, 2008; Michael J Page and Di Cera, 2008). SerineMaulana proteinases Azad are compris Library,ed ofAligarh more than Muslim one-third University of all known proteinases which have been grouped into 13 clans and 40 families. The name of the family arises from the nucleophilic Ser residue in the active site of the enzyme which acts on the carbonyl residue of the peptide bond to form an acyl-enzyme intermediate (Hedstrom, 2002). The of Asp 102, His 57 and Ser 195 residues is responsible for the nucleophilicity of the catalytic Ser residue which is also called as the charge relay system (Blow et al., 1969). Subtilisin, trypsin, ClpP and prolyl oligopeptidase employs the Asp-

2 | P a g e

Introduction

His-Ser catalytic triad in an identical configuration to catalyze the hydrolysis of peptide bonds of the substrate (M. J. Page and Di Cera, 2008). Most of the serine proteinases follow a dyad mechanism in which Lys or His residue is paired with the catalytic Ser residue. Rest of the serine proteinases catalyze the hydrolysis of peptide bonds through novel triads of residues such as a pair of His residue combined with the nucleophilic Ser residue. Trypsin under a major genetic expansion resulted in a proteinase responsible for diverse functions such as digestion, fibrinolysis, apoptosis, fertilization, development, and immunity (Di Cera, 2009).

1.2. ASPARTATE PROTEINASES (EC 3.4.23)

Aspartate proteinases are also called as acidic proteinases and belong to pepsin family. These proteinases are present in bacteria, fungi, viruses, plants, and animals. They are endopeptidases which include digestive enzymes such as chymosin, pepsin, lysosomal D and fungal proteinases. The proteinases have two conserved aspartate residues at their catalytic site which participate in the formation of catalytic triad required for the hydrolysis of a peptide bond. Moreover, it also needs an activated water molecule bound to one or more aspartate residues for the breakdown of peptides and proteins (Suguna et al., 1987). These proteinases are highly specific and cleave dipeptide bonds along with hydrophobic residues and a beta-methylene group. These proteinases follow general acid-base mechanism for the proteolysis which involves coordination between a water molecule and aspartate residues Maulana Azad Library, Aligarh Muslim University (Brik and Wong, 2003; Suguna et al., 1987).

1.3. METALLOPROTEINASES (EC 3.4.24)

Metalloproteinases are another group of proteinases which require metal ion for the catalytic process. They are found in fungi, bacteria and higher animals. These proteinases exhibit wide variation in the primary sequence and structure but generally contain a zinc ion which is responsible for the . In some

3 | P a g e

Introduction proteinases, zinc ion is replaced by cobalt or nickel ion without altering the activity of the enzyme.

The proteolysis mechanism involves the generation of a non-covalent tetrahedral intermediary complex after the attachment of a metal-bound water molecule on the carbonyl group. The newly generated complex is decomposed by transfer of the glutamic acid proton to the leaving group (Skiles et al., 2004). A chelating agent such as EDTA binds to metalloproteinase and completely inactivates the activity of metalloproteinases. The members of this family include carboxypeptidases A, carboxypeptidases B and thermolysin (Skiles et al., 2004).

1.4. THREONINE PROTEINASES (EC 3.4.25)

Threonine proteinase is one of the families of proteinases which bear threonine (Thr) residue at their active sites. The catalysis required secondary alcohol of the N-terminal threonine as a nucleophile (Brannigan et al., 1995).

1.5. GLUTAMIC PROTEINASES (EC 3.4.23.32)

Glutamic proteinases are the proteolytic enzymes containing glutamic acid residue at the active site of catalysis. It was firstly reported in the year 2004 and emerged as the sixth group of proteinases (Fujinaga et al., 2004). It was thought that glutamic proteinases are only present in filamentous fungi (Sims et al.,

2004). HoweverMaulana they have Azad been Library, now reported Aligarh in archaea Muslim as Universitywell as bacterial species (Jensen et al., 2010). These proteinases have been identified in the fungi Scytalidium lignicola (Fujinaga et al. 2004) and Aspergillus niger (Sims et al., 2004). The catalysis is done by two enzymes scytalidoglutamic and aspergilloglutamic proteinase. Glutamic proteinases form a catalytic dyad with the help of glutamic acid and glutamine which play a vital role in the catalysis.

4 | P a g e

Introduction

Table 1: Classification of the proteinases on the basis of their catalytic mechanism, amino acid residue present at the

active sites and their specific inhibitor.

Catalytic group Specific present Other Example of Type of proteinase Inhibitors at the Inhibitors proteinase active site Antipain Trypsin, DIFP Chymostatin Serine proteinases Ser, His, chymotrypsin, PMSF Leupeptin Cys subtilisin, (EC 3.4.21) TPCK elastases TLCK N-ethyl Leupeptin, Cysteine maleimide TLCK, Papain, bromelain, Iodoacetate Cys Antipain, proteinases ficin, cathepsins (EC 3.4.22) Iodoacetamide TPCK, Heavy metals Chymostatin Diazoacetyl- DL-norleucine methyl ester, Aspartic Pepstatin proteinases S-PI (acetyl Asp, Tyr D and (EC 3.4.23) pepstatin), E, pepsin, renin 1,2-Epoxy-3(P- nitrophenoxy) propane, EDTA, Ethylene glycol-bis- (β- Maulana Azadaminoethyl Library, Aligarh Muslim University

ether)- N, N- Metalloproteinases Carboxypeptidases tetraacetic A and B, (EC 3.4.24) acid, Metal Ions Thermolysin o- phenanthroline, α, α’- DIPYRIDYL,

5 | P a g e

Introduction

1.6. ASPARAGINE PEPTIDE LYASES (EC 4.3.2)

Asparagine peptide lyases are the seventh group of catalytic proteinases and have an asparagine residue at their catalytic site. The proteolysis mechanism requires an asparagine residue which functions as a nucleophile and performs nucleophilic elimination reaction (Rawlings et al., 2011). They are synthesized as pro-peptides or precursors which can be auto-cleaved (Tajima et al., 2010). They are present in microorganisms, insects as well as in mammals. The proteolysis mechanism was deciphered with the discovery of the crystal structure of the self-cleaving precursor of the Tsh autotransporter from E. coli. (Tajima et al., 2010). There are ten families of asparagine peptide lyases which are classified in three different kinds of proteins viz. viral coat proteins, autotransporter proteins and intein containing proteins. There are two families of autotransporter, three families of intein-containing proteins and five families of viral coat proteins (Rawlings et al., 2016).

1.7. CYSTEINE PROTEINASES (EC 3.4.22)

Cysteine proteinases are also known as thiol proteinases and contain a cysteine residue at their active site which plays essential role in the hydrolysis of proteins. The representation of the mechanism involved in the cleavage of the peptide bond by cysteine proteinases is given in figure 1. The catalytic site of cysteine proteinase comprises of conserved residues which include cysteine, , andMaulana asparagine Azad (Fabien Library, Lecaille Aligarh et al., 2002)Muslim. The University Cys residue is positioned in a highly conserved peptide sequence, CGSCWAFS (Fabien Lecaille et al., 2002). Of all the families of cysteine proteinases discovered so far, half of them belong to viruses (Otto and Schirmeister, 1997). The mode of catalysis is somewhat different in these proteinases which involves the dissociation of a proton from a sulfhydryl group of cysteine at physiological pH much below the pK value, for example, bromelain and papain. Papain is the best-characterized family of cysteine proteinases. This family consists of proteinases which are structurally similar to papain e.g. lysosomal cathepsins.

6 | P a g e

Introduction

Papain is characterized by a two-domain structure in which the active site is located between the domains. Cys 25 and His 159 are the evolutionary preserved catalytic residues preserved in all cysteine proteinases.

Recently, cysteine proteinases have gained importance because of their numerous essential roles in the physiological processes. They are responsible for lysosomal protein degradation and play a role in different metabolic disorders of vertebrates (Cheng et al., 2012; Tryselius and Hultmark, 1997). They act as digestive enzymes in invertebrates such as nematodes and mites (Pernas et al., 1998; Rawlings and Barrett, 1994). The catalytic activity is due to the presence of highly reactive thiol group (-SH) of the cysteine residue at the catalytic site. They include both endopeptidase and exopeptidase, e.g. dipeptidyl peptidase and cathepsin present in the of cells of higher animals (Nagler et al., 1999; Stoka et al., 2005).

1.8. MAMMALIAN CYSTEINE PROTEINASES

The cysteine proteinases present in the mammalian system is referred to as mammalian cysteine proteinases. They are responsible for intracellular protein catabolism, extracellular protein degradation like bone resorption and macrophage function. They also regulate the signaling pathways by selective activation of signaling molecules such as interleukin, enkephalin, and protein kinase C (Rakash et al., 2012). They are present in the cytosol as , in lysozyme as cathepsins B, L, H, and S and can also be found in extracellular Maulana Azad Library, Aligarh Muslim University secretion under pathological conditions. Willstatter and Bamann coined the term “cathepsin” in 1929 for proteinases which are active at acidic pH but different from pepsin (Willstatter, R., & Bamann, E. 1929). They are the widespread proteinases present in lysosomes of the mammalian system. Fruton et al. identified three different enzymes in cathepsin preparations viz. cathepsin I, II and III (Fruton et al., 1941). The cysteine proteinase has four main groups which are lysosomal cathepsins, calpains, , and legumain.

7 | P a g e

Introduction

Maulana Azad Library, Aligarh Muslim University Figure 1: Mechanism of action of proteinases. N is a nucleophile, B is a base acting as a nucleophile and AH is a proton donor.

8 | P a g e

Introduction

LYSOSOMAL CATHEPSINS

Lysosomal cathepsins are cysteine proteinases belonging to clan CA and family C1. These proteinases are primarily found in the cell but recent investigations have identified them in other parts of the cell, and few members are ubiquitous in the human body. They showed variety based on the difference in the catalytic residue such as cathepsins A and G are serine proteinase, cathepsins D and E belong to aspartate proteinase while the others are lysosomal cysteine proteinases which include human isoforms B, C, F, H, K, L, O, S, V, X and W (Turk et al., 2012).

Cathepsins B, H, L, C, X, F, O, and V are ubiquitously expressed in human tissues while lysosomal is present in osteoclasts, epithelial cells and synovial fibroblasts of rheumatoid arthritis joints (Salminen- Mankonen et al., 2007). Recent advancements have shown that cysteine cathepsins are also present in nucleus, cytoplasm and plasma membrane where active cathepsin L variants play vital role in the regulation of cell-cycle progression and the proteolytic processing of the N-terminus of histone H3 tail (Duncan et al., 2008; Goulet et al., 2004; Maubach et al., 2008; Santos-Rosa et al., 2009; Turk et al., 2012). Most of the cathepsins are small monomeric proteins of molecular weight in the range of 24-35 kDa except which is an oligomeric protein of around 200 kDa. They are optimally active in slightly acidic medium except for which is active at neutral or slightly basic medium (Kirschke et al., 1989; V. Turk et al., 2002). Except for Maulana Azad Library, Aligarh Muslim University cathepsins S, all mature cathepsins are generally glycosylated. They are involved in terminal protein degradation, cell proliferation and adhesion, apoptosis, lipid metabolism and immune response (Cheng et al., 2011; Turk et al., 2001).

Human cathepsins play a vital role in processing and activation of different proteins which also includes proteinases, intracellular protein turnover, bone remodeling and in antigen processing and presentation. The

9 | P a g e

Introduction lysosomal cysteine cathepsins perform various specific functions and involved in regulation of different physiological processes viz. bone remodeling, keratinocyte differentiation, MHC-II-mediated antigen presentation, prohormones activation (Turk et al., 2000; V. Turk et al., 2002; Vasiljeva et al., 2006), osteoarthritis, osteoporosis, rheumatoid arthritis (Hashimoto et al., 2001; YASUDA et al., 2005), pancreatic pathologies (van Acker et al., 2002), cardiovascular diseases (CVD) (Lutgens et al., 2007), cancer (Gocheva and Joyce, 2007; Keppler, 2006) and neurological disorders (Nakanishi, 2003) etc. The cathepsins actively participate in apoptosis, but their role is still undiscovered (Stoka et al., 2005; Turk and Stoka, 2007). Numerous evidence suggest its role in genetic disorder due to mutations in genes of cathepsins and their specific inhibitors.

CALPAINS

Calpains are Ca2+-dependent cytosolic cysteine proteinases which are neutral. They are ubiquitous and tissue-specific isoforms; the total number of isoforms of calpains are fifteen in which eleven are identified in humans (Huang and Wang, 2001; Sorimachi et al., 2011). The members of this family were emerged in 1964 and termed as calcium-activated neutral proteinase (CANP) (Guroff, 1964). family was comprised of two ubiquitously distributed mammalian calpains: calpain 1 (calpain I, μ-calpain and CAPN1) and calpain 2 (calpain II, m-calpain and CAPN2) (Yoshimura et al., 1983). Calpains sever numerous cytosolicMaulana proteins Azad and Library, have been Aligarh embroiled Muslim in fundamental University cellular processes along with cell multiplication, apoptosis, and differentiation (Perrin and Huttenlocher, 2002). Calpains are involved in cell migration as they can change the architecture of cell adhesions and cytoskeletal apparatus, and also has shown its association in intracellular flagging pathways. Neurological disorders like traumatic brain injury and stroke have been linked to overexpression and activation of calpain 1 and calpain 2. Alzheimer’s disease has been recently linked with the malfunctioning of calpains. Similarly limb-

10 | P a g e

Introduction girdle muscular dystrophy 2A is also linked with the results due to the loss-of- function mutations of the calpain 3 gene (Huang and Wang, 2001).

CASPASES

Caspases belong to the family of proteinases found in all metazoans, and a dozen of them are present in humans which regulate the terminal stages of apoptosis, cellular remodeling, and inflammatory response. They are highly conserved cysteine-dependent aspartate-specific proteinase which utilizes cysteine residue as their catalytic nucleophile (Alnemri et al., 1996). They are also known as the cytoplasmic interleukin-1β-converting enzyme which is highly homologous to C.elegans cell death gene CED-3 (Fan et al., 2005). The members of this family were firstly recognized as the proteinases which have been found to be responsible for the proteolytic maturation of pro-IL-1β (interleukin-1β -converting enzyme) (Black et al., 1989; Kostura et al., 1989) which was then purified and cloned as -1(Cerretti et al., 1992; Thornberry et al., 1992). Around 14 caspases have been identified and contain a conserved pentapeptide active site QACXG (X can be R, Q or D), along with this they also share other common characteristics among them. They all are synthesized as zymogens which are called procaspases and are involved in activation of other caspases along with self-activation (Launay et al., 2005). Caspases are involved in the development of tumors (Olsson and Zhivotovsky, 2011), while overexpression of caspases leads to enhanced programmed cell death Maulana which is Azadthe implication Library, Aligarhof numerous Muslim neurodegenerative University diseases (Friedlander et al., 2000; Li et al., 2000).

LEGUMAIN

The term ‘Legumain’ was referred to an endopeptidase which is present in leguminous and other seeds. It was isolated and characterized from Vigna aconitifolia (moth bean), Phaseolus vulgaris (Csoma and Polgar, 1984; Shutov

11 | P a g e

Introduction et al., 1982), while the mammalian legumain was identified as putative cysteine proteinase PRSC1 in humans (Tanaka et al., 1996). They have been now widely identified in plants, mammals, trematodes, blastocysts and ticks which are available on the MEROPS database (Rawlings et al., 2010). Mammalian legumain has shown wide tissue distribution and most abundantly present in testis and kidney (Chen et al., 1998, 1997). Legumain is a cysteine endopeptidase exhibiting high specificity for the cleavage of asparagine bonds. The peptidase belongs to peptidase family C13 however they are not related to the cysteine proteinase of the C1 family of papain (Rawlings and Barrett, 1994). Recent, advancements have shown that legumain which is also called as asparagine endopeptidase (AEP) or vacuolar processing enzyme (VEP) is usually connected with its cysteine endopeptidase activity in lysosomes where it contributes to antigen processing for class II MHC presentation (Dall and Brandstetter, 2016). They are involved in the processing and clearance of hemoglobin from the gut lumen of ticks (Grandjean and Aeschlimann, 1973), activation of TLRs, receptors of the innate immune system (Maschalidi et al., 2012), inhibition of osteoclast formation thereby restricting bone resorption (Choi et al., 1999; Kubota et al., 2003), thereby exhibiting that majority of the functions are related to its proteinase activity.

1.9. PLANT CYSTEINE PROTEINASES

Cysteine proteinases are widely distributed in the plant system and play a central role Maulana in different Azad proteolytic Library, pathways Aligarh (Grudkowska Muslim University and Zagdanska, 2004). They are involved in multi-functions ranging from programmed cell death, germinating seeds, seedling growth, and development (Turk et al., 1997). The cysteine proteinase plays a crucial role in protein maturation, housekeeping function, and protein degradation. These functions require controlled proteolysis which cannot be achieved without a highly regulated system, thereby confirming that cysteine proteinases work in a highly regulated manner. The regulation is achieved by cysteine proteinase inhibitors which

12 | P a g e

Introduction control the malfunctioning of cysteine proteinases. Different kinds of inhibitor are present in the system which is expressed during different stages of growth and development and regulate the proteolysis. The cysteine proteinase activity increases significantly in response to different internal and external factors and can ascend to 90% of the total proteolysis activity (Wiśniewski and Zagdańska, 2001).

In plants, papain is the most studied member of the cysteine proteinase and is monomeric. Papain and actinidin are cysteine proteinases found in papaya and kiwifruit whose complete primary structure and tertiary structure has been deciphered. They are important intracellular proteinases, having cysteine and histidine residue at their catalytic site as a key amino acid. The cysteine works as a nucleophile while histidine performs as a general base for delocalization of proton. The catalytic process involves contact of the amino acid residues present at the active site with the residues of the substrate in order to advance the catalytic process. Few of the important plant proteinases are discussed below:

Papain (EC 3.4.22.2)

Papain is a plant cysteine proteinase extracted from the latex of Carica papaya. It is the most widely investigated member of the cysteine proteinase class of enzyme and has been considered archetype of cysteine proteinases and accordingly, family C1 is referred to as the papain family of cysteine proteinases.Maulana It has Azad been Library,in use for Aligarhlong ages Muslimand is considered University as a traditional cysteine proteinase. It is stable in the pH range of 5 to 9 and withstands heat up to 80°C to 90°C in the presence of its substrate. Papain is a single chain non- glycosylated polypeptide of around 212 amino acids along with three disulfide bridges and molecular weight of 23406 Da. The protein is basic with a pI of 8.75. The catalytic triad is made up of three highly conserved residues viz. Cys, His and Asn which are responsible for maintaining an active enzyme conformation. The polypeptide forms a globular structure with two interacting

13 | P a g e

Introduction domains delimiting a cleft at the surface of the enzyme where substrates can bind. The catalytic residues His159 & Cys25 are present at this interface on opposite domains. It is considered that the active papain consists of a thiolate- imidazolium ion pair formed by the active site amino acid His 159 & Cys25 (Cstorer and Ménard, 1994).

Bromelain (EC 3.4.22.32)

Bromelain is a cysteine proteolytic enzyme derived from pineapple (Ananas comosus) and shows an anti-inflammatory effect. The term bromelain is collectively used for a peptide hydrolyzing enzyme which is found in plant parts such as stem, leaves and fruit peels of the Bromeliaceous family. However, stem bromelain is used for the proteinase present in extracts of the plant stem, and fruit bromelain is the major proteinase in the fruit. Stem bromelain and fruit bromelain is a single-chain polypeptide of 24.5 kDa with a pI value of 9.55 and 4.6 respectively Harrach et al., 1995; Murachi, 1976; Ota et al., 1964). They contain seven cysteine residues which may result in three disulfide linkages (Napper et al., 1994). Stem bromelain is a glycosylated protein while the presence of carbohydrate content is unclear in case of fruit bromelain. The secondary structure of stem bromelain is relatively unchanged between pH 7 to 10 but loses its structure above pH 10 (Dave et al., 2010). Sulfhydryl group is responsible for the proteolysis activity of the bromelain enzyme isolated from stem pineapple. The complete primary sequence of stem bromelain has been deciphered and found to be a member of the papain family. Maulana Azad Library, Aligarh Muslim University Both of them are also immunologically distinct from one another (Ota et al., 1964).

Ficin (EC 3.4.22.3)

Ficin is a generic term coined by Robbins and purified from any member of the genus Ficus (ROBBINS, 1930). It is a cysteine endopeptidase with multiple isoforms and anti-helminthic property (Neuberger and Brocklehurst, 1987). It is most commonly used in blood group antigens differentiation (Hill et al.,

14 | P a g e

Introduction

2017). The enzyme is a single polypeptide chain and the amino acid composition is similar to that of papain but has an additional Cys residue (Englund et al., 1968). The amino acid sequences have been deciphered around the catalytic site and have shown high similarity to those found in and around the catalytic sites in papain (Husain and Lowe, 1970).

1.10. BACTERIAL AND VIRAL CYSTEINE PROTEINASES

Potempa et al. discovered that Staphylococcal aureus produces many proteinases which include cysteine proteinases (Potempa et al., 1988). is a cysteine proteinase isolated from Clostridium histolyticum. It possesses specificity for the carboxyl peptide bond of Arg and requires a cysteine thiol group along with calcium ions for its activity. It shows optimum activity in the pH range 7.6–9.0 and pI of the enzyme is around 4.8. It is a heterodimer made up of two chains with molecular weight 45 kDa and 12.5 kDa (Gilles et al., 1984). This group of cysteine proteinases belongs to the MEROPS peptidase family C11. The picornaviruses are small and non- enveloped viruses containing a positive-sense single-stranded RNA as genetic material (Fields et al., 2001). This family includes numerous human and animal pathogens viz. foot-and-mouth disease virus, hepatitis A virus, polioviruses, and rhinoviruses. The viral genome encodes a single polypeptide, which is further processed by virally encoded 3C and 2A cysteine proteinases (Fields et al., 2001). The 3C proteinase is primarily involved in the processing of the polypeptide. The catalytic site of 2A proteinase is located at the N-terminus of Maulana Azad Library, Aligarh Muslim University the proteinase and catalyzes the release of the structural polypeptide (Toyoda et al., 1986). Crystal structure of 2A and 3C proteinases from various picornaviruses such as hepatitis A virus, human rhinovirus and poliovirus revealed that both the proteinases are chymotrypsin-like cysteine proteinases (Allaire et al., 1994; Bergmann et al., 1997; Mosimann et al., 1997).

15 | P a g e

Introduction

1.11. MECHANISM OF ACTION OF PROTEINASES

Proteinases hydrolyze the amide bonds of proteins and polypeptides. The hydrolysis of the peptide bond is done by the polarisation of the amide bond which takes place by nucleophilic attack on the carboxyl bond, which is aided by the contribution of a proton to the amide nitrogen. There is generally two kinds of mechanism which can be followed by proteinases for hydrolyzing the amide bonds. A water molecule is activated by aspartic, glutamic and metalloproteinases which act as a nucleophile and catalyzes the hydrolysis of amide bonds. The second mechanism of amide hydrolysis is followed by threonine, serine and cysteine proteinases which exploit amino acid residues as a nucleophile which is usually present in the catalytic triad. The eponymous residue presents at the active site with a proton withdrawing group plays an important role in enhancing the nucleophilic attack of cysteine and serine proteinases. Nonetheless, all the mechanisms that hydrolyze peptide bonds are fundamentally the same for almost all proteinases. All the serine proteinases have a catalytic triad present at their active sites with few exceptions like intermembrane serine proteinases which have dyad in spite of triad (Figure 2) (Erez et al., 2009). The triad at the catalytic site is made up of three amino acid residues aspartic acid, serine, and histidine. The histidine along with aspartic acid withdraws a proton of a serine residue which helps in a nucleophilic attack on the carbonyl carbon of the substrate. The salient feature of a serine proteinase is the stabilization of tetrahedral complex transition state through the generation ofMaulana an oxyanion Azad hole. Library, Aligarh Muslim University

The cysteine proteinase follows the similar mechanism of catalysis which is followed by serine proteinase with a little bit difference in the nucleophile (Figure 2). The nucleophile of the cysteine proteinases is a sulfur atom of a cysteine instead of oxygen atom of a serine residue. The formation of the accompanies cysteine proteinases. Serine and cysteine proteinases form a covalent intermediate with the substrate while aspartyl proteinases do not form a covalent intermediate.

16 | P a g e

Introduction

Figure 2. The catalytic triad/dyad in the active site of a) serine proteinase b) cysteine proteinase c) aspartyl proteinase and d) metalloproteinase which acts as proton donor or acceptor to stabilize the enzyme-substrate complexMaulana in peptide Azad bond Library, hydrolysis Aligarh (Erez Muslim et al., 2009) University. The polypeptide is well represented with grey lines while the small pale blue curve in “a” and “b” represents oxyanion holes and the large curve indicates the enzyme schematically. Delocalization of electron pairs is indicated with red arrows while the dotted blue lines show hydrogen bonds or electrostatic bonding.

17 | P a g e

Introduction

Aspartyl proteinases catalyze the hydrolysis of a substrate by a general acid-base mechanism which requires the coordination of a water molecule between two aspartic acids at the catalytic site (Figure 2) (Brik and Wong, 2003; Suguna et al., 1987). A water molecule gets activated upon removal of a proton by one of the aspartates at the catalytic site of aspartyl proteinase thereby promoting the water molecule to attack the carbon of the substrate scissile bond and generation of a tetrahedral oxyanion intermediate. The newly generated intermediate undergoes rearrangement process and leads to the formation of from the substrate. Metalloproteinase is a self-explanatory term, which exploits metal ions and mostly requires zinc ion for the catalysis of the substrate (Figure 2). They require two or three histidine residues along with an acidic side chain for maintaining most of the soluble metalloproteinases.

1.12. REGULATION OF CYSTEINE PROTEINASES

The cysteine proteinases are widely distributed in animal and plant system and play important physiological functions. In the animal system, they are responsible for intracellular protein catabolism, extracellular protein degradation like bone resorption, macrophage function, and also regulate the signaling pathways by selective activation of signaling molecules such as interleukin, enkephalin, and protein kinase C. They are also involved in pancreatic pathologies (van Acker et al., 2002), cardiovascular diseases (CVD) (Lutgens et al., 2007), cancer (Gocheva and Joyce, 2007; Keppler, 2006) and neurological Maulanadisorders (Nakanishi, Azad Library, 2003). InAligarh plant systems, Muslim cysteine University proteinases play multi-functions ranging from programmed cell death, germinating seeds, seedling growth and development, protein maturation, housekeeping function, and protein degradation. Hence, it is immensely important to regulate or control the proteolysis by cysteine proteinases. The proteinase regulation is achieved in vivo through zymogen activation, gene expression, post- translational modifications, compartmentalization, accessibility to the location of a susceptible peptide bond in the substrates and by the action of their

18 | P a g e

Introduction endogenous inhibitors or by employing all the factors together (Lopez-Otin and Bond, 2008; Neurath, 1999). The regulation is also achieved by pH as in case of cathepsins. Most of the cathepsins are active and stable at slightly acidic pH; however, they get inactive at neutral pH (Almeida et al., 2001; Turk et al., 1995a). The endogenous protein inhibitors play a significant role in the inhibition of proteinases. The major proteinase inhibitors include cystatins, thyropins and serpins (Abrahamson et al., 2003; B. Turk et al., 2002; Turk et al.,1997; Turk and Bode, 1991). They are highly efficient at their physiological concentrations even at neutral pH, thus signifying their inhibitory potential under such conditions (Turk et al., 1994). Cystatins are the largest group of cysteine proteinase inhibitors which are ubiquitously found in all animals and plants. They regulate the proteolysis of cysteine proteinases and maintain proteinase-anti proteinase balance thereby limiting the potentially inaccurate activity of their target proteinases.

2. MAMMALIAN PROTEINASE INHIBITORS

Proteinases are regulated through their specific proteinase inhibitors, which serve the function to inhibit the proteolysis when required. They hamper normal functioning of the enzyme when binds to an active site or other than active site, thereby controlling the catalysis process. The proteinase inhibitors have shown a potential usage for exploiting them in various field of study such as pharmacology and agriculture (Ahn et al., 2004; Bode and Huber, 2000; Copeland, 2005; Imada, 2005). There may be different kinds of proteinase inhibitors based on their specificity for the substrate. The Maulana Azad Library, Aligarh Muslim University specific inhibitor includes cystatins while non-specific inhibitors are alpha-2 macroglobulin. Alpha-2 macroglobulin (α2M) is a tetrameric polypeptide and shows inhibitory effect towards all catalytic classes. In some cases, synthetic proteinase inhibitors also inhibit the proteolysis, for instance, cysteine proteinases isolated from larvae of insect are inhibited by synthetic as well as naturally occurring inhibitors (Wolfson and Murdock, 1987). Inhibitors isolated from animal sources inhibit some of the cysteine proteinases of bacteria (Blankenvoorde et al., 1998) and virus (Bjorck et al., 1990).

19 | P a g e

Introduction

3. PLANT PROTEINASES INHIBITORS

Proteinase inhibitors are present in almost all parts of the plant. They are ubiquitous and control the malfunctioning of proteinases. Many proteinase inhibitors have been isolated and investigated from plants (Bijina et al., 2011; Green and Ryan, 1972; Joshi et al., 1998). The serine proteinase inhibitors are the most investigated member of the family and have been isolated from Leguminosae seeds (Macedo et al., 2002; Mello et al., 2001; Oliva et al., 2000; Souza et al., 1995). The legume seeds contain different inhibitors which are classified as Bowman-Birk-type, Kunitz-type, potato I, potato II, cereal superfamily, squash, Ragi A1 and thaumatin-like inhibitors. The plant proteinase inhibitor halts the metabolic activities within the gut of insects resulting in the poor nutrient uptake, retarded growth, and development which ultimately leads to death (Gatehouse et al., 1999). The proteinase inhibitors of plants contribute 1 to 10% of the total protein content present in most of the storage organs and are responsible for the inhibiting the activity of different enzymes (Ryan and Walker-Simmons, 1981). The inhibitors also perform a defensive role against abiotic stress and hazards during germination and growth in plants (Pernas et al., 1998). Isolation and biochemical characterization of Kunitz inhibitor have emphasized on the defensive role of plant proteinase inhibitors against pathogen assaults. After this, the soybean trypsin inhibitor was elucidated and found to be highly toxic against the larvae of flour beetle, Tribolium confusum (Lipke et al., 1954). Maulana Azad Library, Aligarh Muslim University Several reports also confirmed the potential toxic effect of proteinase inhibitors on pathogens. In vitro and in vivo assay against insect gut proteinase confirmed retardation of growth in insect species (Koiwa et al., 1997; Pannetier et al., 1997; Samac and Smigocki, 2003; Urwin et al., 1997; Vain et al., 1998). Different kind of plant proteinase inhibitors have been isolated from various plant sources viz. trypsin inhibitors from Hyptis suaveolens (Aguirre et al., 2004); chymotrypsin inhibitors from Solanum tuberosum (Valueva et al., 2003); cysteine inhibitors from chestnut fruit (Pernas et al., 1999); serine

20 | P a g e

Introduction inhibitors from Archidendron ellipticum (Bhattacharyya et al., 2006). The proteinase inhibitors restrict the attachment of substrate to the active site of proteinases through steric hindrance thereby inhibiting the catalysis process.

4. SPECIFIC INHIBITOR OF CYSTEINE PROTEINASES: CYSTATINS

Cystatins are cysteine proteinase inhibitors ubiquitously found in animals and plants. They play essential roles in a living system such as protect the host from different kinds of assaults and damages due to malfunctioning or overexpression of cysteine proteinases (Bjorck et al., 1990; Bobek and Levine, 1992; Jarvinen, 1978; Sloane and Honn, 1984). The cysteine proteinase inhibitor was first identified as a heat stable inhibitor of in a rat liver tissue (Finkenstaedt, 1957). The first cysteine proteinase inhibitor was isolated and partially characterized from chicken egg white and showed inhibitory activity effects towards papain, ficin, cathepsin B and C (Finkenstaedt, 1957; Keilova and Tomasek, 1975). A J Barret coined the term cystatin in 1986 for the inhibitors which show specificity towards cysteine proteinases and inhibit their activity. Since then the isolation, purification, and characterization of cysteine proteinase inhibitors have been carried forward till now. The inhibitor was isolated and purified from different animal and plant sources. They have been isolated from animal’s body fluids, different organs and tissues (Barrett and Salvesen, 1986). They have also been isolated from rice and soybean (Abe et al., 1988; Brzin et al., 1990).

MaulanaThe cysteine Azad proteinase Library, inhibitors Aligarh isolatedMuslim and University characterized from different sources exhibited functional and structural homology. They all are active inhibitors of cysteine proteinases and hence evolved as a superfamily of related evolutionary proteins which restricts the functioning of cysteine proteinases (Barrett, 1981). For the first time, the intracellular cysteine proteinase inhibitors of papain, cathepsin B and H were extracted and partially characterized from pig spleen and leucocytes (Kopitar et al., 1978). The human stefin and chicken cystatin extracted from polymorphonuclear granulocytes

21 | P a g e

Introduction inhibit cysteine proteinase activity, but the primary sequence does not show homology between them (Machleidt et al., 1983; Turk et al., 1983). The cysteine proteinase inhibitors have also been extracted from sera of patients ailing from autoimmune disorders and show complete homology of its 47 amino acid N-terminal sequence with a small basic protein human γ-trace which is the first report of the functional human cystatin (Grubb et al., 1983; Turk et al., 1983). It was observed that the amino acid sequence shares homology with chicken cystatin, hence the name human cystatin was introduced, and later it was renamed to human cystatin C (HCC) (A J Barrett et al., 1984; Brzin et al., 1984).

The “Cystatin Superfamily” has been classified on the basis of size, structure, and complexity into three families namely stefins, cystatins, and kininogens (Barrett and Salvesen, 1986; Barrett 1986b). A large number of cysteine proteinase inhibitors have been characterized by the animal as well as plant sources. A new family has been introduced for the plant cysteine proteinase inhibitors which are called as “phytocystatins.” Phytocystatins are cysteine proteinase inhibitors present in plants and share structural and functional homology with animal cystatins. Their salient features are intermediate to that of stefins and cystatins family (Brown and Dziegielewska, 1997; Margis et al., 1998; Pavlova and Sveriges, 2003; Rawlings and Barrett, 1990). It can be now concluded that the cystatin superfamily is comprised of four different families viz. stefins, cystatins, kininogens, and phytocystatins. Few reports Maulana also include Azad cystatin Library, related Aligarh protein within Muslim this University superfamily, for instance, histidine-rich glycoprotein and human α 2SH-glycoprotein (fetuin) (Brown and Dziegielewska, 1997).

4.1. CLASSIFICATION OF THE CYSTATIN SUPERFAMILY Cystatins are cysteine proteinase inhibitors and belong to cystatin superfamily. The cystatin superfamily has been classified into four families on the basis of inhibitory activity towards specific proteinase, sequence homology,

22 | P a g e

Introduction carbohydrate content and disulfide bonds (Barrett and Salvesen, 1986). However, the major families which emerged after diversified classification are stefins, cystatins, kininogens, phytocystatins and cystatin related proteins (Turk et al., 1997; Turk and Bode, 1991). The four families are stefins, cystatins, kininogens, and phytocystatins. Among them, stefins and cystatins are comprised of the single domain while kininogens are multi-domains polypeptide in which two of them are inhibitory domains (Figure 3). Stefins are the smallest member of the family having a molecular weight of about 11 kDa and are devoid of disulfide as well as carbohydrate content. Cystatins are low molecular weight protein of around 13 kDa – 14 kDa and contain disulfide bonds but devoid of glycan moiety. Kininogens are large glycoproteins having a molecular weight of 60 kDa – 120 kDa with multiple cystatin-like domains. Phytocystatins are plant cysteine proteinase inhibitors with a molecular weight of around 8 kDa – 25 kDa and devoid of disulfide bridges as well as glycan moiety. Phytocystatins are divided into three groups viz. Group I, Group II and Group III. Group I phytocystatins are low molecular weight proteins of 12 kDa – 16 kDa. Group II phytocystatins are 23 kDa protein which can inhibit the Legumain (C13) family of proteinases. Group III phytocystatins are high molecular weight phytocystatins and devoid of disulfide bridges. Phytocystatins contain highly conserved QXVXG sequence which is a characteristic of the cystatin superfamily. Phytocystatins contain a dipeptide sequence (proline-tryptophan) in the C-terminal region along with a conserved glycine residue in the N-terminal region. Besides, they also contain a consensus Maulana Azad Library, Aligarh Muslim University sequence LARFAV which is located upstream of the QXVXG site.

Generally, the cysteine proteinase inhibitory domain is made up of 100 amino acid residues which fold to form a five-stranded β-sheet that intertwine around a central α-helix (Figure 4). Addition of a large number of cysteine proteinase inhibitors from different sources has diversified the classification system into various subfamilies, clans, and sub-clans (Rawlings et al., 2015). The different families along with their respective members are discussed below. 23 | P a g e

Introduction

4.2. FAMILY I TYPE I CYSTATIN: THE STEFINS

Stefins are cysteine proteinase inhibitors and the simplest of all cysteine proteinase inhibitors. They are also called as family I cystatins. Structurally, they are single chain polypeptide made up of 100 amino acid residues, do not have disulfide linkages or any attached carbohydrate moiety and also lack signal sequence (Sato et al., 1990). They are monomeric proteins of 11000 Da and acidic. They are found in different mammalian species and plants like barley and potatoes (Gruden et al., 1997; Martínez et al., 2005). Stefins are primarily present as intracellular cytoplasmic proteins but also found in extracellular fluids as well (Turk et al., 1993). They are also called as Family I cystatin and their members like stefin A and stefin B have been identified in bovine, rat, porcine and humans. Stefin C and stefin D were found to be present in bovine thymus and pigs respectively (Lenarcic et al., 1993; Turk et al., 1993). They are heat stable inhibitors and are optimally active in the wide range of pH. They bind to proteinase reversibly and show competitive inhibition. The value of inhibition constant for the papain-human stefin A complex was 1.9 x 10-11 M (Turk and Bode, 1991).

MEMBERS OF THE STEFINS FAMILY

They are intracellular proteins but have also been found in extracellular fluids (Abrahamson et al., 1986). The members of this family are monomeric, acidic and devoid of disulfide linkages and carbohydrate moieties (Barret, 1986b). The members of Stefin family are Stefin A, Stefin B and Stefin C. Maulana Azad Library, Aligarh Muslim University

Stefin A

Stefin A is a potent inhibitor of cathepsin B which is present in human skin (Fräki, 1976). The inhibitor was named as stefins after extensive research by different scientific groups (Brzin et al., 1983; Jarvinen, 1978). Machleidt et al. (1983) identified the primary sequence of Stefin in 1983. Green et al. (1984) isolated and characterized similar proteins from the human liver which was later renamed as cystatin A. 24 | P a g e

Introduction

Family I (Type 1 cystatins: The Stefins)

Family II (Type 2 cystatins: The Cystatins)

Family III (Type 3 cystatins: The Kininogens)

Figure 3: Representation of the chain structure of cysteine proteinase Maulana Azad Library, Aligarh Muslim University inhibitors viz. stefins, cystatins, and kininogens. The loops indicate the presence of disulfide linkages whereas arrows specify the potential site of glycosylation.

25 | P a g e

Introduction

Figure 4. Structure of human cystatin F with five anti-parallel beta sheet Maulana Azad Library, Aligarh Muslim University and one alpha helix (PDBID: 2CH9).

26 | P a g e

Introduction

It is found in multiple isoelectric forms with the pI values in the acidic range of 4.5-5.0. (Brzin et al., 1983). Rat cystatin α and human stefin A are found in surplus quantity in polymorphonuclear leucocytes and different epithelial cell (Fraki, 1976). The presence of stefin A in epithelium and skin was confirmed through immuno-histochemical assays which advocates the function of stefin A in defense against uncontrolled proteolysis by cysteine proteinases. Cystatin A was found in the extracts of squamous epithelium of esophagus (Rinne et al., 1978). It was also present in dendritic reticulum cells of the lymph nodes, bovine skin, seminal plasma and epidermoid carcinomas (Alavaikko et al., 1985; Rinne et al., 1984). Chromosome 3 bears the gene which encodes for human cystatin A (Abrahamson et al., 1992). Jarvinen et al. (1976) discovered cystatin α which is a variant of cystatin A in a rat. Cystatin α is a cysteine proteinase inhibitor isolated from rat skin with a molecular weight of 13 kDa. Human Stefin A present in skin epithelia protects from malfunctioning of cysteine proteinases (Barrett, 1986a). The high level of stefin A was reported in patients suffering from cardiovascular complications. The stefins or type I cystatins belong to the subfamily I25 A (Rawlings et al., 2004).

Stefin B

Stefin B is another member of the stefins family and has been isolated from human liver and spleen (Green et al., 1984; Jarvinen and Rinne, 1982). It was found to be a potent inhibitor of cathepsin B and H in human tissues thereby controllingMaulana their Azad inhibitory Library, activity Aligarh (Lenney Muslimet al., 1979) University. Stefin B is widely distributed in different cells, and tissues like epithelial cells (Hopsu-Havu et al., 1985), monocytes (Rinne et al., 1985), lymphocytes (Barrett et al., 1984), and few reports also showed their presence in seminal plasma at a lower concentration. Stefin B was thought to be as intracellular endogenous cysteine proteinase inhibitor; however, the recent reports confirmed its presence in extracellular fluids. It is an important serum marker in the prognosis of colorectal and hepatocellular cancer (Lee et al., 2008; Ma et al., 2017). Stefin B

27 | P a g e

Introduction is an active monomer with a molecular mass of 12000 Da, and an inactive dimer of molecular mass of 24000 Da (Turk et al., 1992). Stefin B is a basic protein with the pI value in the range of 6.25-6.35. It also contains a cysteine residue in the polypeptide chain, which facilitates it in forming an inactive dimer in non-reducing conditions. The inactive dimer can shuffle into an inhibitory active monomer under reducing conditions. Cystatin β is slightly acidic with the pI value of 5.04-5.6 (Finkenstaedt, 1957; Lenney et al., 1979). In comparison with cystatin α, cystatin A is more abundant in all tissues except skin however both the variants are present in all tissues and cell.

Stefin C

Stefin C is present in different forms because of cleavage of Asn 5 – Leu 6 bond. It was the first member of the stefin family which has tryptophan residue and a prolonged N terminus (Turk et al., 1993). It has only one methionine residue at the N terminus while there are two residues in stefin B. The primary sequence of stefin C showed substantial sequence similarity with other members of the stefin family. It shows sequence homology of 47.9% with human stefin A, 84.7% with bovine stefin B and 72.4% with human Stefin B. It is made up of 101 amino acid residues with a molecular weight of 11,546 Da. It is an acidic protein with pI value of 4.5 – 5.0 (Turk et al., 1993). They bind to cysteine proteinase and inhibitor their activity reversibly.

4.3. FAMILYMaulana II TYPE Azad II Library,CYSTATIN: Aligarh THE Muslim CYSTATIN UniversityS

Cystatins are also referred to as family II cystatins of the cystatin superfamily. They are the most complex family of cysteine proteinase inhibitors. The members of this family are composed of 115 amino acids with molecular mass of around 13000 Da (Oliveira et al., 2003). The salient feature of the members of this family is the presence of two intra-chain disulfide linkages near the carboxy-terminal end. The members are usually not glycosylated except cystatin E/M and cystatin F (Laber et al., 1989; G Sotiropoulou et al., 1997). They contain a conserved tripeptide sequence

28 | P a g e

Introduction

(Phe-Ala-Val near the C-terminus), a conserved dipeptide (Phe-Tyr) near the N- terminus and a conserved QXVXG region in the central part of the molecule. The conserved sequences are responsible for binding to the specific proteinases.

Chicken cystatin and human cystatin C are the most essential and founding members of the cystatins family (Barrett, 1986a; Turk and Bode, 1991). The pI value of the members of this family ranges from 4.4 – 9.3. They contain 20 – 26 amino acid long signal sequence which helps in crossing the cell membrane to the extracellular space (Abrahamson et al., 1987). They are abundantly present in biological fluids such as cerebrospinal fluid (5.8 mg/L) and human cystatin C in seminal human plasma (51 mg/L) and at a lower concentration in saliva, urine, and plasma (Abrahamson et al., 1986). The significant concentration of cystatin in biological fluids revealed that they contain cysteine proteinase inhibitors which might be responsible for different essential functions. However, the concentration of cystatin changes in diseased conditions. The cystatin family includes human cystatins viz. C, D, E, F, M, S, SA, and SN. The homologs of human cystatin have also been identified in aves and other mammals (Fossum and Whitaker, 1968; Sen and Whitaker, 1973). The crystal structure of chicken cystatin was determined for the study of the mechanism involved in the inhibition of cysteine proteinase by cystatins (Bode et al., 1988) which is shown in Figure 5. The genes responsible for the expression of human cystatin are present on chromosome 20. The member of cystatin family S, SA and SN show high sequence homology up to 90% and are made up of 121 amino acid residues with a molecular weight of 14.2-14.4 kDa. The post-translational phosphorylation leads to the formation of numerous isoforms of cystatin S, and SA. Expression of cystatin SN is specific and limited to saliva and tears only whereas variants of SA and S are Maulana present in seminalAzad Library, fluid (Isemura Aligarh et al., Muslim 1991). The University human cystatins are further classified in subfamily I25 B of the cystatin family (Rawlings et al., 2004). MEMBERS OF THE CYSTATINS FAMILY

The members of the cystatin family are a monomeric polypeptide containing two disulfide bonds but lack carbohydrate moieties. The members of the cystatin family are chicken cystatin, cystatin C, cystayin D, cystatin S and its variants, cystatin E/M and cystatin F.

29 | P a g e

Introduction

Chicken cystatin

Chicken cystatin was primarily isolated from egg white and some chicken tissues and was found to inhibit papain as well as ficin (Barrett, 1986b; Fossum and Whitaker, 1968). Sen et al. carried out biochemical characterization while Turk et al. determined the amino acid sequence of chicken cystatin (Sen and Whitaker, 1973; Turk et al., 1983). It also inhibits cathepsin B and dipeptidyl peptidase I in a similar fashion it inhibits papain and ficin (Keilová and Tomášek, 1975). It contains two disulfide linkages but lacks carbohydrate moieties. It is present in high concentration in chicken egg white (~60 mg/ml) and at a lower concentration in chicken serum (1 mg/ml) (Anastasi et al., 1983; Barrett, 1981). It can withstand high temperature and stable up to 80-100°C quite well (Hass and Ryan, 1980).

Cystatin C

Cystatin C is a cysteine proteinase inhibitor of extracellular proteinases. It was earlier known as γ-trace or post-γ-globulin because of its characteristic γ- electrophoretic mobility (Brzin et al., 1984). It was firstly identified in cerebrospinal fluid and urine of patients ailing from renal tubular failure (Butler and Flynn, 1961). Cystatin C is abundantly present in seminal plasma, milk, synovial fluid, cerebrospinal fluid and blood plasma (Abrahamson et al., 1986). It has also been detected in normal and neoplastic neuroendocrine cells in the adrenal medulla (Löfberg et al., 1982), cortical brain nerves (Lofberg et al., 1981), pituitaryMaulana and Azad thyroid Library, glands (LAligarhofberg etMuslim al., 1983; University Moller et al., 1985). Human cystatin C is widely distributed and present in most body fluids (Grubb, 2000). Cystatin C is a polypeptide made up of 120 amino acid residues with four conserved cysteine residues which are responsible for a disulfide linkage. Cystatin C is devoid of any carbohydrate moieties and is not glycosylated (Turk 2008). However, it has also been found as dimer, oligomer and in fibril states (Janowski et al., 2001).

30 | P a g e

Introduction

Figure 5. Representation of the proposed “Trunk model” for the interactionMaulana between Azad chicken Library, egg whiteAligarh cystatin Muslim and papainUniversity (Turk and Bode, 1991).

31 | P a g e

Introduction

The most salient feature of cystatin C is that it is used as a biomarker of kidney dysfunction. It is used to measure glomerular filtration rate (GFR) which is an index of kidney health (Grubb et al., 2014). Its low molecular weight and easy detection make it a powerful biomarker.

Cystatin D

Cystatin D is another member of the cystatin family which is found only in human saliva and tears (Freijes et al., 1993). The precursor of cystatin D polypeptide contains 142 amino acid residues and the first 20 amino acid residues correspond to signal peptide. The maturation of cystatin D takes place after cleavage of the first 20 amino acid residues. The molecular weight of cystatin D is around 13.8 kDa, and the pI value ranges from 6.8-7.0 which signifies that it is a neutral protein (Freije et al., 1991). Cystatin D emerges from duplication of cystatin C gene segment and also shows significant homology to human cystatin C. The cystatin D shows homology in the range of 51% - 55% with the salivary cystatin (i.e., cystatin S and SA). Several reports suggest that cystatin D and cystatin S are weak inhibitors of cathepsins as compared to cystatin C (de Sousa-Pereira et al., 2014). Cystatin D preferentially inhibits cathepsin S over and L (BalbinSO et al., 1994). Recent reports identified that cystatin D is present within the nucleus at specific active chromatin sites and involved in regulating gene transcription (Manconi et al., 2017). Maulana Azad Library, Aligarh Muslim University

Cystatin S and its other variants

Cystatin S is an acidic cysteine proteinase inhibitor isolated from human saliva. Earlier, it was called as salivary acid proteinases (SAP-1). Isemura et al. sequenced the inhibitor and showed sequence homology with γ-trace protein (cystatin C) (Isemura et al., 1984). It also showed cysteine proteinase inhibitory activity hence renamed as “Cystatin S” (Isemura et al., 1984). Several other isoforms of cystatin S were also identified viz. cystatin SA and SN, however,

32 | P a g e

Introduction they differ in amino acid sequence at N-terminal end and pI values (Isemura et al., 1987, 1986). Chicken cystatin and cystatin C showed 54% - 41% sequence identity with cystatin S (Isemura et al., 1984). It was also reported that a variant of cystatin S contains phosphoserine residues. Cystatin S, D, SA, and SN are weak inhibitors of cathepsins (Ramasubbu et al., 1991).

Cystatin E/M

Cystatin E/M is the secretory cysteine proteinase inhibitors of the cystatin family. They are structurally related to other members of the family and contains intra-chain disulfide linkages. They are present in glycosylated as well as non-glycosylated forms (Soh et al., 2016). The glycosylated E/M cystatin bears an N-linked carbohydrate chain at position 108 (Georgia Sotiropoulou et al., 1997). The gene encoding cystatin E/M is present on a chromosome 11q13 (Stenman et al., 1997), thereby indicating that the inhibitor is distantly related to other members of the cystatin family. Cystatin E/M is synthesized as a precursor protein of 149 amino acids and converts into an active inhibitor after cleavage of a signal sequence of 28 amino acids (Ni et al., 1997). The malfunctioning or absence of this inhibitor leads to abnormal skin development (Zeeuwen et al., 2002). Cystatin E/M inhibits tumor growth, but the molecular mechanism is yet to be discovered. The biochemical characterization of cystatin E/M showed that it binds reversibly and non- competitively to cathepsin V and L. It is expressed in breast tissue (Qiu et al., 2008) Maulanaand restricts Azad the membrane Library, digestion Aligarh by Muslim cathepsin University (Cheng et al., 2006).

Cystatin F

Cystatin F is a cysteine proteinase inhibitor of cystatin family which targets lysosome proteinases. It is selectively expressed in the immune system cells like T cells, dendritic cells, and natural killer (NK) cells. It is involved in different processes which are associated with immune response (Obata-Onai et al., 2002). Three different groups identified cystatin F, two of them isolated the

33 | P a g e

Introduction inhibitor by cDNA cloning while the third group identified by overexpressing mRNA encoding cystatin F in metastatic liver tumors and called it as CAMP (cystatin-like metastasis-associated protein) (Morita et al., 2000). Cystatin F shows low sequence similarity with other members of the cystatin family. The disulfide-linked dimer of cystatin F is an inactive form, but it gets activated once converted into monomeric form (Langerholc et al., 2005). Cystatin F is a potent inhibitor of cathepsin K, V, F but a poor inhibitor of cathepsin H and S. does not inhibit cathepsin X (Langerholc et al., 2005). The crystal structure of cystatin F revealed that the dimeric form is stabilized by two inter- subunit disulfide linkages between Cys 26 in the extended N terminus of one monomer and Cys 63 on the other monomer (Schuttelkopf et al., 2006). Human cystatin F is made up of 145 amino acids as a precursor protein with 19 residues long signal peptide (Ni et al., 1998). However, the mature human cystatin F has a molecular mass of 14.5 kDa as determined by sequence analysis (Ni et al., 1998). It is abundantly present in peripheral blood leukocytes and spleen while moderately in small intestine and thymus.

4.4. FAMILY III TYPE III CYSTATIN: THE KININOGENS

The kininogens belong to family III of cystatin superfamily. They are single chain multifunctional glycoprotein present in plasma and other biological fluids of the mammalian system (Zhou et al., 2006). Two different kinds of kininogens are present in a mammalian system based on their molecular weight; low molecularMaulana weight Azad kininogen Library,s Aligarh(LMWK) Muslimand high molecularUniversity weight kininogens (HMWK). A different kind of kininogen is present in rat plasma which is called as T-kininogens (DeLa Cadena and Colman, 1991) having a molecular weight in the range of 50 kDa – 120 kDa (Bobek and Levine, 1992). Kininogens contain an N-terminal heavy chain, kinin segment, and C-terminal light chain. The light chain and heavy chain are connected through disulfide bridges. The heavy chain and kinin segment of human LMWK and HMWK bears identical amino acid sequence however light chains do not share

34 | P a g e

Introduction sequence homology (DeLa Cadena and Colman, 1991; Salvesen et al., 1986). The heavy chain of kininogens contains three tandemly repeated type 2- like cystatin domains (D1, D2, and D3) which resulted from gene duplication. Kininogens contain eight disulfide linkages with six conserved and two additional disulfide bonds at the beginning of cystatin domain D2 and D3. The carbohydrate binding sites are not identified in the cystatin domains of kininogen family of cystatin, although the inhibitors are glycosylated. They contain an extending polypeptide sequence at the C terminus which corresponds to bradykinin and can be cleaved by kallikrein (Bobek and Levine, 1992). Kallikreins cleave kininogens into two side chains by freeing kinin segment. Only cystatin domains D2 and D3 contain cysteine proteinase inhibitory activity, and both the domains are grouped in subfamily I25B of the cystatin superfamily (Rawlings et al., 2004).

Kininogens are widely distributed in blood plasma, synovial and amniotic fluids. They perform numerous functions such as inhibition of cysteine proteinase, induction of the endogenous blood coagulation cascade and mediation of the acute phase response. They are strong inhibitors of cathepsin L and papain but weak inhibitors of cathepsin B and H (Müller-Esterl et al., 1985). The level of kininogens have been determined by radioimmunoassay, and found to be 69-116 μg/ml (H-kininogens) and 109 – 217 μg/ml (L-kininogens) (Adam et al., 1985). Low molecular weight kininogen binds to two molecules of cathepsin S, L & papain and high molecularMaulana weight Azad kininogen Library, also bindAligarh to two Muslim molecules University of , papain and cathepsin S (Turk et al., 1995b, 1997).

4.5. FAMILY IV THE PHYTOCYSTATINS

The phytocystatin family is the fourth family of cystatin superfamily and has been emerged after the identification of proteinase inhibitors in plants. Phytocystatins are cysteine proteinase inhibitors specifically present in plants. They are counterparts of animal cystatins which inhibit cysteine proteinases in

35 | P a g e

Introduction plants. They are responsible for controlling and regulating protein degradation by cysteine proteinase activity in plants. The database available at MEROPS (http://merops.sanger.ac.uk) have all the repository data which records all the peptidases and their inhibitors into families and clans (Rawlings et al., 2015). The members of 21 families out of 78 families have been reported from plant source in the latest MEROPS 10.0 version (Santamaria et al., 2014). The salient features and phylogenetic relationship suggested the evolution of phytocystatin as a new plant cystatin family. They inhibit the activity of target cysteine proteinases by restricting its accessibility to its substrate. They share common characteristic features of the cystatin family I and II. Oryzacystatin was the first phytocystatin isolated from rice. It is structurally and functionally related to egg white lysozyme (Oliveira et al., 2003). Afterwards, phytocystatins have been isolated from numerous plant sources including monocots and dicots species viz. potato (Waldron et al., 1993), cowpea (Fernandes et al., 1993), carrot (Ojima et al., 1997), pineapple (Shyu et al., 2004), strawberry (Martinez et al., 2005), cacao (Pirovani et al., 2010), latex tree (Bangrak and Chotigeat, 2011).

5. CHARACTERISTIC FEATURES OF PHYTOCYSTATINS

Phytocystatins are plant cysteine proteinase inhibitors containing more than 80 members (Bateman et al., 2002). Earlier, it was found to be responsible for regulating protein turnover in developing and germinating seeds. However, it has been now found to be involved in numerous physiological processes like Maulana Azad Library, Aligarh Muslim University inhibition of extracellular cysteine proteinases of pathogenic nematodes and arthropods (Arai et al., 2002) and controlling endogenous proteolysis. Recent reports have manifested their exploitation in developing pathogen resistant plants (Soares-Costa et al., 2002; Ussuf et al., 2001). They are involved in leaf senescence (Diaz-Mendoza et al., 2014), programmed cell death (Belenghi et al., 2003), defense against biotic and abiotic stress (Delledonne et al., 2001; Siqueira-Júnior et al., 2002) and fruit development (Neuteboom et al., 2009). The genome of Arabidopsis encodes seven phytocystatin isoforms namely

36 | P a g e

Introduction

AtCYS1, AtCYS2, AtCYS3, AtCYS4, AtCYS5, AtCYS6 and AtCYS7 which are comprised of two distantly related gene clusters (Martinez et al., 2005). AtCYS3 and AtCYS6 provide resistance to salt and drought stress as well as defense against cold and oxidative stress (Yue et al., 2008). Oryzacystatin showed homology with egg white lysozyme and grouped into stefin like cystatin having no disulfide linkages. However, it was found that oryzacystatin exhibits sequence homology with cystatins family. The distinguishing features observed for plant proteinase inhibitors from the rest of the members of cystatin superfamily directed towards the proposal for a new cystatin family that is phytocystatins (Wu and Haard, 2000). Therefore, the phylogenetic tree of cystatin represents phytocystatin as an independent superfamily which is devoid of disulfide linkages and carbohydrate moieties (Rawlings et al., 2007).

Phytocystatins have been classified into three distinct groups based on the molecular mass and conserved domains namely group I phytocystatin, group II phytocystatin and group III phytocystatin. Group I phytocystatin contains a single cystatin domain and has a molecular weight of 12-16 kDa. The members of the group I phytocystatin are found in chestnut and sugarcane (Pernas et al., 1999; Soares-Costa et al., 2002). Chicken egg white cystatin is the animal counterpart of group I phytocystatin. Group II phytocystatins have a highly conserved cystatin domain at N -terminal and an additional extension at C-terminal of 10 kDa along with a conserved SNSL motif. The group II phytocystatin has a molecular weight of around 23 kDa (Martínez et al., 2005; ValdesMaulana-Rodriguez Azad et al., Library,2010). Group Aligarh III phytocystatins Muslim University contain multi-domains and are of high molecular weight. The members of group III phytocystatins have been identified in tomato and potato which is comprised of eight cystatin domains and molecular mass of around 85 kDa (Wu and Haard, 2000). The typical basic structure and classification of phytocystatin are represented in figure 6 and 7 respectively. Few phytocystatins are enlisted in Table 2 along with their molecular mass and a total number of amino acid residues. Animal and plant cystatins have well-conserved motifs of cystatin superfamily such as a Gln-Xaa-Val-Xaa-Gly motif at the central region of the polypeptide chain 37 | P a g e

Introduction

(Xaa is any amino acid), a Pro-Trp (or Leu-Trp) dipeptide motif at the C- terminal region, and a conserved Gly residue at the N-terminal region (Barrett 1986b; Turk and Bode, 1991). NMR spectroscopy was employed to study the tertiary structure of oryzacystatin in solution which revealed that it consists of five stranded antiparallel beta-sheet wrapped around central alpha-helix. The structure was found to be similar to animal cystatins like human stefin A and chicken egg white (shown in Figure 8) (Nagata 2000).

Phytocystatin along with three conserved motifs of typical cystatins, it also contains an N-terminal conserved motif [LVI]-[AGT]-[RKE]-[FY]-[AS]- [VI]-X-[EDQV]-[HYFQ]-N, known as LARFAV-like motif which constitutes the alpha helix in the cystatin structure (Chu et al., 2011; Reis and Margis, 2001; Zhang et al., 2008). Many studies have been conducted to analyze the functional role of motifs present in cystatins. It was observed that the transition and transversion of an amino acid within the conserved motif significantly reduces the inhibitory activity of cystatin (Martínez et al., 2003). Few members of phytocystatin family contain an additional N-terminal signal peptide for transport into the lumen of the endoplasmic reticulum (Martínez et al., 2005; Valdes-Rodriguez et al., 2010).

Phytocystatins particularly inhibit papain-like proteinases; however group II phytocystatin bearing an extended polypeptide region at C-terminal can also inhibit legumain-like proteinases (Martinez et al., 2007). Figure 8 shows the alignment between conserved elements of phytocystatins along with Maulana Azad Library, Aligarh Muslim University their predicted secondary structure. The structure-function model of animal cystatin concludes that the activity of proteinase is inhibited by three different structural domain of cystatin which forms a tripartite wedge that slides into enzyme active site cleft (Figure 5) (Bode et al., 1988; Stubbs et al., 1990). The first Gln-Xaa-Val-Xaa-Gly motif forms a surface hairpin loop which binds to the active site, another conserved element at the C-terminal region contains Pro (Leu)-Trp motif also interacts with the active site.

38 | P a g e

Introduction

FigureMaulana 6. Structure Azad of phytocystatin Library, purified Aligarh from Muslim sesame indicum University (PDBID:2MZV).

39 | P a g e

Introduction

Cystatin Superfamily

Mammalian cystatins Phytocystatins

Group I Group II Group III Stefins Cystatins Kininogens phytocystatins phytocystatins phytocysytatins

Figure 7: Scheme representing the classification of cystatin superfamily.

Maulana Azad Library, Aligarh Muslim University

40 | P a g e

Introduction

Table 2. Molecular weight and number of amino acid residues present in different phytocystatins.

No of Molecular Plant amino Phytocystatin weight Reference source acid (kDa) residues

Saccharum (Souza et al., 106 11.9 officinarum 2017)

Dianthus (Sugawara et 98 10.8 caryophyllus al., 2002)

Group I

Hordeum (Abraham et al., 107 11.8 vulgare 2006)

Actinidia (Rassam and 116 11 deliciosa Laing, 2004)

Fragaria (Martinez et al., 235 23.1 ananassa 2005)

Maulana Azad Library, Aligarh Muslim University

Group II Triticum (Dutt et al., 243 23* aestivum 2010)

(Walsh and Solanum 756 86.8 Strickland, Group III tuberosum 1993) * Predicted signal peptides were not included in the analysis.

41 | P a g e

Introduction

Figure 8: Alignment of known phytocystatin representing the conserved Maulana Azad Library, Aligarh Muslim University regions N-terminal G/GG, the QXVXG sequence in the central part, the P/PW sequence at the C-terminal end and the unique LARFAV motif found in all phytocystatin. CAH60163.1: Fa-CPI-1 from Fragaria ananassa; NP_001237734.1: Soya cystatin from Glycine max; CAA11899.1: Phytocystatin from Castanea sativa; AAK15090.1: SiCYS from Sesamum indicum (Lima et al., 2015).

42 | P a g e

Introduction

However, the role of the third element at the N-terminal region is still not known, but it is also essential for the inhibitory activity of cystatin (Bjork et al., 1995; Turk and Bode, 1991). The functional analysis of motifs has been done by site-directed mutagenesis. Any change or malfunctioning in any of the conserved motifs will disrupt the cysteine proteinase inhibitory activity. Several reports documented experimental evidence for the interaction of proteinase and its specific inhibitor such as the structure of human stefin B – papain complex (Stubbs et al., 1990) and human stefin A - cathepsin H complex (Jenko et al. 2003). Nissen et al. deciphered the crystal structure of potato multi-cystatin 2 which is a member of group III phytocystatin (Nissen et al., 2009).

6. EVOLUTION OF PHYTOCYSTATINS AS SEPARATE SUPERFAMILY

Animal and plant cystatins share many common features such as conserved motifs and the mechanism of proteinase inhibition in particular papain. However, plant cystatins show few distinguishing features which are absent in animal cystatins thereby raising a strong need for a separate family for plant cystatins (Kondo et al., 1989; Margis et al., 1998). The animal cystatins have been classified into three families namely stefins, cystatins, and kininogens. The stefins are single chain polypeptides devoid of carbohydrate and disulfide linkages. The molecular weight of stefins member is around 11000 Da. The cystatin family members are comprised of 115 amino acid residues with a Maulana Azad Library, Aligarh Muslim University molecular mass of 13000 Da and contain two intra-molecular disulfide linkages but lacks carbohydrate content. The kininogens or family III cystatins are multi-domain cystatin with high molecular mass and repeated stefin-like domains. The amino acid sequence of phytocystatins showed homology with the members of the cystatin family. The complex and unique gene organization diverging from the structure of animal cystatin genes have been readily identified as a possible sign of preliminary evolutionary divergence of plant and animal cystatins (Margis et al., 1998; Waldron et al., 1993). Such as the

43 | P a g e

Introduction presence of a specific consensus sequence [LVI]-[AGT]-[RKE]-[FY]-[AS]- [VI]-x-[EDQV]-[HYFQ]-N of unknown function placed in α-helix of most plant cystatins. The functionally divergent relatives of cystatins have been documented in few plant families. Phytocystatins have also been also reported with similarities of type II family cystatin and type I family cystatin (Nissen 2009). The observations for the similarity and dissimilarity among animal and plant cystatin resulted into the emergence of a dynamic evolutionary scheme for phytocystatin which is parallel to the evolutionary scheme of animal cystatins (Benchabane et al., 2010). A specific fold of plant cystatins with distinct features like Cys residues at the active site has been identified such as Cys residue in algae cystatin. These findings support the formation and emergence of an independent and parallel evolutionary scheme for plant cystatins, encompassing a shared ancestor appeared well before the evolutionary break between plants and animals (Benchabane et al., 2010). The common ancestor derived from phytocystatins would have undergone a number of variations, with the help of complementary evolutionary processes which include gene duplication, alternative splicing, and adaptive evolution, in order to raise different cystatins and cystatins related members encoded by plant genome (Christeller et al., 2006; Martinez and Diaz, 2008).

7. GARLIC PLANT

Garlic (Allium sativum) is a perennial plant of the genus Allium and family amaryllis (Amaryllidaceae).Maulana Azad The Library, garlic plantAligarh is native Muslim to centralUniversity Asia and northeastern Iran; however it grows wild in southern France and Italy (Block, 2010). It has been used as a food flavoring agent as well as traditional medicine. It was also known to ancient Egyptians and Romans as a food among the poor. The garlic plants grow up to 1.2 m in height. Phyllotaxy depends on the variety of garlic plant. The garlic is grown as an annual crop and can be propagated by planting top bulbils or cloves or seeds. The cultivation of garlic crop is easy and can be harvested throughout the year in mild climates. The

44 | P a g e

Introduction planting of cloves is done in autumn season in a colder region and harvested in late spring or early summer. Garlic plants prefer to be grown in high organic content soil. Garlic crops are affected by numerous pathogens such as nematodes and fungi. Garlic suffers from leek rot, downy mildew, and pink root diseases. The principal bioactive compound present in the aqueous extract of garlic is allicin (allyl 2-propenethiosulfinate or diallyl thiosulfinate). Allinase enzyme gets activated upon chopping and crushing of garlic and produces allicin from allinin which is present in garlic. There are numerous other active compounds which are present in garlic extract such as 1 -propenyl allyl thiosulfonate, allyl methyl thiosulfonate, y-L-glutamyl-S-alkyl- L-cysteine and (E, Z)-4,5,9-trithiadodeca- l,6,11-triene 9- oxide (ajoene)(Bayan et al., 2014).

Fresh garlic cloves contain different organosulfur compounds, phenolic and steroidal compounds, trace elements along with proteins, carbohydrates and fibers (Lanzotti, 2006; Shukla and Kalra, 2007). The compounds are grouped into two groups on the basis of solubility; lipid soluble and water- soluble compounds. Diallyl disulfide (DADS) and diallyl trisulfide (DATS) are lipid soluble whereas g-glutamyl S-allyl cysteine is a water-soluble compound (Thomson and Ali, 2003).

7.1. MEDICINAL SIGNIFICANCE OF GARLIC

Garlic is consumed as a flavoring agent in food as well as a source of medicine in differentMaulana ways. Azad The garlic Library, extract Aligarh has shown Muslim antimicrobial University activity against bacteria, fungi, and viruses. The chemical compounds present in garlic are involved in the treatment of cardiovascular ailments, diabetes, cancer, blood pressure, hyperlipidemia, and atherosclerosis, etc.

EFFECT OF GARLIC IN CARDIOVASCULAR DISEASES

Garlic and its extract are an active agent for the prevention and treatment of cardiovascular diseases. The clinical and experimental studies showed that the

45 | P a g e

Introduction consumption of garlic has significantly reduced blood pressure, a decline of serum cholesterol and triglyceride, inhibition of atherosclerosis, increased fibrinolytic activity and inhibition of platelet aggregation (Chan et al., 2013). Reduction in both systolic and diastolic pressure has been observed upon intravenous administration of garlic extracts in the animal experiment (Sial and Ahmad, 1982). Similarly, oral ingestion of garlic extract brought the blood pressure back to normal level in hypertensive animals. Several clinical reports documented that administration of garlic reduces blood pressure in 80% of the patients suffering from high blood pressure (Omar, 2013; Petkov, 1979; Stabler et al., 2012). The antihypertensive property of garlic is because of its prostaglandin-like effects which decrease peripheral vascular resistance (Rashid and Khan, 1985). Aged garlic is more effective in reducing the systolic blood pressure of patients ailing from high blood pressure. A dosage of aged garlic containing 0.6 – 2.4 mg S-allyl cysteine reduces blood pressure by 12 mmHg (Ried et al., 2013).

Similarly, the anti-atherosclerotic effect of garlic is due to its ability to reduce lipid content in the arterial membrane. The anti-atherosclerotic effect is primarily due to the presence of allicin, S-allyl cysteine and diallyl-disulfide (Gebhardt and Beck, 1996; Yeh and Liu, 2001). The plasma fibrinolytic activity in healthy individuals as well as in acute myocardial infarction patients increases when the diet is supplemented with garlic (Mirhadi et al., 1991). It was observed that pre-administration of garlic significantly inhibited 2+ thromboxaneMaulana-A2 synthesis, Azad intracellular Library, CaAligarh mobilization Muslim thereby University protecting against thrombocytopenia induced by collagen or arachidonate application in rabbits. It was also reported that garlic decreases the risk of plasma viscosity, peripheral arterial occlusive diseases, unstable angina and increases elastic property of blood vessels and capillary perfusion (Sumiyoshi and Wargovich, 1990).

46 | P a g e

Introduction

GARLIC AND DIABETES MELLITUS

Garlic shows the hypoglycemic effect, but the effect of garlic on human blood glucose is still not known. Studies have shown that garlic can reduce blood glucose level in streptozotocin and alloxan-induced diabetes mellitus mice and rats (Sheela et al., 1995). Garlic reduces total serum cholesterol, and LDL cholesterol however increases HDL cholesterol (Ashraf et al., 2005). A bioactive compound S-allyl cysteine from garlic restores erectile functions in diabetic rats by inhibiting reactive oxygen species formation through modulation of NADPH oxidase subunit expression (Yang et al., 2013). Fasting blood glucose reduces substantially when metformin treatment is supplemented with garlic as compared to metformin alone (Kumar et al., 2013). Chronic administration of garlic extracts showed an exponential decline in blood glucose level. Some reports suggest no reduction in the blood glucose level in case of human. Therefore it is essential to further investigate the role of garlic in diabetic conditions (Banerjee and Maulik, 2002). The credit for the hypoglycemic effect in diabetes mellitus goes to the numerous volatile sulfur compounds present in garlic such as allicin, alliin, diallyl sulfide, diallyl trisulfide, S-allyl cysteine, allyl mercaptan, and ajoene. The extracts of garlic have been documented to be effective in decreasing insulin resistance (Padiya and Banerjee, 2013).

ANTI-TUMOR EFFECT OF GARLIC Maulana Azad Library, Aligarh Muslim University Many studies have been done to investigate the anticancer effects of garlic, which suggested positive results and showed anticancer properties of garlic extract and its constituents. Allylsulfide derivatives are the primary compounds in garlic extract which showed anticancer properties. The garlic components have been found to regulate a number of molecular mechanisms in carcinogenesis viz. mutagenesis, cell proliferation, DNA adduct formation, scavenging of free radicals, differentiation and angiogenesis. Garlic reduces the growth rate of cancer cells by cell cycle blockade which occurs in the G2/M

47 | P a g e

Introduction phase (Capasso, 2013). It shows different anti-tumor properties such as tumor cell growth inhibition and chemopreventive effects. Garlic and its components have been reported to restrict the development of chemically induced tumors in the colon (Knowles and Milner, 2003), liver (Kweon et al., 2003), bladder (Lau et al., 1986), prostate (Hsing et al., 2002), mammary gland (Amagase and Milner, 1993), lung (Sparnins et al., 1986), stomach (Wattenberg et al., 1989) and esophagus (Wargovich et al., 1988) in human as well as rodent studies. Anticancer activity has been shown in vitro and in vivo conditions by diallyl trisulfide (DATS) which is an organosulfur compound isolated from garlic.

Garlic and its constituents show anticancer properties by inhibiting carcinogen activation (Amagase and Milner, 1993), protection of DNA from activated carcinogens (Tadi et al., 1991), enhancement of detoxification (Sumiyoshi and Wargovich, 1990), and excretion (Tadi et al., 1991). The constituents of garlic block the covalent binding of carcinogens to DNA, have anti-oxidative & free radical scavenging potential, increase degradation of carcinogens, as well as regulate apoptosis, cell proliferation, and immune responses. It also inhibits the proliferation of colon and human mammary endometrial cancer cells. The activity of arylamine N-acetyltransferase and 2- aminofluorene-DNA in human promyelocytic leukemia cells is inhibited by diallyl sulfide and diallyl disulfide (Lin et al., 2002). The cell growth of basal cell carcinoma and human melanoma A375 cells is inhibited by diallyl trisulfide (DATS). DATS inhibits the growth by increasing the levels of intracellular Maulana reactive oxygen Azad species Library, and Aligarh DNA damage Muslim along University with inducing mitochondria-mediated apoptosis and endoplasmic reticulum stress (Wang et al., 2012).

ANTI-PROTOZOAL EFFECT OF GARLIC

The garlic extract was found to be effective against Scedosporium prolificans (Davis et al., 2003), Candida albicans (Lemar et al., 2002), Tinea pedis (Ledezma et al., 2000), Balantidium entozoon, Opalina ranarum,

48 | P a g e

Introduction

Trypanosomes, Leptomonas, Leishmania, Crithidia and Entamoeba histolytica (Koch and Lawson, 1996). Garlic has been recommended for the treatment of giardiasis because of its strong inhibitory activity against giardiasis pathogen. The crude extract of garlic (25 pg/mL) showed inhibitory activity against pathogen, and 50 pg/mL was established as a lethal dose. The above results established garlic as an anti-giardia agent and remove all the symptoms within 24 h. It was observed that the constituent of garlic such as ajoene, allicin, and organosulfides are effective antiprotozoans’ compounds (Bayan et al., 2014).

ANTI-MICROBIAL EFFECT OF GARLIC

Garlic has been used for centuries across the globe against infectious diseases. Louis Pasteur had identified the antibacterial activity of garlic in 1858 for the first time. Garlic is found to be effective against some gram-negative, gram- positive and acid-fast bacteria. These include Pseudomonas, Proteus, Staphylococcus aureus (Cavallito and Bailey, 1944), Escherichia coli, Salmonella (Adler and Beuchat, 2002; Johnson and Vaughn, 1969), Clostridium (De Wit et al., 1979), Mycobacterium (Delaha and Garagusi, 1985), Klebsiella, Micrococcus, Bacillus subtilis (Sharma et al., 1977) and Helicobacter (O’Gara et al., 2000). Garlic exerts a differential inhibition between beneficial intestinal microflora and potentially harmful enterobacteria (Rees et al., 1993). The antibacterial activity of garlic is found to be primarily due to allicin. It is reported that allicin has sulfhydryl modifying activity and can inhibitMaulana sulfhydryl Azad enzymes Library, (Wills Aligarh, 1956). Muslim University

ANTI-FUNGAL EFFECT OF GARLIC

Garlic shows antifungal activity against many fungi such as Torulopsis, Trichophyton, Cryptococcus (Fromtling and Bulmer, 1978), Trichosporon, and Rhodotorula (Tansey and Appleton, 1975), Candida (Yousuf et al., 2011). Garlic extracts also reduce the growth of the organism, inhibit the synthesis of lipids, proteins, and nucleic acids (Adetumbi et al., 1986), decrease the oxygen

49 | P a g e

Introduction uptake (Szymona, 1952) and damage membranes (Ghannoum, 1988). The main component of garlic, allicin was shown to be antifungal. However, other components of garlic also showed inhibitory activity such as ajoene against Aspergillus (Yoshida et al., 1987) and diallyl trisulfide against cryptococcal meningitis. Supplementation of ajoene to fungal growth mixtures such as Candida albicans, Aspergillus niger, and Paracoccidiodes inhibits the growth at a lower concentration than that experienced with allicin. It has been reported that aged garlic extract exhibited diminished in vitro antifungal activity. Moreover, garlic is also found to be effective against air-borne pathogen Trichoderma harzianum and Botrytis cinerea (Lanzotti et al., 2012).

ANTI-VIRAL EFFECT OF GARLIC

Garlic extract has shown in vitro activity against rhinovirus, HIV, herpes simplex virus (Tsai et al., 1985), cytomegalovirus (Guo et al., 1993) and influenza A and B (Fenwick and Hanley, 1985), rotavirus and viral pneumonia. Diallyl trisulfide, allicin, and ajoene show inhibitory activity against viral infection (Weber et al., 1992).

8. IMPORTANCE OF PHYTOCYSTATINS

Phytocystatins are cysteine proteinase inhibitors present in plants. The inhibitors are involved in regulating different physiological processes and maintain proteinaseMaulana-anti Azadproteinase Library, balance. Aligarh It is Muslim essential University to maintain the balance between proteinase and antiproteinase activity in order to coordinate different functions of a living system. Phytocystatins are involved in various physiological processes that vary from regulating the endogenous cysteine proteinase activity during seed maturation and germination as well as biotic and abiotic stresses (Irene et al., 2012). They also act as a potent inhibitor of gut proteinases leading to the plant defense against insects (Aguiar et al., 2006) for example over-expressing of phytocystatin CeCPI from taro in transgenic plants increases resistance to bacterial phytopathogens and insects

50 | P a g e

Introduction

(Senthilkumar et al., 2010). Phytocystatins of strawberry (Martinez et al., 2005), taro (Wang et al., 2008), sugarcane (Soares-Costa et al., 2002) and winter wheat (Christova et al., 2006) have an in vitro inhibitory effect on fungal growth. Phytocystatins are also involved in programmed cell death by modulating cysteine proteinase activity in the regulation of protein turn over (Irene et al., 2012). Keeping in view the importance of phytocystatins and lacuna in the knowledge, the present study focuses on purification and characterization of a phytocystatin from a new source that is garlic (Allium sativum). The work of this thesis has been divided into the following five sections:

I. PURIFICATION AND CHARACTERIZATION OF GARLIC PHYTOCYSTATIN

Protein purification is a multi-step process which leads to the isolation of a protein from a complex mixture of proteins. The structure and function of a protein can be determined once it is isolated from the mixture of proteins, thereby giving importance to the purification of proteins. There are numerous factors which governed the process of purification such as molecular weight, binding affinity, physicochemical properties, and biological activity. The initial steps involved in the purification of proteins are crude extract preparation, precipitation, differential solubilization, and ultracentrifugation. The crude extract preparation is done for bringing protein into solution by breakage of cell Maulana Azad Library, Aligarh Muslim University membrane through homogenization. Then, it is subjected to different conditions such as pH and salt concentration. pH treatment is used for precipitating unwanted proteins which may be acidic or alkaline treatment based on the solubility of the protein. Similarly, salt fractionation is a technique used to precipitate out protein on the basis of solubility. The solution is then subjected to centrifugation in order to get rid of precipitated protein from the solution. The precipitated protein can be dialyzed in order to remove the excess salts along with other impurities. Dialysis is done several times in order to

51 | P a g e

Introduction make sure that the solution is completely rid of salts. The mixture of protein obtained after salt fractionation are subjected to a chromatographic technique which separates them by molecular mass. Gel-filtration chromatography is used to separate proteins by size and shape of the protein. A mixture of proteins can also be separated by gel-electrophoresis technique on the basis of size, shape and isoelectric point. Gel-electrophoresis includes Native- PAGE, SDS-PAGE and 2D-PAGE which corresponds to native polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two- dimensional polyacrylamide gel electrophoresis. The protein moves in native PAGE on the basis of protein’s charge and its hydrodynamic radii while SDS- PAGE separates proteins on the basis of molecular mass. Two-dimensional gel electrophoresis separates protein in two different dimensions, hence called as 2D gel electrophoresis. In the first dimension, the proteins are separated horizontally on the basis of their isoelectric point, while in second dimension the protein mixture is separated perpendicularly from the earlier electropherogram based upon the molecular weight of proteins. It is essential to analyze the purity and content of protein at each step of purification in order to choose the most efficient method for purification. Protein’s purity is checked through protein specific assay which describes its activity at each step of purification. The increment in the specific activity of a protein at each step of purification suggests the efficiency of that particular step and purification procedure. Upon reaching maximum specific activity, the protein can be isolated and checked for its homogeneity, and it may be used for further Maulana Azad Library, Aligarh Muslim University studies.

Allium sativum commonly known as garlic (Alliaceae family) is a widely cultivated crop that is used as a flavoring agent and in traditional medicines. Competitive weeds commonly threaten garlic crops. Hence production of weed resistant crops can upsurge the yield potential. Abiotic stresses such as heat, drought, salinity and biotic stresses such as insects attack, fungal, parasite and viral infections are the major players which adversely affect the crop yield (Bita and Gerats, 2013). The role of phytocystatins in pathogenic 52 | P a g e

Introduction resistance (Belenghi et al., 2003), virus attack (Gutierrez-Campos et al., 1999) and protection against biotic and abiotic stresses (Hwang et al., 2010) is well documented in the literature.

The present study focuses on purification and characterization of a phytocystatin from garlic (Allium sativum). This has been achieved by ammonium sulfate fractionation followed by gel-filtration chromatography. The homogeneity of the purified inhibitor was analyzed by gel electrophoresis. The inhibitor was then biochemically and biophysically characterized for its pH stability, temperature stability, molecular weight, secondary structure, and kinetic properties. The study provides essential information about the properties of garlic phytocystatin and adds information into the existing pool of already known phytocystatins.

II. UNFOLDING OF GARLIC PHYTOCYSTATIN IN PRESENCE OF UREA AND GUANIDINE HYDROCHLORIDE

A specific three-dimensional structure of a protein critically defines its conformational stability providing a specific function to that protein. The interplay of physiochemical forces like hydrophobic interactions, ionic interactions, disulfide bonds and van der Waals forces (Dill, 1990; Nick Pace et al., 2014) impart a precise three-dimensional structure to a protein as they are involved at various stages of folding-unfolding. Therefore, the advancement in a profound comprehension of the nature and magnitude of the strength of all Maulana Azad Library, Aligarh Muslim University the physiochemical interaction involved would directly affect the approach to elucidate the transition stages during folding-unfolding of a protein (Ptitsyn et al., 1980; Uversky, 2002). Balanced equilibrium of these forces is primarily required for proper folding of a protein and any disruption in this equilibrium can shift the process of folding-unfolding in either way. Thus, the protein folding studies are stepping stone towards the definitive revelation of principles lying behind the structure – stability relationship of a protein. Several pieces of literature advocate that protein unfolding process involves only two state

53 | P a g e

Introduction transition, from native to a completely unfolded state (Privalov, 1979; Privalov and Khechinashvili, 1974). However, the possibility of intermediate state has now been accepted which does not correspond to either native or completely unfolded state (Tanford, 1968). In addition to these forces, physical parameters such as temperature, entropy, ionic strength, pH and solvent composition significantly play a role in maintaining stable three-dimensional structure of a protein (Priyadarshini et al., 2010). Therefore, it is requisite to have a quantitative determination of these parameters in order to decipher the molecular structure of a protein. It is well documented that attainment of the native structure of a protein should not be a random search, protein attains its native conformation by a properly regulated mechanism that may involve well- defined intermediates (Levinthal, 1968). Also, for a better understanding of protein unfolding dynamics, it is imperative to have an insight into the pathways by which the unfolded state can be recognized (Torrent et al., 2008). Apart from unraveling various intermediate stages, the folding-unfolding study also reveals the amount of free energy involved in stabilization of the three- dimensional structure of a protein.

Amyloidogenic property of animal cystatin C is well documented which indicate misfolding potential of this protein under extreme physiological conditions (Palsdottir et al., 2006). It has been recently discovered that Arabidopsis plant protein exhibited prion behavior inside yeast after misfolding (Chakrabortee et al., 2016). Prions, as well as misfolded proteins, are the major foundations ofMaulana various animalAzad neurodegenerativeLibrary, Aligarh diseases Muslim (Freire University et al., 2013; Soto, 2003). Hence, it is pivotal to identify and characterize conformational intermediates of plant proteins generated during the process of folding- unfolding in order to explore their biological manifestation inside the animal body. It is now our interest to study the folding-unfolding process of garlic phytocystatin. Therefore, in the present study, an attempt is made to investigate garlic phytocystatin under denaturing conditions of urea and guanidine hydrochloride (GdnHCl).

54 | P a g e

Introduction

UREA

Urea is a nitrogenous compound made up of a carbonyl group attached to two amine group. It is the most nitrogenous fertilizers because of its high nitrogen content. It is also used as a cattle feed supplement and in other industrial uses. Urea is a widely used denaturing agent for assessing folding-unfolding as well as the stability of protein (Pace, 1986). However, the molecular mechanism of urea-induced denaturation of protein is still not precise. Several reports suggest direct binding of urea with protein and interfere with the different interactions involved in maintaining the native structure of protein thereby assisting the unfolding process (Tirado-Rives et al., 1997; Klimov et al., 2004; Mountain and Thirumalai, 2003). It has also been proposed that urea functions indirectly by changing the solvent environment, and exerts hydrophobic effect and exposes the residues of the hydrophobic core (Finer et al., 1972). The basic principle for the destabilization of native protein by aqueous urea focused on perturbation of water structure which is referred to as an indirect mechanism. However, this concept did not gain much support from experimental and simulation studies (Soper et al., 2003). Therefore, another concept of direct mechanism was overlaid which suggests the direct binding between urea and polypeptide, which is exhibited in the simulated pathways (Bennion and Daggett, 2003; Hua et al., 2008). Urea increases the aqueous solubility of small hydrocarbons (Watlaufer et al., 1964), which weakens hydrophobic interaction by stabilizing solvation of the unfolded protein state where a higher number of non-polarMaulana side chains Azad are Library, exposed Aligarhto the solution Muslim. The University forces responsible for the solvation of peptide by urea are Van der Waals interactions which are evident in the simulation studies, thereby increasing the availability of urea for hydrophobic hydration (Hua et al., 2008). Furthermore, other studies also supported that electrostatic basis for the action of urea and destabilization of a polypeptide helix by aqueous urea correlated with the binding of urea with polypeptide backbone polar groups and side chains (O’Brien et al., 2007).

55 | P a g e

Introduction

GUANIDINE HYDROCHLORIDE

Guanidine hydrochloride is a strong chaotrope and denaturant which is widely used in research work and laboratories. Chemically, it is hydrochloride salt of guanidine and hygroscopic in nature. The folding of proteins has been investigated through the denaturation process. Hence, the denatured state of protein has gained widespread importance in understanding the folding process (Shortle, 1996).

The major denaturants used for the denaturing the proteins are urea and guanidinium chloride. The thermodynamic parameters associated with protein unfolding do not depend on the denaturing agent, but the structural properties change upon treatment (Makhatadze and Privalov, 1992; Pfeil and Privalov, 1976). In simple words, the net denaturation enthalpies and entropies are intrinsic properties of proteins whereas the changes in secondary structure, compactness as well as burial surface depend on the pathway followed during denaturation of proteins. The protein folding-unfolding is investigated through analysis of structural characteristics and not by direct calorimetric assessment. Therefore, it is essential to analyze the effects of the specific denaturing agent. However, the molecular basis for the denaturation by guanidine HCl and urea is still unknown. As previously described, two models have been put forward based on the direct and favorable interaction between the denaturant and protein (Kresheck and Scheraga, 1965; Schellman, 1955). While, the other mechanism is based on indirect interaction which involves modification of Maulana Azad Library, Aligarh Muslim University hydrogen-bonding of water and weakening of hydrophobic interactions (Frank and Franks, 1968). Midinfrared spectroscopy experiments have suggested that the dynamics of hydrogen bonding is poorly affected in the presence of urea (Rezus and Bakker, 2006). However, end-to-end diffusion of unstructured peptides suggests that urea and guanidine hydrochloride interact homogeneously with all the amino acids with binding constants 0.26 M−1 and 0.62 M−1, respectively (Moglich et al., 2005).

56 | P a g e

Introduction

UREA

Maulana Azad Library, Aligarh Muslim University GUANIDINE HYDROCHLORIDE

57 | P a g e

Introduction

III. INTERACTION OF PESTICIDES WITH PURIFIED GARLIC PHYTOCYSTATIN

Pesticides are chemical compounds that have been proved as a backbone for increased agricultural productivity and are widely used in agricultural lands to avoid the menace created by pest and pathogens. Pesticides are classified on the premise of their ability to target destructive agents like insects, fungi and unwanted plants and so consequently sorted as insecticides, fungicides, and herbicides respectively (Cohen, 2007). The prolonged usage and persistence of pesticides amplified its availability in the environment. Hence, it exerted potential detrimental effects on humans, animals, bees, and birds when inhaled or ingested. Eventually, pesticides have emerged as a major health concern worldwide for their malicious effects on animals and humans (de Souza et al., 2011; Mostafalou and Abdollahi, 2013). Bio-concentration and biomagnification of pesticides enable their deposition in fatty tissues which lead to a marked increment in its concentration in the food chain. Therefore, the ubiquitous nature of pesticides must be checked in a way to minimize its detrimental impact on biodiversity and the ecosystem. Hazards of pesticides like teratogenicity, mutagenicity, and carcinogenicity are very well documented (Bignold et al., 2006; Mdegela et al., 2010).

Living organisms are composed of a number of biomolecules; among them, protein is one of the most important and fundamental biomolecules. Proteins playMaulana an important Azad role Library,beginning Aligarhfrom the fertilizationMuslim University of germ cell to senescence; hence, the proper functioning of protein is necessary to sustain life. The functioning of a protein depends on its native conformation, so a little modification in it can disrupt the functionality of a protein. High binding efficiency of pesticides may affect essential proteins whose normal functioning is necessary for sustaining and maintaining the physiological and biological balance within plants and animals. A number of physiological processes have been reported to be disrupted by the interaction of pesticides with plant proteins viz. root-growth retardation, regulation of proteolytic activity, defects in the

58 | P a g e

Introduction storage of proteins, xylogenesis, defense against pathogens and apoptosis (Cercos and Carbonell, 1993; Chauhan et al., 2001; Lamb and Dixon, 1997; Solomon et al., 1999) . Therefore, it is requisite to have an insight into the structural and functional modification of essential proteins on interaction with pesticides.

Numerous pesticides are utilized for the elimination of agricultural and household pests. They enable the improvement of a qualitative and quantitative facet of crops by limiting various diseases in humans disseminated by pests (Lewis et al., 1997). Usually, these pesticides are extremely toxic and are retained in the environment due to high stability. Imperceptive and indiscreet applications of pesticides have resulted in environmental toxicity, pest resurgence, and residual concentrations in food, soil, and water (Gaines, 1969). Their presence in food and crops has severe detrimental effects on both human species and environment (van der Werf, 1996). The bioaccumulation of these pesticides in plants, fishes, animals, and humans through bioresources can induce diseases and adversely stunt the development and growth of the organisms (Margni et al., 2002). The application of pesticides alters physiological, biochemical, enzymatic and non-enzymatic functions of plants from early germination phase to growth of the plant which affects the yield (Parween et al., 2016).

The chemicals in physiological system target proteins which are present in the living organisms as important biological macromolecules. The Maulana Azad Library, Aligarh Muslim University abundance of proteins in components of cellular metabolism presents them as primary targets of these chemicals (Mokarizadeh et al., 2015). The agricultural practices devise considerable usage of pesticides creating ecological and toxicological complications. The pesticides are taken up by the plant system and then enter animal metabolism through the food cycle (Yusa et al., 2015). The exposure of pesticides to humans through different routes result in the increased incidence of various ailments such as Parkinson, Alzheimer, amyotrophic lateral sclerosis, bronchitis, asthma, congenital disabilities,

59 | P a g e

Introduction infertility, autism and diabetes (Mostafalou and Abdollahi, 2017). Pesticides hinder the functioning of cholinesterase enzymes (Jaga and Dharmani, 2003), reduction in insulin secretion, affects the cellular metabolism of carbohydrates, fats, and proteins, and also hampers mitochondrial function which results in oxidative stress thereby affecting nervous and endocrine system (Karami- Mohajeri and Abdollahi, 2011). Therefore, it is essential to assess the toxic potential of pesticides in order to understand their role in the development of plants and animals. The outcome of synthetic pesticides on symbiotic nitrogen fixation is enhanced dependence on fertilizers, diminished soil fertility and unsuitable long-term yield of crops. Also, pesticides contaminate soil, surface water, groundwater, soil microorganisms and plant physiology posing significant risks to the environment (Aktar et al., 2009). Following pesticides were chosen for their interaction with garlic phytocystatin.

CARBENDAZIM (a fungicide)

Carbendazim (CAR) [methyl N-(1H-benzimidazol-2-yl)carbamate] is an example of carbamates that belongs to the group of fungicides which is extensively used in many countries (Yamamoto et al., 2007). It is a broad spectrum fungicide which was found to be effective against fungal infections and control foot-rot and damping-off diseases (Prasad and Hiremath, 1985). It was also found to be potent against various foliar diseases of cereals, fruits, flower bulbs, sugar and fodder beet (Tortella et al., 2013). All these features lead to excessiveMaulana usage Azadof carbendazim Library, i nAligarh fields which Muslim resulted University in its harmful manifestation on humans as well as wildlife. Organisms get exposed to carbendazim by consuming contaminated food products or by fume inhalation. Deposition of this pesticide upon various mammalian tissues such as adipose tissue, skin, liver, and gonads creates life-threatening problems in many living organisms (Grogan and Jukes, 2003), like in case of rats (Adedara et al., 2013). Carbamates have been reported to affect immunocompetence and possess immunotoxic effects (Colosio et al., 1999). Reportedly, carbendazim is to be involved in immune disorders of humans (Corsini et al., 2013). In another 60 | P a g e

Introduction literature, carbamates showed associations with various immune diseases (Duramad and Holland, 2011) by disrupting the regulation of the immune system thereby, rendering host prone to allergies (Corsini et al., 2013), infectious diseases (Blakley et al., 1999), autoimmune disorders (Kassi and Moutsatsou, 2010) and cancers (Alavanja and Bonner, 2012; Dietert, 2011).

OXYFLUORFEN (a herbicide)

Oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl) benzene] is an important herbicide used in fields to control the invasion of pathogens. Chemically, it is fluorinated diphenyl ether utilized in restraining broadleaf and grassy weeds in food crops. The oxyfluorfen present in soil gets transported through run-off surface water from agricultural fields to marine ecosystems (Peixoto et al., 2006). Oxyfluorfen is steady and relatively stationary in the soil. The rate of microbial degradation of this pesticide is prolonged and practically insoluble in water. Oxyfluorfen alters chlorophyll biosynthesis in plants by hindering the functioning of enzyme protoporphyrinogen oxidase which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX (Geoffroy et al., 2002). On application to foliage, oxyfluorfen causes necrosis and chlorosis while seed germination is inhibited through the pre-emergent application. It also induces lipid peroxidation & light intensity dependent bleaching. Oxyfluorfen is extremely pestilential to aquatic invertebrates, fish, flora, and fauna (Hassanein, 2002; Riley et al., 1994).

Maulana Azad Library, Aligarh Muslim University

61 | P a g e

Introduction

CARBENDAZIM

Maulana Azad Library, Aligarh Muslim University OXYFLUORFEN

62 | P a g e

Introduction

IV. AGGREGATION OF GARLIC PHYTOCYSTATIN ASSISTED BY TRIFLUOROETHANOL (TFE)

Protein aggregation is a complex process which mostly arises due to misfolding of the proteins. It is governed by sequence or some factors when combined results in aggregation of the protein (Chiti and Dobson, 2006). Aggregation- prone regions (APRs) present on polypeptide are of 5-15 amino acids in length and are responsible for the protein aggregation (Betti et al., 2016; Goldschmidt et al., 2010; Rousseau et al., 2006). The APRs self-associate in a specific pattern to form beta sheet structures (Betti et al., 2016). The probability of aggregation is higher during the folding process, and the partially unfolded intermediate serve as a precursor for the aggregation. Aggregation of protein also occurs in the presence of external driving forces such as organic solvents. Aggregation process is often irreversible, and aggregates are characterized by the presence of non-native intermolecular beta-sheet structures. Some neurodegenerative diseases in human beings are linked with amyloid formation such as Parkinson’s disease, Alzheimer’s diseases, type II diabetes mellitus and spongiform encephalopathies (Armen et al., 2004; Tartaglia et al., 2008). Understanding the mechanism of aggregation will empower to develop novel therapeutics in order to prevent the outcomes of deposition of aggregates in tissues which causes toxicity.

Aggregation study is not limited to animal proteins only; however, it has also been reported in plant proteins. Amyloid kind of aggregates have been Maulana Azad Library, Aligarh Muslim University found in transgenic tobacco plants formed by maize transglutaminase (Villar- Pique et al., 2010). Another study describes that environmental stresses viz. heat stress, ultraviolet (UV) radiation, and cold stress induce aggregation in plant cells (Nakajima and Suzuki, 2013). The environmental stress subsequently develops endoplasmic reticulum stress which facilitates the aggregation process of misfolded proteins (Nakajima and Suzuki, 2013). APRs can be selectively used to induce aggregation-linked phenotype within the plant system to knock down protein function. A significant increase in starch content

63 | P a g e

Introduction can be achieved in maize (Zea mays) by the use of APRs which induces aggregation of target proteins (Betti et al., 2016).

A number of organic solvents are exploited nowadays in the laboratory to unravel the mechanism of aggregation by imitating the aggregation process. However, such high concentration does not exist in the biological system but the use of such agents aided in understanding the aggregation process in vitro. Trifluoroethanol is one such organic solvent which is used in the laboratory. It is simple ethanol along with CF3 group. It mainly affects the tertiary structure of the protein leading to the formation of aggregates (Gast et al., 2008). The intermolecular H-bonding is also responsible for the aggregation. It is generally observed that the final product of aggregation is cross-β conformation irrespective of the kind of the protein from which it initiates. β-strand interactions are possible due to the hydrogen bonding facilitated by the peptide backbone of the aggregate. However, the side chains provide stability to β- strands by aligning and close packing to the identical sequence of the adjacent strand (Makin et al., 2005; Sawaya et al., 2007). Ultimately, a clump of protein emerges which is regarded as an aggregated state. Amorphous kind (aggregated state) of a protein is a robust and insoluble state. The transition of native protein to amorphous nature disrupts the normal functioning of the biological system thereby rendering the system defective. However, researchers are going ahead to come up with the strategies for anti-aggregation. The present study employed trifluoroethanol (TFE) as an aggregating agent to investigate the nature and extentMaulana of structural Azad Library, changes Aligarh that accompany Muslim or University promote loss of biological activity upon aggregation in garlic phytocystatin.

V. EFFECT OF HEAVY METAL IONS (ZINC AND CADMIUM) ON GARLIC PHYTOCYSTATIN

Metals are considered as a natural resource, but their toxicity has always been a concern. Increased exposure to heavy metals has led to high concern for the health and well-being of animals and plants. Human beings are also exposed to

64 | P a g e

Introduction heavy metals via the food chain. Heavy metals are discharged from various sources such as industrial wastes, chemical laboratories, volcanic eruptions, fossil burning, and automobile exhaust etc (Zhang et al., 2005a). The heavy metals dumped by the sources gets accumulated and pose a serious threat to animals and plants. The increased concentration of heavy metals in soil and water bodies raised a major concern to the plant growth and severely stunted productivity. However, at various incidence, it was found that heavy metals are responsible for the increased stability and functioning of enzymes. They act as cofactors for the enzyme, responsible for disease resistance and during seed production. Although, it is necessary for cells to regulate the concentration of metals within the system to carry out normal and healthy functioning of various components of the living system if not regulated could lead to growth inhibition.

Excessive exposure of metals can induce a number of abnormalities such as necrosis, chlorosis, alteration in plant growth along with damage to tissues and cells. Hence, the ubiquitous nature of metals can be a stress factor for the growth and development of plants. However, plants have acquired a number of mechanisms as self-defense to regulate the uptake and accumulation of metals such as the formation of the proteinaceous complex via chelation of metals to ligands (Zhang et al., 2005b). Phytochelatins and metallothioneins are the two heavy metal binding ligands present in plants which take part in the chelation and sequestering of heavy metals (Zenk, 1996). Cysteine-rich peptidesMaulana are major Azad components Library, Aligarh of phytochelatins Muslim whichUniversity forms chelation complex between heavy metals (cadmium, zinc, copper) and thiol subunits in the presence of phytochelatin synthase (Cobbett, 2000; Zenk, 1996). Metallothioneins are characterized by metal binding and cysteine-rich domains which give them a dumbbell shape. Both metal binding proteins are present in yeast (Vatamaniuk et al., 2002), plants (Cazale and Clemens, 2001; Kohler et al., 2004; Murphy and Taiz, 1995) and animals (Yu et al., 1994). The expression of metallothioneins gene in Festuca rubra and Arabidopsis increases on the treatment with heavy metals like cadmium and copper (García- 65 | P a g e

Introduction

Hernández et al., 1998; Ma et al., 2003). There are numerous incidences where metallothioneins and phytochelatins are responsible for regulating the concentration of heavy metals such as detoxification of trivalent arsenic in seaweed Fucus vesiculosus (Merrifield et al., 2004). There are other strategies which are also used to regulate the accumulation of heavy metals such as the use of microbes, plants and both for remediation of heavy metals. Heavy metals absorbed by the plants hinders the metabolism of plants by affecting the normal functioning of crucial enzymes and proteins. They can induce structural and conformation changes which generally results in loss of function. Hence, it is necessary to regulate the uptake and accumulation of heavy metals.

Zinc is an important micro-nutrient required for the normal functioning of enzymes and proteins. It acts as a catalyst in many physiological reactions and the second most abundant transition metal (Barak and Helmke, 1993; Vallee and Auld, 1990). The plant's uptake zinc as Zn+2 with the help of rhizospheres present in the roots; however, the grasses uptake the metals in complexed form (Bashir et al., 2012). The metals get distributed after uptake and make complexes with various ligands, proteins and metal transporters (Kramer, 2010). The toxicity of zinc arises due to its high concentration in the plant system or inefficient remediation of zinc. The high concentration of zinc shows a direct relationship with the increased reactive oxygen species (Remans et al., 2012). It also displaced essential metals such as Fe and Mn which act as metal in enzymes thereby hampering the normal functioning of biomolecules.Maulana Zinc toxicity Azad in Library, sugar beet Aligarh includes Muslim iron deficiency University induced chlorosis as well as changes in the mineral composition (Sagardoy et al., 2009). Zinc increases leaf respiration rate thereby decreasing the photosynthesis rate, alteration in the structure and functioning of stomata, changes in mesophyll structure, reduces the conductance of carbon dioxide to stomata and mesophyll cells (Sagardoy et al., 2010). It also affects the carboxylate metabolism which is observed in iron-deficient plants (Sagardoy et al., 2011). Consequently, elevated levels of zinc can result in growth inhibition and toxic symptoms.

66 | P a g e

Introduction

Cadmium is a heavy metal and a major environmental pollutant that adversely affects animals and plants. Its chronic exposure via respiratory or gastrointestinal tracts majorly affects the kidney in human beings (Johri et al., 2010). Cadmium has been classified as an intermediate toxic element by Duxbury (Duxbury, 1985). Accumulation of cadmium in plants reduces photosynthesis, reduction in water and nutrient uptake induces many injuries to plants such as chlorosis, browning of root tips and growth inhibition (Drążkiewicz et al., 2003; Kahle, 1993). High concentration of cadmium induces oxidative stress within plants thereby causing the lipid peroxidation and affecting the tissues and cells of plants. The high solubility of cadmium makes it the most phytotoxic among heavy metals. It is readily absorbed by plants and enters the food chain which further causes a serious threat to human life (Buchet et al., 1990). The cadmium can be absorbed even at low concentration which gets transported to different parts of plants and exhibits a negative effect on mineral distribution, homeostasis, growth, and development of plants (Farinati et al., 2010; Metwally et al., 2004). The cadmium toxicity also includes leaf rolling & chlorosis. It affects stomatal opening and closure along with the altered water uptake (Clemens, 2006). Furthermore, it also affects the photosynthetic machinery and reduction in chlorophyll and carotenoid content (Sanita Di Toppi and Gabbrielli, 1999). It reduces the efficiency of various enzymes involved in carbon assimilation thereby reduces the carbon dioxide fixation (Perfus-Barbeoch et al., 2002). Exposure of high concentration of cadmium results in abnormal embryos and enhanced mutation Maulana Azad Library, Aligarh Muslim University rate in Arabidopsis species and can also result in chromosomal aberrations and changes in the cell cycle (Benavides et al., 2005; Ernst et al., 2007). Therefore, the present study investigates the heavy metal stress on a phytocystatin isolated from garlic cloves. The study employed different biochemical and biophysical methods to analyze the effects of zinc and cadmium on the structural and functional status of garlic phytocystatin.

67 | P a g e

Introduction

9. SCOPE OF THE THESIS

Phytocystatins have shown their importance in different physiological processes of the plant which includes regulation of endogenous or heterologous proteinases. They are also involved in defensive functions. Its significance has compelled the researchers to consider phytocystatins as proteins of immense importance with a high potential to be included as the latest tool in pest control management. The biochemical characteristics of phytocystatins such as small size and stability have attracted the interest of researchers for their production as recombinant molecules. Furthermore, the large-scale production of phytocystatins has been done for their use as nutraceuticals, food additives and stabilizers of other recombinant proteins. The phytocystatins are being evaluated as a potential acaricide and insecticide in order to increase the crop yield. Therefore, phytocystatins have been isolated and characterized from various sources such as rice (Chen et al., 1992), potato (Annadana et al., 2003), pineapple (Shyu et al., 2004) and strawberry (Martinez et al., 2005) in order to study the role of phytocystatins in detail in plant system.

The protection of garlic crop from environmental hazards owing to its endogenous phytocystatin needs to be studied. Thus, it was thought to be necessary to study the structure and function of phytocystatin specially to improve garlic resistance to various stresses and fertility under abiotic and biotic stress conditions. The proven antioxidative, hypocholesterolemic, antithrombotic and antihypertensive properties of garlic make it a key player in several diseaseMaulana pathologies Azad (Petrovska Library, and Aligarh Cekovska, Muslim 2010). AlliumUniversity sativum), is thus, a novel source to explore salient features of phytocystatin in order to understand its role in various physiological processes. Moreover, as garlic is involved in reducing parameters associated with cardiovascular disease (Rahman and Lowe, 2006), diabetes mellitus and anti-tumor effects, it would be an interesting study to explore the role of garlic phytocystatin in various ailments. Therefore, the present study focuses on the purification of phytocystatin from garlic cloves along with its biochemical and biophysical

68 | P a g e

Introduction characterization. The study also investigated the structural and functional transition of garlic phytocystatin under the effect of extrinsic factors such as denaturants, pesticides, aggregating agent and heavy metals. This study will serve as a model for providing knowledge about garlic phytocystatin along with the structure-function relationship upon exposure of extrinsic factors. The study about garlic phytocystatin will add knowledge to the existing pool and will provide a better understanding of the diverse roles of phytocystatin. The thesis contains the following chapters.

Chapter 1 focuses on the purification and characterization of garlic phytocystatin (GPC). The phytocystatin has been purified from garlic cloves using ammonium sulfate fractionation and gel-filtration chromatography. The homogeneity of the purified protein was confirmed by gel electrophoresis. A single band was obtained under both reducing and non-reducing conditions with a molecular weight of 12.5 kDa. The protein inhibitor was stable under a broad range of temperature and pH. Immunological studies confirmed the purity of epitopes as a single precipitin line obtained in immunodiffusion Kinetic studies suggested that garlic phytocystatin has the highest affinity for papain as compared to ficin and bromelain. UV and fluorescence spectroscopy shed light on the conformational features of the inhibitor. The secondary structure of garlic phytocystatin was determined by circular dichroism spectroscopy, and it was found to have 33.9% alpha-helical content.

ChapterMaulana 2 Azaddescribes Library, the chemical Aligarh denaturation Muslim University of garlic phytocystatin

(GPC) with the help of urea and guanidine hydrochloride. Various biophysical technique such as fluorescence spectroscopy, circular dichroism, and Fourier transform infrared spectroscopy were employed to investigate the unfolding of garlic phytocystatin which describes structure-function relationships on the basis of conformational changes caused by denaturants. The increase in the denaturant concentration adversely affects the cysteine proteinase inhibitory activity of GPC along with the alteration in the conformation of GPC. The

69 | P a g e

Introduction study showed changes in the secondary structure of GPC upon treatment with denaturants (urea and guanidine hydrochloride). The study also sheds light on the different mode of denaturation followed by urea and guanidine hydrochloride.

Chapter 3 sheds light on the possible interaction of pesticides

(carbendazim and oxyfluorfen) with garlic phytocystatin. Herein, the study has been designed to probe the possible alteration in the structure and function of a critical regulatory protein of plant origin, i.e. garlic phytocystatin. Carbendazim (a fungicide) and oxyfluorfen (a herbicide) are the pesticides whose effects have been investigated on garlic phytocystatin by employing various biophysical techniques which include circular dichroism, FTIR, isothermal titration calorimetry and fluorescence spectroscopy.

Chapter 4 characterizes the aggregates of garlic phytocystatin formed upon treatment with trifluoroethanol. The cysteine proteinase inhibitory activity declines at subsequent addition of TFE thereby suggesting the functional inactivation of GPC. Fluorescence spectroscopy showed the conformational changes within GPC upon the increasing concentration of TFE. Congo red and ThT assays were carried out to confirm the formation of aggregates in the presence of TFE. The secondary structural changes within GPC upon treatment with TFE was studied with the help of circular dichroism and Fourier transform infrared spectroscopy. The morphological examinations Maulana Azad Library, Aligarh Muslim University of aggregates were done with the help of scanning electron microscopy. The study showed the formation of garlic phytocystatin aggregates at 60% (v/v) TFE and confirmed the transition of garlic phytocystatin from native to non- native conformation when incubated in the presence of trifluoroethanol.

Chapter 5 evaluates the in vitro effect of heavy metals on garlic phytocystatin. The study aimed at deciphering the toxic effect of heavy metals upon its exposure to GPC. The varying concentrations of heavy metals (Zn+2

70 | P a g e

Introduction and Cd+2) were incubated with purified protein in order to probe the structural and functional transition of garlic phytocystatin. The study also sheds light on the implication of abiotic stress on the garlic protein. Different biochemical and biophysical parameters such as proteinase inhibitory activity and secondary structures were analyzed using ultraviolet absorption spectroscopy, fluorescence spectroscopy, and circular dichroism to study the effect of heavy metals on purified phytocystatin.

Maulana Azad Library, Aligarh Muslim University

71 | P a g e

Materials & Methods

Maulana Azad Library, Aligarh Muslim University

Materials and Methods

MATERIALS AND METHODS

A. MATERIALS

All the chemicals and other consumables used were of the best quality and purchased from multinational brands given below:

1. Merck [Sigma-Aldrich Chemical Co. (St. Louis)], USA

8-Anilinonaphthalene-1-sulfonic acid, 2-mercaptoethanol, anti-rabbit alkaline phosphatase (conjugate), blue dextran, bromelain, carbendazim [methyl 1H- benzimidazol-2-ylcarbamate], Congo red, cytochrome C, chymotrypsin, ficin, guanidine hydrochloride (GdnHCl), L-cysteine, ovalbumin, oxyfluorfen [2- chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl) benzene], papain, soybean trypsin inhibitor, Sephacryl S 100-HR and Thioflavin T.

2. GeNei Pvt. Ltd. Bangalore, India

Low range molecular weight markers and Freund’s incomplete and incomplete adjuvants.

3. Sisco research lab (SRL.), India

5, 5’-Dithio-bis-2-nitrobenzoic acid (DTNB), 2, 2, 2 trifluoroethanol (TFE), acetic acid, acrylamide, casein, copper sulphate, Coomassie Brilliant Blue R- 250, dimethylMaulana sulpho Azadxide Library, (DMSO), Aligarh Folin’s reagent,Muslim hydrochloric University acid (HCl), N, N' methylene-bis-acrylamide, sodium azide, sodium carbonate, sodium chloride, sodium hydroxide, sodium potassium tartrate.

4. Bio-rad laboratories, USA

Gel electrophoresis assembly.

72 | P a g e

Materials and Methods

B. METHODS

I. PURIFICATION AND CHARACTERIZATION OF GARLIC PHYTOCYSTATIN (GPC)

1. Homogenate preparation

Purification of garlic phytocystatin was based on the modification of the method described earlier by Siddiqui et al., (2016) and Khan et al., (2016). Fresh cloves of garlic (100 g) were taken and peeled off. It was then homogenized in 50 mM sodium phosphate buffer (300 ml), pH 7.5 containing 0.15 M sodium chloride (NaCl), 3 mM ethylene diamine tetraacetic acid and filtered through a chilled muslin cloth. Then, the crude homogenate was centrifuged at 5,000 rpm for 15 min at 4°C in a Sigma 3K30 centrifuge. The pellet obtained after centrifugation was discarded and the left-over supernatant was further used for purification.

2. Ammonium sulfate fractionation

The supernatant was supplemented with 30% ammonium sulfate fractionation. This mixture was then kept for 3 h. at 4 °C and centrifuged at 8000 rpm for 15 min. The supernatant thus collected after centrifugation was fractionated by 60% ammonium sulfate saturation. After incubation of 3 h at 4°C, the mixture was centrifuged at 8000 rpm for 15 min. Pellets obtained after centrifugation Maulana Azad Library, Aligarh Muslim University was dissolved in a minimum amount of 50 mM sodium phosphate buffer (pH 7.5) and dialyzed four times against the same buffer to remove the ammonium salts.

3. Gel filtration chromatography

A Sephacryl S 100-HR column was used for the gel filtration chromatography. The column was assembled as described by Peterson et al. (Peterson and Sober, 1962). The slurry suspended in ethanol was washed several times with double

73 | P a g e

Materials and Methods distilled water in order to ensure the absence of smell of ethanol. The glass column was fixed on strong vertical support, and glass wool was fixed from inside at the bottom opening of the column. The slurry was then poured with the help of a long glass rod. The flow rate was then adjusted according to the requirement, and column was washed thoroughly with buffer. The void volume of the column was calculated by passing 2% (w/v) solution of blue dextran through the column. The dialyzed pellet sample was then applied to gel filtration chromatography on Sephacryl S-100 HR column equilibrated with 50 mM sodium phosphate buffer, pH 7.5. The flow rate of the column was fixed at 16 ml per hour. Fractions of 5 ml were collected and assayed for phytocystatin inhibitory activity and protein concentration.

4. COLORIMETRIC ANALYSIS

4.1 Determination of protein concentration

Protein concentration was determined by the method of Lowry et al., (1951). Aliquots of the protein solution were pipetted in a test tube, and volume was makeup to 1 ml with distilled water. Then, 5 ml alkaline copper reagent (prepared by adding 2% sodium carbonate, 0.4% sodium hydroxide, 1ml of 1% (w/v) copper sulfate and 1 ml 2% (w/v) sodium potassium tartrate) was added after 10 min of incubation at room temperature. Lastly, 0.5 ml of Folin- Ciocalteau’s phenol reagent (1:1 diluted with distilled water) was added, and tubes were finally vortexed. The color developed was read after 30 minutes at a wavelengthMaulana of 660 Azad nm Library, against theAligarh reagent Muslim blank. University A standard curve was prepared using BSA as standard.

4.2 Carbohydrate estimation

The carbohydrate content was determined by the method of Dubois (DuBois et al., 2002) using glucose as a standard. The 2 ml aliquots of protein solution were pipetted into a set of test tubes, and 0.05 ml of 80% phenol was added to test tubes. Then, 5 ml of concentrated sulfuric acid is poured into sample test

74 | P a g e

Materials and Methods tubes. The sample test tubes were left on standby for 10 min at 30°C. The color developed was measured at 490 nm for the quantification of hexose content.

4.3 Thiol group estimation

Thiol group estimation was done by the method of Ellman (Ellman, 1969). The appearance of free thiol groups is induced by sodium dodecyl sulfate (SDS) and β-mercaptoethanol (β- ME). The sample aliquots of 0.2 ml GPC alone, GPC treated with SDS and GPC treated with β- ME were mixed with 0.1 ml of DTNB reagent in a total volume of 3.1 ml. The absorbance of the sample was read after 15 min at 412 nm. The estimation of free thiol concentration was done from the absorbance using the molar extinction coefficient of 13,600 M- 1cm-1for the released thionitrobenzoic acid. A standard plot was prepared using L-cysteine. DTNB reagent is prepared by dissolving 40 mg DTNB in 100 ml of 0.05M Tris- EDTA buffer, pH 8.0.

4.4 Cysteine proteinase inhibitory activity assay

The inhibitory activity assay was done by the method of Kunitz based on its ability to inhibit the caseinolytic activity of papain (Kunitz, 1947). Papain was activated in the presence of 0.14 M cysteine and 0.04 M EDTA for 10 min prior to incubation with the inhibitor (GPC). The volume of the reaction mixture was made 1.0 ml by the same buffer and inhibitor-papain (GPC- papain) complex was incubated at 37◦C for 30 min. After incubation, 1.0 ml of ◦ 2% (w/v) of Maulanacasein (substrate) Azad wasLibrary, added Aligarhand re-incubated Muslim for University 30 min at 37 C. The reaction was stopped by the addition of 1.0 ml of 10% (w/v) of trichloroacetic acid. Tubes were then centrifuged at 2000 rpm for 15 min. An appropriate amount of aliquot was developed by Lowry’s procedure and the blue color was read at 660 nm. The control was prepared without an inhibitor (GPC). The inhibitory activity of GPC (inhibitor) was defined as the decrease in casein hydrolyzing activity per mL of inhibitor solution per minute reaction time. One unit of inhibitor activity of the purified garlic phytocystatin was defined as the amount of inhibitor bringing about 0.001 change in absorbance

75 | P a g e

Materials and Methods per minute per ml. GPC was also assessed for its inhibitory activity against bromelain, ficin, trypsin, and chymotrypsin using casein as a substrate. The method of Lowry et al., (1951) was used for the estimation of protein content in every sample.

5. GEL ELECTROPHORESIS

Native and SDS-PAGE were performed in the presence and absence of 2- mercaptoethanol as described by Laemmli (1970) to examine the homogeneity of purified protein. Native (7.5%) and SDS-PAGE (15 %) were run for GPC.

5.1 Polyacrylamide gel electrophoresis (PAGE)

The Polyacrylamide gel electrophoresis was done by the method of Laemmli (1970) using the gel apparatus. The required gel concentration was obtained by mixing 30% acrylamide solution along with 0.8% N’N’ methylene-bis- acrylamide and 1.5 M Tris (pH 8.8). The solution was then poured gently in order to avoid bubble formation into the mould formed by glass plates (8.5x10 cm) separated by 1.5 mm thick spacers. A comb with a template for seven wells was inserted into the stacking gel solution before the onset of polymerization. The comb was removed instantaneously after the polymerization of gel and wells are filled with running buffer. The sample solution containing 40-60 μg of protein and one-fourth volume of 0.0001% bromophenol blue (used as tracking dye) is then loaded into wells.

ElectrophoresisMaulana was Azad performed Library, at Aligarh 50 – 100 Muslim V in the University electrophoresis running buffer containing 192 mM glycine and 25 mM Tris-HCl (pH 6.8) until the tracking dye reaches the bottom of the gel.

5.2 SDS Polyacrylamide gel electrophoresis (SDS-PAGE)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the method reported by Laemmli (1970), using a gel electrophoresis apparatus. The required gel concentration was obtained by mixing 30% acrylamide solution along with 0.8% N’N’ methylene-bis-

76 | P a g e

Materials and Methods acrylamide, 1.5 M Tris (pH 8.8) and 10% SDS. The sample solution containing 40-60 μg of protein, 2% SDS (w/v), 5% (v/v) β- mercaptoethanol and 0.001% (w/v) bromophenol blue is incubated at 100°C for 5 min then loaded in to wells. Electrophoresis was carried out at 50 - 100 V till the tracking dye reaches the bottom of the gel. Running buffer contains 1% SDS in addition to 192 mM glycine and 25 mM Tris-HCl (pH 6.8).

5.3 Staining of the gel: Coomassie Brilliant Blue staining

The gel bands were analyzed by staining the gel with 0.25% Coomassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid for 4 h The de- staining was done by keeping the gel in a solution containing 5% methanol and 7.5% glacial acetic acid at room temperature for overnight.

6. MOLECULAR MASS DETERMINATION

The molecular mass of GPC was determined by gel electrophoresis (SDS- PAGE) and gel filtration chromatographic technique. The molecular mass was determined under reducing and non-reducing conditions on SDS-PAGE as given below:

6.1 Molecular mass determination by SDS-PAGE

The molecular mass of GPC was determined under reducing and non-reducing conditions by employing the method of Weber and Osborn (1969). The molecular massMaulana of protein Azad was determinedLibrary, Aligarh by running Muslim standard University marker proteins along with purified protein on SDS-PAGE. The low range standard protein marker was used which contain ovalbumin (45 kDa), carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20.1 kDa), lysozyme (14.3 kDa) and aprotinin (6.5 kDa). The mobilities of molecular mass standards determined under identical conditions were plotted against the logarithms of molecular mass and the plot was used for calculating the molecular mass of purified GPC.

77 | P a g e

Materials and Methods

6.2 Molecular mass determination under native condition by gel filtration chromatography

The molecular mass of the purified GPC was determined by measuring elution volume on a Sephacryl S 100-HR column. The column was calibrated by determining the elution volume of some standard protein markers such as ovalbumin, trypsin, cytochrome c, and aprotinin. The data obtained were analyzed by the method of Andrews (Andrews, 1964). The linear relationship between Ve/Vo and log M was used for determining the molecular mass of

GPC. Ve is the elution volume of the protein and Vo is the void volume of the column.

7. DETERMINATION OF HYDRODYNAMIC PROPERTIES

Stokes radius (r) was calculated by the method reported earlier by Andrews (Andrews, 1964). The estimation was done using Sephacryl S100-HR column calibrated with globular proteins of already known Stokes radii such as BSA (Stokes radii 35 Å); B, ovalbumin (Stokes radii 29 Å); lysozyme (Stokes radii 18 Å); D, Cytochrome c (Stokes radii 17 Å) and aprotinin (13.5 Å,). The data were analyzed according to the equation of Laurent and Killander (1964).

Stokes radius

1/2 Stokes radii of GPC was determined by plotting a graph of - log (Kav) vs. Stokes radius (r) of standard protein markers: Maulana Azad Library, Aligarh Muslim University

Kav= (Ve-Vo) / (Vt-Vo)

Where, Ve = elution volume, Vo = void volume, Vt = bed volume of the column.

78 | P a g e

Materials and Methods

Diffusion coefficient

The diffusion coefficient was calculated by the equation:

D=KbT/f where, Kb = Boltzmann constant, T = temperature in Kelvin, f = frictional coefficient. 8. pH stability

GPC (50 µg) was incubated with different pH buffers viz. 50 mM sodium acetate buffer (pH 3.0, 4.0, 5.0 and 6.0), 50 mM sodium phosphate buffer (pH 7.0 and 8.0) and 50 mM Tris-HCl buffer (pH 9.0 and 10.0) for 30 min at 37 °C. Remaining inhibitory activity was measured by the method of Kunitz (1947) described in section 2.4.

9. Thermal stability

GPC (50 µg) of purified inhibitor was incubated at different temperature (30-90 °C) for 30 min and then kept in an ice-cold water bath to bring the temperature down. Remaining inhibitory activity was measured by the method of Kunitz (1947) described in section 2.4.

10. INHIBITION KINETICS

10.1 Determination of Michaelis constant (K ) Maulana Azad Library, Aligarh Muslimm University Increasing concentration of casein ranging from 20 µM to 200 µM with 0.2 mg/ml of a different cysteine proteinase (papain, ficin, and bromelain) was used to determine the value of Km. Lineweaver-Burk plot was plotted for determination of Km (Lineweaver and Burk, 1934).

79 | P a g e

Materials and Methods

10.2 Determination of inhibition constant (Ki)

Increasing concentration of GPC ranging from 0.06 µM to 0.36 µM was allowed to react with a constant concentration (0.2 mg/ml) of different protease viz. papain, bromelain, and ficin. Residual activity of different proteases was measured by the method of Kunitz (1947). The values obtained were analyzed by Lineweaver-Burk plot (Lineweaver and Burk, 2002), and Ki was determined by the Dixon plot (Dixon, 1972; Dixon, 1953).

10.3 Determination of dissociation rate constant (K−1)

Determination of dissociation constant K-1 was done before the reaction gets shifted towards dissociation. The conditions for the maximum association between proteinase and the inhibitor was achieved by adding an excess of substrate, which binds all the free enzyme molecules. Substrate-induced dissociation was monitored by adding excess substrate (6% casein) to the reaction volume for varying time periods (0–30 min). The residual activity was then determined.

10.4 Determination of association rate constant (K+1)

Association rate constant was calculated using the method described by Abrahamson et al. (1986).

K+1= K-1/Ki Maulana Azad Library, Aligarh Muslim University where, K+1 =association constant; K-1= dissociation constant; Ki = inhibition constant.

10.5 Half-life of complex

The enzyme-inhibitor complex half-life was calculated by the following equation:

80 | P a g e

Materials and Methods

t1/2=0.693/K-1

where t1/2= half-life of the enzyme-inhibitor complex; K-1= dissociation constant.

10.6 IC50 value

The IC50 value was calculated from the following equation:

Ki=IC50/1+[S]/Km

where, Ki= inhibition constant; [S]= substrate concentration; Km= Michaelis constant.

11. IMMUNOLOGICAL STUDIES

11.1 Production for antisera

Antibodies were raised against garlic phytocystatin (GPC) by injecting 300 µg of purified GPC in Freund's complete adjuvant subcutaneously into a healthy male albino rabbit. The booster dose was given repeatedly every week in Freund's incomplete adjuvant and the rabbit was bled every second week. The blood collected was allowed to coagulate at room temperature. The antisera were then collected and stored at -20 °C in aliquots.

11.2 Immunodiffusion Maulana Azad Library, Aligarh Muslim University Immunodiffusion experiment was performed on the basis of the procedure described by Ouchterlony (1962). 1% agarose in normal saline along with 0.2% sodium was poured on a petridish and allowed to solidify at room temperature. The antiserum is poured in a central well while the surrounding wells are loaded with the antigen (GPC). The plate is left for the antigen-antibody reaction in for 24 – 48 h.

81 | P a g e

Materials and Methods

11.3 Direct binding enzyme-linked immunosorbent assay (ELISA)

Direct binding ELISA was performed according to the method reported by Voller et al. (1978). The ninety-six wells of a microtiter plate (Immulon 2 HB Flat Bottom, Dynex, USA) were coated with 100 μl of antigen (GPC) and kept for overnight incubation at 4°C. After then, the plate was washed three times with TBS-T buffer [Tris buffered saline-Tween 20 (pH 7.4), 200 mg potassium chloride, 14.3 mM sodium chloride, 20 mM Tris, and 5 ml Tween 20 dissolved in 1 liter of distilled water and pH was adjusted to 7.4 by 1 N HCl]. The left- over spaces were covered by incubation with 150 μg/200 ml of 1.5% milk in TBS [Tris-buffered saline (pH 7.4), 20 mM Tris, 150 mM sodium chloride] for 5-6 h at room temperature. The plates were washed with TBS-T after the completion of incubation. The control and test wells were then loaded with 100 μl of serially diluted serum. The plate was further incubated for 2 h at room temperature. 100 μl of an appropriate conjugate of anti-rabbit alkaline phosphatase (1:3000) was then coated in each well and left for 2 h at room temperature. After further washing with TBS-T and distilled water, the substrate p-nitrophenyl phosphate (5 μg/100 ml of 50 mM bicarbonate buffer, pH 9.5, containing 0.02% sodium azide) was added to each well and incubated for 30 - 45 min. The reaction was finally stopped by the addition of 100 μl of 3 M NaOH to each well. The absorbance of each well was recorded at 405 nm on an ELISA reader. Maulana Azad Library, Aligarh Muslim University

12. SPECTROSCOPIC ANALYSIS

12.1 Ultraviolet absorption spectroscopy

The ultraviolet absorption spectrum of GPC was recorded on a UV-1800 Shimadzu spectrophotometer in a cuvette of 1 cm path length. The absorption spectra were recorded in the region of 200 – 340 nm (Ahmed et al., 2016). An equimolar concentration of GPC and papain was used in GPC-papain complex

82 | P a g e

Materials and Methods study. The concentration of GPC and papain were 2 µM and the samples were prepared in 50 mM sodium phosphate buffer (7.5)

12.2 Fluorescence spectroscopy

The fluorescence spectra were recorded on a Shimadzu RF-5301 spectrofluorophotometer (Tokyo, Japan). The protein sample was excited at a wavelength of 280 nm and slits were set at 5 nm. The emission spectrum was recorded in the range of 300 - 400 nm with the path length of 1 cm (Ahmed et al., 2016) . An equimolar concentration of GPC and papain was used in GPC- papain complex. The concentration of GPC and papain was 2 µM and the samples were prepared in 50 mM sodium phosphate buffer (7.5).

12.3 Circular dichroism spectroscopy

Circular dichroism spectra were recorded on a JASCO J-810 spectropolarimeter equipped with a Jasco Peltier-type temperature controller (PTC-424S/15) and calibrated with ammonium D-10-camphorsulfonate. The path length of the cell was fixed at 1 cm and the spectra were recorded in the range of 250-190 nm at 25 °C (Khan et al., 2016). Average of four scans were taken for the good signal to noise ration. An equimolar concentration of GPC and papain was used in GPC-papain complex. The concentration of GPC and papain was 8 µM and the samples were prepared in 50 mM sodium phosphate buffer (7.5). MaulanaThe results Azadwere expressed Library, as Aligarh MRE (Mean Muslim Residue University Ellipticity) in deg cm2dmol−1.

θ MRE = θ (mdeg)/ 10 x n x C x l

θobs = observed ellipticity in millidegrees; C = molar concentration; n = number of amino acid residues; l = path length in centimeters.

83 | P a g e

Materials and Methods

12.4 Fourier transform infrared (FTIR) spectroscopy

FTIR measurements were performed at room temperature on a Perkin Elmer Spectrum. An equimolar concentration of GPC (8 µM) and papain (8 µM) was used in GPC-papain complex. The samples were prepared in 50 mM sodium phosphate buffer (pH 7.5). The absorbance of the buffer solution was subtracted from that of GPC solution. The spectra were recorded in the region 1700-1600 cm-1 (Ahmed et al., 2016). Each spectrum was an average of three scans.

II. UNFOLDING STUDIES OF GARLIC PHYTOCYSTATIN IN PRESENCE OF DENATURANTS

13. Unfolding of GPC in the presence of urea and guanidine hydrochloride

Native GPC (4 µM) was incubated for 2 h with a varying concentration of urea (0 – 8 M) at 37° C in 50 mM sodium phosphate buffer (pH 7.5) to examine the denaturation process. Similarly, GPC was incubated with increasing concentration (0 - 4 M) of GdnHCl to monitor the denaturation of GPC.

14. Cysteine proteinase inhibitory assay of GPC in the presence of urea and guanidine hydrochloride

CysteineMaulana proteinase Azad inhibitory Library, activity Aligarh of garlic Muslim phytocystatin University was determined in the presence of urea and guanidine hydrochloride. Native GPC (4 µM) was incubated with a varying concentration of urea (0 – 8 M) and GdnHCl (0 – 4 M) at 37° C in 50 mM sodium phosphate buffer (pH 7.5) for 2 h. The assay was performed by the method of Kunitz based on its ability to inhibit the caseinolytic activity of papain (Kunitz, 1947). Papain was activated in the presence of 0.14 M cysteine and 0.04 M EDTA for 10 minutes prior to incubation with inhibitor (GPC). The volume of the reaction mixture was made

84 | P a g e

Materials and Methods

1.0 ml by the same buffer and inhibitor-papain (GPC-papain) complex was incubated at 37°C for 30 minutes. After incubation, 1.0 ml of 2 % (w/v) of casein (substrate) was added and re-incubated for 30 minutes at 37°C. The reaction was stopped by the addition of 1.0 ml of 10% (w/v) of trichloroacetic acid. After 15 minutes, tubes were centrifuged at 2000 rpm for 15 minutes. An appropriate amount of aliquot was developed by Lowry's procedure and the blue color was read at 660 nm. The control does not contain inhibitor (GPC). The inhibitory activity of GPC (inhibitor) was defined as the decrease in casein hydrolyzing activity per mL of inhibitor solution per minute reaction time. One unit of inhibitor activity of the purified phytocystatin was defined as the amount of inhibitor bringing about 0.001 change in absorbance per minute per mL.

15. Intrinsic fluorescence spectroscopy

The Fluorescence measurements were performed on a Shimadzu spectrofluorophotometer (RF-5301, Japan). The intrinsic fluorescence emission refers to the cumulative emission of all the aromatic amino acid residues. Native GPC (4 µM) was incubated with a varying concentration of urea (0 – 8 M) and GdnHCl (0 – 4 M) at 37° C in 50 mM sodium phosphate buffer (pH 7.5) for 2 h. The protein sample was excited at a wavelength of 280 nm and the emission spectra were recorded in the range of 300 - 400 nm at 25° C (Sharma et al., 2006). The path length was set at 1 cm and the width of slits were set at 5 nm. Maulana Azad Library, Aligarh Muslim University 16. ANS fluorescence analysis

ANS fluorescence was performed using a Shimadzu fluorescence spectrofluorophotometer (Tokyo, Japan). ANS is a hydrophobic dye which binds to hydrophobic patches present in the proteins and used to unravel the presence of intermediate states in the folding or unfolding pathways of proteins. The slit width of excitation and emission were set at 5 nm and the path length was set at 1 cm. Native GPC (4 µM) was incubated with a varying

85 | P a g e

Materials and Methods concentration of urea (0 – 8 M) and GdnHCl (0 – 4 M) at 37° C in 50 mM sodium phosphate buffer (pH 7.5) for 2 h. The ANS fluorescence study was carried out post incubation of GPC with urea and GdnHCl at an excitation wavelength of 380 nm and emission spectra were recorded in the range of 400 - 600 nm (Shamsi et al., 2016). A fresh stock solution of ANS was prepared and filtered with a 0.22 µm Millipore filter. The post incubated GPC - urea, and GPC - GdnHCl solutions were supplemented with ANS dye for 30 min. The concentration of ANS dye was 400 µM.

17. Acrylamide quenching experiment

Acrylamide quenching experiments were done using 5 M acrylamide stock solution. The excitation wavelength was set at 295 nm in order to probe tryptophan fluorescence only. The emission spectrum was recorded in the range of 300 - 400 nm (Iram et al., 2013). The slit width was set at 10 nm for excitation as well as emission. Native GPC (4 µM) was incubated with a varying concentration of urea (0 – 8 M) and GdnHCl (0 – 4 M) at 37° C in 50 mM sodium phosphate buffer (pH 7.5) for 2 h. Aliquots of 5 M acrylamide stock solution were added in GPC to achieve the desired acrylamide concentration. Stern-Volmer equation F0/F=1+KSV [Q], was used to quantify the decrease in fluorescence intensity at λmax. F0 and F are the fluorescence intensity at an appropriate wavelength in the absence and presence of acrylamide, respectively. Ksv is the Stern –Volmer constant for the collisional quenchingMaulana process, Azad and [Q] Library, is the concentration Aligarh Muslim of the acrylamide. University

18. Circular dichroism (CD) analysis

Circular dichroism study was done to analyze the secondary structure of proteins. Circular dichroism measurements were performed on a JASCO J-815 spectropolarimeter equipped with a Jasco Peltier-type temperature controller (PTC–424S/15) and calibrated with ammonium D-10-camphorsulfonate. Native GPC (4 µM) was incubated with a varying concentration of urea (0 – 8

86 | P a g e

Materials and Methods

M) and GdnHCl (0 – 4 M) at 37° C in 50 mM sodium phosphate buffer (pH 7.5) for 2 h (Rashid et al., 2005). Post incubated samples were scanned in the range of 190 - 250 nm in a cuvette of 1 mm path length. Each spectrum was an average of three scans. The CD results are expressed as the mean residual ellipticity (MRE) in deg cm2 d mol−1 which is defined as the following equation:

MRE= θ (mdeg)/ 10 x n x C x l

θobs = observed ellipticity in millidegrees; C = molar concentration; n = number of amino acid residues; l = path length in centimetres.

19. Fourier transform infrared (FTIR) spectroscopy

FTIR spectra were recorded on Spectrum 100 FTIR spectrometer (Perkin Elmer). Native GPC (4 µM) was incubated with a varying concentration of urea (2 M, 4 M, 6 M, and 8 M) and GdnHCl (1 M, 2 M, 3 M, and 4 M) at 37° C in 50 mM sodium phosphate buffer (pH 7.5) for 2 h. Post incubated protein samples were scanned from 1600-1700 cm-1 (Ahmed et al., 2016). Each spectrum was an average of three scans.

20. Equilibrium denaturation experiment of GPC

The thermodynamic parameters of GPC were calculated assuming a two-state denaturation process. The data are expressed in terms of the fraction unfolded (Fu) calculatedMaulana from the Azadstandard Library, equation Aligarh (Creighton, Muslim 1997; Pace, University 1986).

Fu= Fobserved – Fnative / Funfolded – Fnative

where, Fobserved is the observed value of the signal at a given denaturant concentration and Fnative and Funfolded are the values of native and unfolded protein, respectively. The difference in free energy between the folded and the unfolded state, ΔG0, was calculated by the following equation.

87 | P a g e

Materials and Methods

ΔG0 = − RTln K = − RT ln Fu where K is the equilibrium constant, R is the gas constant, and T is the absolute temperature. If the unfolding proceeds through two state transition then urea and GdnHCl are fitted to the equation.

ΔGN−U = ΔG(H2O) −m[D]

where, ΔG(H2O) and ΔGN−U are the free energy of the folding in water and at a denaturation concentration D, respectively. m is the slope of the ΔGN−U vs [denaturant] plot, and D is the denaturant concentration (Creighton, 1997).

III: INTERACTION OF PESTICIDES (CARBENDAZIM AND OXYFLUORFEN) WITH GARLIC PHYTOCYSTATIN

21. Sample preparation

Carbendazim and oxyfluorfen stock solution was prepared in DMSO (Dimethyl sulphoxide) and was makeup with the same buffer (50 mM sodium phosphate buffer, pH 7.5) in order to avoid solution effect. Serial dilutions were performed to obtain working solutions. Three sets of individual experiments were performed with increasing concentrations of pesticides (carbendazim & oxyfluorfen) and incubation was done at 37°C in 50 mM sodium phosphate Maulana Azad Library, Aligarh Muslim University buffer for 4 h. All the solutions were filtered with a 0.22 µm Millipore filter.

22. Cysteine proteinase inhibitory assay of GPC in the presence of carbendazim and oxyfluorfen

Cysteine proteinase inhibitory activity of GPC was determined in the presence of pesticides (carbendazim and oxyfluorfen). Native GPC (4 µM) was incubated with different concentration of carbendazim and oxyfluorfen (20 µM,

88 | P a g e

Materials and Methods

40 µM, 60 µM, 80 µM, and 100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. The assay was performed by the method of Kunitz (1947) based on its ability to inhibit the caseinolytic activity of papain.

23. Ultraviolet absorption spectroscopy

UV–visible spectra were recorded on a UV-1800 Shimadzu spectrophotometer using a cuvette of 1 cm path length. The absorption spectra of the GPC and GPC-pesticide complex were recorded in the range of 190-300 nm (Ahmed et al., 2018). Native GPC (4 µM) was incubated with different concentrations of carbendazim and oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. Baseline was corrected with the appropriate solvent buffer. Proper controls of pesticides (carbendazim & oxyfluorfen) were taken and subtracted from the GPC- pesticide complex in order to cancel out the absorbance of pesticide.

24. Intrinsic fluorescence spectroscopy

Fluorescence emission spectra were recorded on a Shimadzu RF-5301PC spectrofluorophotometer (JAPAN) equipped with xenon flash lamp using 1.0 cm quartz cells. The emission spectra of GPC and GPC-pest were recorded in the range of 300 - 400 nm (Ahmed et al., 2018) . Excitation was done at 295 nm and both the slits were set at 5 nm along with the path length of 1 cm. Native GPC (4 µM) was incubated with different concentration of carbendazim and oxyfluorfenMaulana (20 µM, Azad 40 µM, Library, 60 µM, Aligarh80 µM, and Muslim 100 µM) University at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. Appropriate controls of pesticides (carbendazim and oxyfluorfen) were taken and spectra were corrected for the intrinsic fluorescence of pesticides. The corrected spectra of GPC-pesticide were then plotted after canceling out the fluorescence of pesticide. The decrease in fluorescence intensity was then analyzed with the Stern-Volmer equation (Lakowicz, 2006) .

89 | P a g e

Materials and Methods

Fo/F=1+Ksv[Q]

Where, Fo and F are the fluorescence intensities at an appropriate wavelength in the absence and presence of quencher (pesticide), respectively; Ksv is the Stern– Volmer constant and [Q] is the concentration of the quencher. The number of binding sites and binding constant was calculated by the modified Stern- Volmer equation.

log (Fo-F)/F= log Kb + n log [Q] where K is the binding constant and n is the number of binding sites.

25. Synchronous fluorescence spectroscopy

Synchronous fluorescence spectra were obtained at a constant concentration of GPC (4 µM) by adding carbendazim and oxyfluorfen (0 – 100 µM) in successive increments in 50 mM sodium phosphate buffer (pH 7.5). The excitation wavelength was set at 240 nm and emission spectra were recorded in the range of 255 – 400 nm and Δλ was set at 15 nm for tyrosine residues. Similarly, Δλ was set at 60 nm for tryptophan residues along with excitation wavelength of 240 nm and emission spectra were recorded from 300 – 400 nm.

26. Isothermal titration calorimetry

The cMaulanaalorimetric Azad experiment Library, was Aligarh carried Muslim out using University a VP-ITC titration microcalorimeter (MicroCal Inc., Northampton, MA). GPC (10 μM), carbendazim (20 μM), oxyfluorfen (100 μM) and buffer solutions (50 mM sodium phosphate buffer, pH 7.5) were filtered and then degassed for 15 minutes at 37°C using the Microcal Thermovac attachment of the instrument before the start of the experiment. The sample cell and reference cell were filled with the GPC and buffer solution respectively. The protein solution (GPC) in the sample cell was stirred at 307 rpm and titrated with 29 consecutive injections (10 μl) of carbendazim and oxyfluorfen. The spacing 90 | P a g e

Materials and Methods between each injection was of 180 s and reference power was set at 16 μCal s−1 (Rahman et al., 2017) Control experiments involve titration of pesticide with buffer alone and GPC with DMSO. The integrated heat data were then subtracted from data obtained from GPC-pesticide interaction to eliminate heat of dilution. The integrated heat data were then analyzed by the MicroCal analyzer (Rahman et al., 2017).

27. Circular dichroism (CD) analysis

Circular dichroism experiments were performed on a JASCO J-815 spectropolarimeter equipped with a Jasco Peltier-type temperature controller (PTC–424S/15) and calibrated with ammonium D-10-camphorsulfonate. Far UV CD spectra were recorded in the range of 190 nm – 260 nm at 25°C in a cuvette of 1 mm path length (Ahmed et al., 2018). Native GPC (4 µM) was incubated with different concentration of carbendazim and oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. The CD results are expressed as the mean residual ellipticity (MRE) in deg cm2 dmol−1 which is defined as the following equation:

MRE= θ (mdeg)/ 10 x n x C x l

θobs = observed ellipticity in millidegree; C = molar concentration; n = number of amino acid residues; l = path length in centimeters.

Maulana Azad Library, Aligarh Muslim University IV: AGGREGATION STUDY OF GARLIC PHYTOCYSTATIN ASSISTED BY TRIFLUOROETHANOL (TFE)

28. Cysteine proteinase inhibitory assay of GPC in the presence of TFE

Cysteine proteinase inhibitory activity of GPC was determined by the method described by Kunitz (1947). Native GPC (4 µM) was incubated with varying

91 | P a g e

Materials and Methods concentrations of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h, and then the assay was performed.

29. Intrinsic fluorescence spectroscopy

Intrinsic fluorescence was recorded on a Shimadzu RF-5301 spectrofluorophotometer (Tokyo, Japan). Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Excitation was done at 280 nm and both the slits were set at 5 nm along with the path length of 1 cm. The emission spectra of GPC and GPC treated with TFE were recorded in the range of 300 - 400 nm (Bhat and Bano, 2014).

30. ANS fluorescence measurements

The ANS fluorescence spectra were recorded on a Shimadzu spectrofluorophotometer (Tokyo, Japan). The slit width was set at 5 nm along with the path length of 1 cm. ANS fluorescence was measured at an excitation wavelength of 380 nm and recorded in the range of 400 – 600 nm (Bhat and Bano, 2014). Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Pre-incubated GPC with TFE (0-90% v/v) was treated with ANS (1 mM) dye and spectra were recorded.

31. Circular dichroism (CD) analysis Maulana Azad Library, Aligarh Muslim University Circular dichroism experiments were performed on a JASCO J-815 spectropolarimeter equipped with a Jasco Peltier-type temperature controller (PTC–424S/15) and calibrated with ammonium D-10-camphorsulfonate. Far UV CD spectra were recorded in the range of 190 nm – 260 nm at 25°C in a cuvette of 1 mm path length (Bhat and Bano, 2014). Native GPC (4 µM) was incubated with varying concentrations of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. The CD spectra were recorded after

92 | P a g e

Materials and Methods incubation of GPC with TFE. Each spectrum recorded is an average of three scans.

32. Turbidity measurement

Turbidity measurements of samples were performed on a UV-1800 Shimadzu UV spectrophotometer using a cuvette of 1 cm path length (Shamsi et al., 2016). Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. The absorbance of pre-incubated GPC with TFE (0-90% v/v) was recorded at 350 nm (Shamsi et al., 2016).

33. Rayleigh scattering measurement

The Rayleigh measurement was performed on a Shimadzu RF-5301 spectrofluorophotometer (Tokyo, Japan) using a 1 cm path length quartz cell. Native GPC (4 µM) was incubated with increasing concentration of TFE (0- 90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. The excitation wavelength of the experiment was set at 350 nm and emission range was 300 – 400 nm (Shamsi et al., 2016). The excitation and emission slit width was fixed at 5 nm.

34. Thioflavin T (ThT) assay

ThT assay was done analyze the presence of aggregates of GPC upon treatment with TFE. MaulanaThT fluorescence Azad Library, spectra Aligarh were recorded Muslim onUniversity a Shimadzu spectrofluorophotometer (RF-5301). The excitation wavelength was set at 440 nm and emission spectra were recorded in the range of 450 - 600 nm (Bhat and Bano, 2014). The excitation and emission slits were set at 5 nm along with the path length of 1 cm. Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. It was then treated with ThT dye in 1:5 ratio and incubated in the dark chamber for 30 minutes. The ThT stock solution was prepared in 50

93 | P a g e

Materials and Methods mM sodium phosphate buffer, pH 7.5 and the final concentration of ThT was 20 μM. All the solutions were filtered using a 0.22 μm Millipore filter.

35. Congo red assay

Congo red assay was performed to probe the formation of aggregates by taking absorbance in the range of 400 nm - 600 nm on a UV-1800 Shimadzu UV spectrophotometer using a cuvette of 1 cm path length (Shamsi et al., 2016). Native GPC (4 µM) was incubated with increasing concentration of TFE (0- 90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h, and then treated with Congo red dye for 20 minutes. The stock solution of Congo red was prepared in 50 mM sodium phosphate buffer and the solutions were filtered through a 0.22 μm Millipore filter. The concentration of Congo red was 100 μM.

36. Scanning electron microscopy

Native GPC (4 µM) was incubated with TFE (60%) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. The post-incubated sample was then adsorbed on to cellulose ultrafiltration membrane and then placed on a gold- palladium grid for fine image tuning and imaged using a JSM-6510LV (JEOL JAPAN) scanning electron microscope at a voltage of 10 kV (Shamsi et al., 2016).

V: EFFECTMaulana Azad OF HEAVYLibrary, Aligarh METALS Muslim (Zn University+2 and Cd+2) ON GARLIC PHYTOCYSTATIN

37. Cysteine proteinase inhibitory assay of GPC in the presence of zinc and cadmium

The cysteine proteinase inhibitory assay of GPC was assessed in the presence of zinc and cadmium. The GPC (4 µM) was incubated with 20 μM, 40 μM, 60 μM, 80 μM and 100 μM of zinc and cadmium, respectively at 37ºC in 50 mM

94 | P a g e

Materials and Methods sodium phosphate buffer (pH 7.5) for 4 h and then the inhibitory assay was performed according to the method described by Kunitz (1947).

38. Ultraviolet absorption spectroscopy

The UV-vis absorption spectroscopy of GPC and GPC treated with metals was performed on a UV-1800 Shimadzu spectrophotometer. The native GPC (4 µM) was incubated with 20 μM, 40 μM, 60 μM, 80 μM, 100 μM of zinc at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and the similar protocol was followed for the cadmium (Khan et al., 2017). The absorption spectra were recorded in the range of 240-300 using cuvette with a path length of 1 cm. Baseline was corrected with the 50 mM sodium phosphate buffer. Proper controls for the metals were taken and subtracted from the complex to cancel out absorbance of metals.

39. Intrinsic fluorescence spectroscopy

The steady-state fluorescence spectroscopy of native GPC and GPC treated with metal was performed on a Shimadzu spectrofluorophotometer (RF- 5301PC). The excitation wavelength was set at 280 nm, whereas the emission range was fixed at 300-400 nm (Khan et al., 2017). The width of both the slits was fixed at 5 nm, and the path length was 1 cm. The native GPC (4 µM) was incubated with 20 μM, 40 μM, 60 μM, 80 μM, 100 μM of zinc at 37°C in 50 mM sodium Maulana phosphate Azad buffer Library, (pH 7.5) for Aligarh 4 h and Muslim the similar University protocol was followed for the cadmium. Proper controls were recorded for the metals and corrected spectra were plotted. The quenching constant was determined by the Stern-Volmer equation (1) (Lakowicz, 2006) and the binding constant was calculated by the modified Stern-Volmer equation (2).

Fo/F=1+Ksv[Q]……………………….(1)

95 | P a g e

Materials and Methods

Fo and F represent the fluorescence intensities at an appropriate wavelength in the absence and presence of quencher, respectively. Ksv denotes Stern-Volmer quenching constant and [Q] is the concentration of the quencher (zinc & cadmium).

log (Fo-F)/F= log Kb + n log [Q]……………..(2)

Kb is the binding constant and n is the number of binding sites.

40. Synchronous fluorescence spectroscopy

The synchronous fluorescence spectra were recorded for the native GPC and

GPC treated with metals. The λex was set at 240 nm and λem was 255-400 nm, thereby satisfying the Δλ=15 nm for tyrosine residues. A similar protocol was followed for the tryptophan residues (Δλ=60 nm) with the λex of 240 nm and

λem was 300-400 nm (Khan et al., 2017). The concentration of native GPC was 4 µM and the concentration of zinc and cadmium was varied from 10 µM-100 µM. Proper controls for zinc & cadmium were taken and corrected spectra were plotted.

41. Three-dimensional fluorescence spectroscopy

Three-dimensional fluorescence spectroscopy was done on a RF 6000 Shimadzu fluorescence spectrophotometer. The spectra of native GPC and GPC treated with zinc were recorded at λ and λ of 200 - 350 nm and 200 - Maulana Azad Library, Aligarhex Muslimem University 600 nm respectively (Rahman et al., 2018). The native GPC (4 µM) was incubated with 100 µM zinc at 37°C in sodium phosphate buffer (pH 7.5) for 4 h before obtaining the spectra. Similarly, GPC was incubated with cadmium (100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h.

42. Circular dichroism (CD) analysis

CD spectroscopy was done on a J-810 Jasco spectropolarimeter using a quartz cell of 1 mm path length. The native GPC (4 µM) was incubated with zinc (100

96 | P a g e

Materials and Methods

μM) and cadmium (100 μM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. The spectra were recorded in the far UV range, i.e. 190-250 nm (Khan et al., 2017). The results obtained are expressed as CD (mdeg) and each spectrum is the average of three scans.

43. Thioflavin T (ThT) assay

Thioflavin-T fluorescence spectra were recorded on a Shimadzu (RF-5301) spectrofluorophotometer. The excitation wavelength was set at 440 nm and emission spectra were recorded in the range of 450 - 600 nm (Bhat and Bano, 2014). The excitation and emission slits were set at 5 nm along with the path length of 1 cm. The GPC was incubated with Zn+2 (100 µM) and Cd+2 (100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. It was then treated with ThT dye in 1:5 ratio and incubated in the dark chamber for 30 minutes. The ThT stock solution was prepared in 50 mM sodium phosphate buffer, pH 7.5 and the final concentration of ThT was 20 μM. All the solutions were filtered using a 0.22 μm Millipore filter.

44. Congo red assay

Congo red assay was performed to probe the formation of aggregates by taking absorbance in the range of 400 nm - 600 nm on a UV-1800 Shimadzu UV spectrophotometer using a cuvette of 1 cm path length (Bhat and Bano, 2014). +2 +2 GPC was incubatedMaulana with Azad Zn (100 Library, µM) and Aligarh Cd (100 Muslim µM) at University 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and then treated with Congo red dye for 20 minutes. The stock solution of Congo red was prepared in 50 mM sodium phosphate buffer and the solutions were filtered through a 0.22 μm Millipore filter. The concentration of Congo red was 100 μM.

97 | P a g e

Materials and Methods

45. Scanning electron microscopy

Scanning electron microscopy was done on a JSM-6510LV (JEOL JAPAN) at a voltage of 10 kV. The GPC was incubated with zinc (100 µM) and cadmium (100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. After incubation, the samples were then air dried and then placed on a gold- palladium grid for image fine-tuning (Shamsi et al., 2016).

46. Statistical analysis

The experiments were carried out three times to document reproducibility. The data have been expressed as mean ± SEM. Significance of difference in mean values was evaluated using one-way analysis of variance (ANOVA). A probability level of p<0.05 was selected as an indicator of statistical significance.

Maulana Azad Library, Aligarh Muslim University

98 | P a g e

Conclusion

Maulana Azad Library, Aligarh Muslim University

Conclusion

CONCLUSION

Phytocystatins have shown ubiquitous nature in the plant system and bind non- covalently to the cysteine proteinases. They are non-competitive and reversible inhibitors of cysteine proteinases. They have been classified into three distinct groups based on the molecular mass and conserved domains, namely group I, group II and group III phytocystatins. The group I members contain a single cystatin domain and has a molecular weight of 12-16 kDa. The group II and group III members have a molecular weight of around 23 kDa and 85 kDa, respectively. Group II contains conserved cystatin domain at N -terminal and an additional extension at C-terminal of 10 kDa along with a conserved SNSL motif however, group III phytocystatins contain multi-domains. They are biologically active substances playing an essential role in various metabolic functions of plants such as regulating protein turnover in developing and germinating seeds, tolerance against environmental stress as well as various defense mechanisms. They have been isolated from various sources such as potato, cowpea, chickpea, almond, and pineapple. Recent advancements in genetic engineering have garnered significant attention towards cysteine proteinase inhibitors as a potential alternative for developing resistant crops. There is also rapidly growing information about the importance of cysteine proteinase inhibitors in many pathological diseases. In this study a phytocystatin has been isolated from garlic cloves and characterized structurally, biochemically and kinetically. In the present study, a phytocystatin of 12.5 kDa has been purified from garlic Maulana Azad Library, Aligarh Muslim University cloves which is named as GPC. The purified garlic phytocystatin has shown a broad range of temperature and pH stability. The kinetic analysis of the purified inhibitor showed strong inhibition against papain followed by ficin and bromelain. The secondary structure analysis showed the presence of 33.9% alpha-helical content in the purified GPC.

Insight into the molecular mechanism underlying the unfolding of protein is one of the major challenges in the field of protein biochemistry. Conformational stability of a protein is primarily required for its maximum

276 | P a g e

Conclusion functionality. In a way to understand the dependence of function of a protein on its conformational stability, phytocystatin was chosen for detailed study. It plays a crucial role in the various physiological process and maintains the protease-antiprotease balance of cell owing to their inhibitory activity. In the present study, a phytocystatin was isolated, and the chemical denaturation was done with the aid of urea and guanidine hydrochloride. Normal functioning of phytocystatin is required to maintain the physiological balance and development of plants. Therefore, phytocystatin must sustain functional and structural conformation for its proper functioning. The study suggested that denaturants bring structural and functional alterations, thereby affecting the normal functioning of crucial proteins which could ultimately disrupt the growth and development of plants. The unfolding pattern of phytocystatin was monitored by intrinsic fluorescence, circular dichroism and FTIR spectroscopy which showed the complete unfolding of garlic phytocystatin at 8 M urea and 4 M GdnHCl. The results confirmed the loss in the inhibitory activity of garlic phytocystatin along with structural alteration, which is due to the exposure of denaturants.

Pest and pathogens are the primary cause in declining quantity and quality of crop yield. Hence, it is practically impossible to forbid the use of pesticides against pest and pathogens. However, extensive usage of pesticides is a major health concern worldwide due to its negative impact on living organisms. Therefore, it is required to assess the toxic potential of pesticides which are extensivelyMaulana used Azad in the Library, fields. pesticides. Aligarh TheMuslim present University study examines the effects of fungicide (carbendazim) and herbicide (oxyfluorfen) on a phytocystatin isolated from garlic. The study employed different biophysical techniques to elucidate the binding of carbendazim and oxyfluorfen to garlic phytocystatin. The UV-visible and fluorescence quenching experiment confirmed the binding of carbendazim and oxyfluorfen to garlic phytocystatin. The synchronous fluorescence and three-dimensional fluorescence have shown the perturbation in the microenvironment around aromatic residues of garlic phytocystatin. The circular dichroism analysis confirmed the alterations in the 277 | P a g e

Conclusion secondary structure of garlic phytocystatin upon binding of carbendazim and oxyfluorfen. The study showed concentration-dependent effects of carbendazim and oxyfluorfen on the function and structure of garlic phytocystatin. Hence, the study confirmed the transition of native garlic phytocystatin to non-native form, thereby disrupting the normal functioning of phytocystatin. The study suggests that it is necessary to explore the action of pesticides in order to draw a broad line between negative and positive impact of pesticides and focusses on the need of developing fast as well as accurate screening techniques which could examine the impact of pesticides in a way to minimize its accumulation in environment and exposure to living organisms.

Protein aggregation is a central topic in the field of biochemistry. It is a well-known fact that a number of diseases are due to misfolding or aggregation of crucial proteins. Also, the occurrence of an insoluble intracellular complex of proteins is a major problem for human health and is symptomatic of several neurodegenerative diseases. Thus, it can be said that the aggregation of proteins has always been a hot topic in the field of protein biochemistry. Moreover, the boundaries are expanding day by day as the research continues due to its crucial role in the pathophysiology of innumerable diseases. Recent studies have proved that protein aggregation is not limited to only animals. Various reports confirmed the presence of amyloid-like protein inclusions in plant proteins along with their implications. Hence, the present study was done to analyze the functional and structural changes in garlic phytocystatin upon aggregation.Maulana Trifluoroethanol Azad Library, was Aligarh used to aggregateMuslim University the purified GPC. The study employed various biophysical techniques such as intrinsic fluorescence, ANS fluorescence, circular dichroism, Thioflavin-T assay, and Congo red assay for the assessment of aggregates formed in the presence of trifluoroethanol. The morphology of the aggregates was studied with the help of scanning electron microscope. The cysteine proteinase inhibitory activity of garlic phytocystatin reduces with increasing concentration of trifluoroethanol. It was also observed that trifluoroethanol induces the transition of garlic phytocystatin towards non-native form and results in aggregation of garlic 278 | P a g e

Conclusion phytocystatin at higher concentration of trifluoroethanol. The morphology of the native GPC also changes upon interaction with trifluoroethanol. The results obtained from the study might be helpful for better understanding and characterizing the amyloid formation within plant proteins.

Accumulation and exposure of heavy metals have raised concern for human and animal health. Heavy metals induce abiotic stress within the plant system and result in growth inhibition. Uptake of high concentration of heavy metals causes toxicity to plants and results in malfunctioning of physiological activities. Zinc is a heavy metal which generally acts as a cofactor for different enzymes and catalyst to various reactions. Similarly, cadmium is also a heavy metal and causes a severe threat to the plants at higher concentration. The present work was done to investigate the effect of zinc and cadmium on garlic phytocystatin. Phytocystatins have been shown to express strongly during abiotic stress. It is found that the cysteine proteinase inhibitory activity of GPC declines upon binding with zinc and cadmium to form complex. The UV- visible absorption spectroscopy and fluorescence quenching experiment showed that GPC binds with zinc and cadmium. Different biophysical techniques suggested functional and structural alteration within garlic phytocystatin upon binding of zinc and cadmium. Thereby, confirming that the exposure of high concentration of zinc and cadmium might alter the normal functioning of GPC, which could further affect the growth and development of plants. Maulana Azad Library, Aligarh Muslim University The importance of work lies in adding knowledge to the existing information in the pool of plant genetic engineering to improve crop yield and quality by targeting the endogenous protease mechanism of pests that harm the vitality and yield of the crop. Furthermore, the work also sheds light on the effect of abiotic stress on the plant system.

279 | P a g e

Results & Discussion

Maulana Azad Library, Aligarh Muslim University

Chapter – 1 Purification and characterization of garlic phytocystatin

Maulana Azad Library,(GPC) Aligarh Muslim University

Chapter -1 Results and discussion

RESULTS

PURIFICATION AND CHARACTERIZATION OF GPC

1. PURIFICATION OF GARLIC PHYTOCYSTATIN (GPC)

In the present study, garlic phytocystatin has been purified from garlic cloves. As explained in the method section, the process involved two steps after homogenization viz. ammonium sulfate fractionation and gel filtration chromatography on Sephacryl S-100 HR column. The crude homogenate of cloves prepared in 50 mM sodium phosphate buffer (pH 7.5) contain a number of impurities which were removed by ammonium sulfate fractionation (30- 60%). The precipitate containing protein of interest obtained after treatment was then dissolved in a minimum volume of sodium phosphate buffer (0.05 M, pH 7.5) and dialyzed several times against same sodium phosphate buffer (0.05 M, pH 7.5). This method of purification resulted in 16.3-fold purification with a yield of 62.7. The summary of purification of GPC is given in Table-3.

2. GEL FILTRATION CHROMATOGRAPHY

The precipitate obtained after ammonium sulfate treatment and dialysis was filtered with Whatman paper and then loaded on Sephacryl S-100HR column pre-equilibratedMaulana with Azad 0.05 Library, M sodium Aligarh phosphate Muslim buffer pHUniversity 7.5. The fractions (5 ml) were collected and analyzed for the cysteine proteinase inhibitory activity along with protein content. Absorbance taken at 660 nm was plotted for protein concentration. A single protein peak with significant inhibitory activity against papain was obtained (Fig. 9). The protocol provided a purification fold of 152.6 and 48.9% yield. The step-wise purification result is summarized in Table-3. The fractions having significant cysteine proteinase inhibitory activity were pooled for further analysis.

99 | P a g e

Chapter -1 Results and discussion

Table 3: Purification table of phytocystatin from garlic (Allium sativum).

Steps of Total Specific Volume Total Fold % activity ٭purification Protein (ml) units† purification Yield of GPC (mg) Units/mg

Crude 250 7025 133.4 0.019 1 100 Homogenate

Ammonium sulfate 270 83.7 0.31 16.3 16.3 62.7 fractionation (30-60%)

Gel filtration 15 22.5 65.25 2.9 152.6 48.9 Sephacryl S100-HR

Maulana Azad Library, Aligarh Muslim University .Protein concentration determined by the method of Lowry et al ٭

† 1 Unit of inhibitor enzyme activity is defined as the amount of inhibitor bringing about 0.001 changes in O.D./min./ml.

100 | P a g e

Chapter -1 Results and discussion

% Inhibition Absorbance@660 nm 70 0.35

60 0.3

50 0.25

nm

0

n

6 o

ti 40 0.2

@6

bi nce nce

Inhi 30 0.15

rba

% o

20 0.1 bs A

10 0.05

0 0 0 2 4 6 8 10 12 14 16 18 20 Fraction Number

Figure 9. Elution profile of GPC on a Sephacryl S-100 HR column. Maulana Azad Library, Aligarh Muslim University The precipitate obtained from 30% to 60% ammonium sulfate fractionation was loaded on a gel filtration column at a flow rate of 16 ml h-1. Fractions of 5 ml were collected, and the inhibitory activity of each fraction was determined. Fractions having maximum inhibitory activity were pooled for further studies.

101 | P a g e

Chapter -1 Results and discussion

3. HOMOGENEITY OF THE PURIFIED GPC

The elution profile of the inhibitor showed a single protein peak having significant inhibitory activity, thus suggesting the homogeneity of the purified inhibitor (Fig. 9) on the basis of molecular weight. The purified cysteine proteinase inhibitor was further run on a 7.5% native polyacrylamide gel electrophoresis. A single band was obtained, which suggested the presence of a single protein that is garlic phytocystatin (Fig. 10).

4. REDUCING (SDS + βME) AND NON-REDUCING SDS- PAGE

The purified garlic phytocystatin was also analyzed for its molecular weight and subunit structure by SDS-PAGE under reducing (in the presence of β- mercaptoethanol) and non-reducing conditions (in the absence of β- mercaptoethanol). The GPC migrated as a single band under reducing as well as non-reducing conditions, thereby confirming the absence of any subunit (Fig 11).

5. PROPERTIES OF THE PURIFIED GPC

5.1 Molecular mass determination

The molecular mass of GPC was determined under native conditions by size exclusion chromatography on Sephacryl S-100HR column using standard Maulana Azad Library, Aligarh Muslim University marker proteins of known molecular weight. The standard molecular weight markers used were aprotinin (6.5 kDa), cytochrome C (12.3 kDa), lysozyme (14.3 kDa), trypsin (23.3 kDa) and ovalbumin (43 kDa). The data was analyzed by the method of Andrews (1964) and showed a linear relationship between log M and Ve/Vo, where Ve is the elution volume and Vo is the void volume of the column.

102 | P a g e

Chapter -1 Results and discussion

1 2

Maulana Azad Library, Aligarh Muslim University Figure 10. Gel electrophoresis of purified garlic phytocystatin. Electrophoresis was performed on 7.5% gel. Lane 1 and 2 showing bands of different fractions having maximum inhibitory activity against papain. 50 µg of protein was applied in each lane.

103 | P a g e

Chapter -1 Results and discussion

A B C

D

E

1 2 3

Figure 11. SDS-PAGE of garlic phytocystatin under non-reducing and reducing conditions. Maulana Azad Library, Aligarh Muslim University Electrophoresis was performed on 12.5% polyacrylamide gel. Lane 1 contains GPC (50 µg) under non-reducing conditions, lane-3 contains GPC (50 µg) under reducing conditions. Lane 2 contains standard protein markers viz. (A) Ovalbumin (43 kDa) at the top, (B) Carbonic anhydrase (29 kDa), (C) Trypsin inhibitor (20.1 kDa), (D) Lysozyme (14.3 kDa) and (E) Aprotinin (6.5 kDa) at the bottom.

104 | P a g e

Chapter -1 Results and discussion

The arrow shows the position of GPC, and the molecular weight was found to be 12 kDa (Fig. 12). The molecular mass of GPC was also analyzed by SDS- PAGE (Fig. 11) under reducing and non- reducing conditions. GPC migrated as a single band under reducing and non-reducing condition suggesting a single polypeptide chain lacking any subunit moiety. The data was used to plot relative mobility (Rm) of standard maker protein against the log molecular weight of standard protein (Fig. 13). The plot showed a linear relationship between relative mobility (Rm) and log molecular weight. The molecular weight of GPC was found to be 12.5 kDa by SDS-PAGE.

5.2 Stokes radius Stokes radius of proteins correlates well with their elution behavior from a gel filtration column. The Stokes radius of the purified GPC was found to be 17.8 Å indicating that the shape of GPC is similar to that of cytochrome C (Fig.14).

5.3 Diffusion coefficient

The diffusion coefficient (D) of the GPC was determined with the help of the equation given below:

D = kT/f where, f is the frictional coefficient and depends on the shape and size of the protein, r = is the Stokes radius of the protein. ɳ = is the coefficient of viscosity of the medium, k = 1.38 × 10-6 erg/deg is Boltzmann’s constant, and T is Maulana Azad Library, Aligarh Muslim University absolute temperature.

The diffusion coefficient was calculated with the help of the Stokes radius and found to be 12.4 x 10-7 m2 sec-1.

5.4 Carbohydrate estimation

The carbohydrate estimation was done by Dubois method. It was found that garlic phytocystatin is devoid of carbohydrate content.

105 | P a g e

Chapter -1 Results and discussion

1.9 GPC A 1.8

1.7 B

1.6 C

o D

1.5

e/V V 1.4 E

1.3

1.2

1.1

1 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 Log M

Figure 12. Molecular mass determination of purified GPC using Sephacryl S-100 HR gel filtration chromatography. Maulana Azad Library, Aligarh Muslim University

The plot of log M vs. Ve/Vo for the determination of the molecular mass of purified GPC using Sephacryl S-100HR column. Markers passed through the column are (A) Aprotinin (6.5 kDa), (B) Cytochrome C (12.3 kDa), (C) Lysozyme (14.3 kDa), (D) Trypsin (23.3 kDa) and (E) Ovalbumin (43 kDa).

106 | P a g e

Chapter -1 Results and discussion

1.8

1.6 A

1.4 B

M C GPC

1.2 g D Lo 1

0.8 E 0.6

0.4

0.2

0 0 0.2 0.4 0.6 0.8 1 1.2 1.4

Relative mobility (Rm)

Figure 13. Log M (molecular weight) of marker proteins against relative mobility (Rm) of molecular weight markers for determination of molecular mass. Maulana Azad Library, Aligarh Muslim University Relative mobility (Rm) of standard protein markers was plotted against the log molecular weight of standard maker protein for determination of the molecular mass of GPC. The molecular weight of standard protein marker is (A) Ovalbumin (43 kDa), (B) Carbonic Anhydrase (29 kDa), (C) Soybean Trypsin Inhibitor (20.1 kDa), (D) Lysozyme (14.3 kDa) and (E) Aprotinin (6.5 kDa).

107 | P a g e

Chapter -1 Results and discussion

0.18

0.16 A

0.14

0.12

B

)1/2 0.1

v a

K 0.08

g C lo

- GPC ( 0.06 D 0.04

0.02 E

0 10 12 14 16 18 20 22 24 26 28 30 Stokes radius (r)

Figure 14. Determination of Stokes radius of GPC by the plot of Laurent and Killander [(log Kav)1/2 vs. r].

Marker proteinsMaulana and the Azad purified Library, GPC Aligarhwere subjected Muslim to Universitygel filtration on Sephacryl S- 100 HR. Stokes radii of the marker proteins were: A- Ovalbumin (27.3Å); B-Trypsin (20.2 Å), C-Lysozyme (18.6 Å), D- Cytochrome C (17.5 Å), E-Aprotinin (13.5 Å). The arrow shows the Stokes radius of purified GPC.

108 | P a g e

Chapter -1 Results and discussion

5.5 Thiol group estimation

The thiol group estimation was done by the Ellman method. It was found that garlic phytocystatin lacks sulfhydryl linkages.

5.6 Effect of pH on the activity of GPC

The garlic phytocystatin was found to be quite stable in the pH range of 6-8 (Fig. 15). The GPC retained about 31.2 % of its inhibitory activity at pH 6 and 56.4 % inhibitory activity at pH 8. The inhibitory activity decreases significantly below pH 6 and above pH 8. The maximum inhibitory activity was observed at pH 7.5. Purified GPC was found to be stable in a broad range of temperature. It was quite stable between 30˚C and 60˚C (Fig. 16). However, the activity decreases significantly from 60˚C to 90˚C.

5.7 Effect of temperature on the activity of GP

Purified GPC was found to be stable in a broad range of temperature. It was quite stable between 30˚C and 60˚C (Fig. 16). The GPC showed 52.3% inhibitory activity at 30˚C and 6.6% inhibitory activity at 90˚C. The result showed that inhibitory activity declines gradually with the increasing temperature. However, the activity decreases significantly from 60˚C to 90˚C.

6. IMMUNOLOGICAL PROPERTIES

6.1 AntibodyMaulana AzadTiter Library, Aligarh Muslim University

The GPC produced an effective immune response which contributes to a high titer of antibody. The titer value was found to be 19952.6 for the resulting antisera, as determined by direct binding ELISA in rabbit antisera (Fig. 17).

109 | P a g e

Chapter -1 Results and discussion

70

60

50

40

ition Inhib

30 % 20

10

0 2 4 6 8 10 pH

Figure 15. pH stability profile of GPC

Fifty µg of the GPC was incubated in 50 mM sodium acetate buffer (pH 3.0-6.0), 50 MaulanamM sodium Azad phosphate Library, buffer Aligarh (pH 7.0 Muslim-8.0) and University 50 mM Tris- HCl buffer (pH 9.0-10.0) for 30 min at 37 °C. After incubation pH of the inhibitor solution was neutralized, and then the remaining % inhibitory activity was determined against 50 µg of papain at 37ºC. The activity of papain in the absence of inhibitor at pH 7.5 should have been taken as 100%.

110 | P a g e

Chapter -1 Results and discussion

70

60

50

ition 40

Inhib

30 %

20

10

0 30 40 50 60 70 80 90 100 Temperature °C

Figure 16. Effect of temperature on the inhibitory activity of GPC. Maulana Azad Library, Aligarh Muslim University Fifty µg of the inhibitor was incubated at a different temperature ranging from 30°C – 90°C for 30 min in sodium phosphate buffer (50 mM, pH 7.5). Samples were rapidly cooled ice-cold water bath, and remaining inhibitory activity was determined against fifty µg papain.

111 | P a g e

Chapter -1 Results and discussion

1.4

1.2

nm

5 1

0.8

nce at 40 at nce rba

0.6 Abso

0.4

0.2

0 0 1 2 3 4 5 -anti log serum dilution

Figure 17. Direct binding ELISA plot Serially dilutedMaulana antisera Azad and Library, pre-immune Aligarh sera Muslim were incubated University with 0.5 μg/100 μl antigen. The curve with solid circles is for post-immunized sera, whereas the curve with solid triangles is for pre-immunized sera.

112 | P a g e

Chapter -1 Results and discussion

6.2 Crossreactivity

The GPC shows high immune response as the antibodies raised against GPC in rabbit serum gives a single precipitin line (Fig. 18), thereby suggesting that the well contains single protein (GPC). Moreover, GPC was also checked for cystatins purified from other sources, but no precipitin line was found suggesting the presence of immunologically pure GPC.

7. KINETIC PROPERTIES OF GPC

7.1 Stoichiometry of inhibition

The inhibition of different proteases was studied by varying their molar concentration at a fixed molar concentration of GPC. The residual activity of protease showed that as its concentration is increased from 0.01- 0.06 μM, it is progressively inhibited by 0.06 μM GPC giving a stoichiometric ratio of 1:1, thus depicting that one molecule of GPC inhibits one molecule of active protease. A similar result was obtained for bromelain and ficin.

7.2 Inhibition of different proteinases by GPC

Purified GPC showed the strongest inhibition against papain followed by ficin and bromelain, which are also cysteine proteinase, thereby confirmingMaulana the natureAzad Library, of the purified Aligarh inhibitor Muslim (Fig. University 19). However, the purified inhibitor (GPC) showed negligible inhibitory activity against trypsin and chymotrypsin, which are serine proteinases.

113 | P a g e

Chapter -1 Results and discussion

Figure 18. Ouchterlony immunodiffusion of GPC. The antiserum was allowed to react with GPC in agarose plates. The Maulana Azad Library, Aligarh Muslim University central well (D) contains antiserum, whereas the other wells, namely A, B, and C, contain purified GPC inhibitor. Single precipitin line was formed.

114 | P a g e

Chapter -1 Results and discussion

Figure 19. Inhibitory activity of GPC against different proteinases.

Fifty Maulana(50 μg) of Azad cysteine Library, proteinases Aligarh papain, Muslim ficin, University bromelain and serine proteases trypsin and chymotrypsin were incubated with varying concentration of GPC (0-70 μg) for 30 min. The inhibitory activity of GPC towards different proteinases was measured using casein as a substrate.

115 | P a g e

Chapter -1 Results and discussion

7.3 Determination of inhibition constant (Ki)

Garlic phytocystatin inhibited papain and ficin more efficiently and specifically. However, the inhibition of serine proteases, such as chymotrypsin and trypsin was found to be insignificant. It has been reported earlier that cystatin binds and inhibits papain more efficaciously (Priyadarshini and Bano, 2010; Shah et al., 2013). Phytocystatins binds reversibly and non-competitively with cysteine proteases (Wang et al., 2008). GPC bound with high affinity to papain and showed potent inhibition. Figure 20 shows the Lineweaver-Burk plot in which Vmax decreases with the increase of inhibition at varying concentrations (0.06 µM – 0.30 µM) of GPC, and the Ki apparent remains the same. These observations suggest the non-competitive inhibition for GPC. The inhibition constant was determined by the method reported earlier. The linear equation is given by Henderson (1972), is described as follows,

[I]o /1˗ (Vi/Vo) = Ki [1+ [S]o /Km] Vi/Vo + [E]o

Where, [E]o [I]o and [S]o are the initial concentrations of enzyme, inhibitor, and substrate, respectively. Vi is the reaction rate in the presence of inhibitor, and

Vo is the reaction rate in the absence of inhibitor. The plot of [I]o /1˗ (Vi/Vo) against Vo/Vi is a straight line, the slope of which gives,

Ki (app) = Ki [1+ [S]o/Km]

True Ki value was obtained from a replot of Ki (app) against [S]o. The Lineweaver-BurkMaulana was plottedAzad Library, for bromelain Aligarh (Fig. Muslim 22) and University ficin (Fig. 24) against the different concentration of GPC. The Ki values obtained for papain (Fig. 21), bromelain (Fig. 23) and ficin (Fig. 25) are 8.5x10-8 M, 1.38x10-7 M, -7 and 1.2x10 M, respectively. The Ki value for papain suggests that GPC has the highest affinity for it, as compared to other cysteine proteinases.

116 | P a g e

Chapter -1 Results and discussion

10

9

8 0.06 µM 7

0.12 µM 6

0.18 µM /[V] 1 0.24 µM 5

0.30 µM 4

3

2

1

0 -10 -5 0 5 10 15 1/[S] (mM)

Figure 20. Lineweaver-Burk plot representing the inhibitory effect of GPC Maulanaon papain. Azad Library, Aligarh Muslim University Papain was used at a final concentration of 0.06 μM with increasing amounts of GPC (0.06-0.30 μM). Measurements for residual activity were made, as described in the methodology section.

117 | P a g e

Chapter -1 Results and discussion

3.5

3

2.5

2

/[V] 1 1.5

1

0.5

0 -0.2 -0.1 0 0.1 0.2 0.3 0.4

[I] (µM)

Figure 21. Dixon plot for determination of apparent Ki of GPC for papain. Maulana Azad Library, Aligarh Muslim University

The plot of 1/Vmax vs. [I] by obtaining Vmax from Lineweaver–Burk plot of papain for determination of apparent Ki.

118 | P a g e

Chapter -1 Results and discussion

4

3.5 0.06 µM 3 0.12 µM

2.5

0.18 µM /[V] 1 2 0.24 µM

0.30 µM 1.5

1

0.5

0 -3 -1 1 3 5 7 1/[S] (mM)

Figure 22. Lineweaver-Burk plot representing the inhibitory effect of Maulana Azad Library, Aligarh Muslim University GPC on bromelain. Bromelain was used at a final concentration of 0.06 μM with increasing amounts of GPC (0.06-0.30 μM). Measurements for residual activity were made, as described in the methodology section.

119 | P a g e

Chapter -1 Results and discussion

1.4

1.2

1

]

/[V 0.8 1

0.6

0.4

0.2

0 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 1/[S] (µM)

Maulana Azad Library, Aligarh Muslim University Figure 23. Dixon plot for determination of apparent Ki of GPC for bromelain. The plot of 1/Vmax vs. [I] by obtaining Vmax from

Lineweaver–Burk plot of bromelain for determination of apparent Ki.

120 | P a g e

Chapter -1 Results and discussion

10

9

8

7

0.06 µM 6

0.12 µM /[V] 1 5 0.18 µM 0.24 µM 4 0.30 µM 3

2

1

0 -5 -3 -1 1 3 5 7 9 1/[S] (mM)

Figure 24. Lineweaver-Burk plot representing the inhibitory effect of Maulana Azad Library, Aligarh Muslim University GPC on ficin.

Ficin was used at a final concentration of 0.06 μM with increasing amounts of GPC (0.06-0.30 μM). Measurements for residual activity were made, as described in the methodology section.

121 | P a g e

Chapter -1 Results and discussion

2.5

2

1.5

/[V] 1 1

0.5

0 -0.2 -0.1 0 0.1 0.2 0.3 0.4 [I] (µM)

Figure 25. MaulanaDixon plot Azad for determination Library, Aligarh of apparent Muslim UniversityKi of GPC for ficin. The plot of 1/Vmax vs. [I] by obtaining Vmax from Lineweaver–Burk plot of ficin for determination of apparent Ki.

122 | P a g e

Chapter -1 Results and discussion

7.4 Determination of dissociation rate constant (K−1)

For the determination of K-1 values of cysteine proteinase-GPC complex, the displacement procedure was undertaken. In this method, the inhibitor released from the enzyme-inhibitor complex was trapped by excess substrate (casein) concentration. The amount of enzyme released was checked continuously through monitoring the enzyme activity. The dissociation of complex [EI] complex obeys first order reaction kinetics. Hence, the integrated form of the dissociation rate equation is given by:

ln ([EI] [EI]o) =K-1t

[EI]o: initial concentration of enzyme inhibitor complex; [EI]: final concentration of enzyme inhibitor complex; K-1: dissociation constant; t: time

The dissociation constant (K-1) values for papain, ficin, and bromelain have been found to be 3.2x10-4 s-1, 4.9x10-4 s-1, and 5.8x10-4 s-1, respectively (Table 4).

7.5 Determination of association rate constant (K+1)

Association rate constant was calculated using the method described by Abrahamson et al. (1986).

K+1= K-1/Ki where,Maulana K+1 =association Azad Library, constant; Aligarh K-1= dissociation Muslim constant; University Ki = inhibition constant. The association constant (K+1) was also calculated for papain, ficin, and bromelain as 3.76 × 103 M-1s−1, 4.08 × 103 M-1s−1 and 4.2 × 103 M-1s−1 (Table 4).

123 | P a g e

Chapter -1 Results and discussion

Table 4: Kinetic parameters obtained upon interaction of GPC with different proteinases: papain, ficin, and bromelain.

Proteinases Ki K-1 K+1 Half-life IC50

-1 complex (M) (s ) (M-1s-1) (µM) (s)

Papain 8.5x10-8 3.2x10-4 3.76x103 2.1x103 0.094

Ficin 1.2x10-7 4.9x10-4 4.08x103 1.41x103 0.128

Bromelain 1.38x10-7 5.8x10-4 4.2x103 1.1x103 0.145

Results represent the mean value calculated from three independent Maulana Azad Library, Aligarh Muslim University experiments.

124 | P a g e

Chapter -1 Results and discussion

8. SPECTROSCOPIC ANALYSIS OF GPC

8.1 Ultraviolet absorption spectroscopy

UV-absorption spectra of GPC showed a characteristic protein absorption peak at 278 nm (Fig. 26). The ratio of absorbance at 280/260 was 1.7, which confirmed the purity of GPC. Similar results were reported for other cystatins (Priyadarshini and Bano, 2010). The UV absorption spectrum recorded for GPC-papain complex showed a marked increase in absorption intensity upon complex formation.

8.2 Fluorescence spectroscopy

The fluorescence emission spectra of GPC and GPC-papain complex were recorded at an excitation wavelength of 280 nm. The fluorescence emission spectrum of GPC showed emission maxima at 337 nm, which can be owed to the non-polar side chains (Fig. 27) (Burstein et al., 1973). The fluorescence intensity of GPC-papain was found to be higher than GPC alone, which suggests the formation of GPC-papain complex. The increment in the fluorescence intensity may be due to the exposure of aromatic amino acid residues upon complex formation.

8.3 Circular dichroism (CD) spectroscopy

Far-UV circular dichroism is used to determine the secondary structural elementsMaulana of the protein. Azad Library,The far-UV Aligarh CD spectr Muslima were recordedUniversity in the range of 190-250 nm. The far-UV CD spectrum of native GPC shows two characteristic negative peaks at 208 nm and 222 nm, which corresponds to alpha-helix (Fig. 28) (Naeem and Khan, 2004). Native GPC has 33.9 % of alpha-helical content as determined by the method of Chen et al. (Chen et al., 1972). The negative ellipticity decreases upon GPC-papain complex formation, thereby confirming the reduction in alpha-helical content of GPC upon complex formation.

125 | P a g e

Chapter -1 Results and discussion

0.14 papain GPC GPC- complex

0.12

0.1

)

.u. a

0.08 ce ( ce

rna 0.06 Abso 0.04

0.02

0 240 260 280 300 320 Wavelength (nm)

Figure 26. UV-visible absorption spectra of GPC and GPC-papain complex. Maulana Azad Library, Aligarh Muslim University Equimolar concentration (2 µM) of GPC and papain was used in the experiment. The spectra were recorded in the region of 240-300 nm. Solutions were prepared in sodium phosphate buffer (50 mM, pH 7.5) for the experiment.

126 | P a g e

Chapter -1 Results and discussion

1000 papain GPC GPC - papain complex

800

(a.u.)

600 enisty

400 escence int escence

200 Fluor

0 300 310 320 330 340 350 360 370 380 390 400 Wavelength (nm)

Figure 27. Fluorescence emission spectra of GPC and GPC-papain complex. Maulana Azad Library, Aligarh Muslim University Equimolar concentration (2 µM) of GPC and papain was used in the experiment. The spectra were recorded in the region 300-400 nm after excitation at 280 nm. Solutions were prepared in sodium phosphate buffer (50 mM, pH 7.5) for the experiment.

127 | P a g e

Chapter -1 Results and discussion

30000 GPC 25000 GPC-papain complex

20000

)

1 15000

-

l o

dm 10000

2

-

cm 5000 . .

(deg 0

E E 190 200 210 220 230 240 250 260 R

M -5000

-10000

-15000

-20000 Wavelength (nm)

Figure 28. Far-UV CD spectra of GPC and GPC - papain complex.

Equimolar concentrationMaulana Azad (8 Library,µM) of GPCAligarh and Muslim papain was University used in the experiment. The spectra were recorded in the far-UV region of 190-260 nm. Solutions were prepared in sodium phosphate buffer (50 mM, pH 7.5) for the experiment. The results were expressed as MRE (Mean Residue Ellipticity) in deg cm2dmol-1.

128 | P a g e

Chapter -1 Results and discussion

8.4 Fourier transform infrared spectroscopy (FTIR)

FTIR measurements were done to analyze the secondary structure of GPC. The secondary structure of a protein is particularly indicated by the amide I and amide II bands in FTIR spectroscopy (Kong and Yu, 2007). Hydrogen bonding and the coupling between dipoles are the prime factors governing conformational sensitivity of amide bands. The amide I band absorbs in the range 1600-1690 cm-1, which mainly represents C=O stretching (Kong and Yu, 2007). Figure 29 shows the FTIR spectra of native GPC and GPC-papain complex. Native GPC showed a characteristic peak at 1654 cm-1, which corresponds to alpha helix (Kong and Yu, 2007). GPC-papain complex spectrum showed a decrease in the intensity of the peak at the same frequency of 1654 cm-1, thus suggesting the reduction in the alpha helix.

Maulana Azad Library, Aligarh Muslim University

129 | P a g e

Chapter -1 Results and discussion

GPC 0.36

GPC - papain 0.34 complex

0.32

) 0.3

.u. (a

0.28

nce nce rba

o 0.26

bs A 0.24

0.22

0.2

0.18 1600 1620 1640 1660 1680 1700 1720 Wavenumber (cm-1)

Figure 29. FTIR spectra of GPC and GPC - papain complex.

Equimolar concentrationMaulana Azad (8 Library,µM) of GPCAligarh and Muslim papain was University used in the experiment. The spectra were recorded in the amide I range of 1600-1700 cm-1. Solutions were prepared in sodium phosphate buffer (50 mM, pH 7.5) for the experiment.

130 | P a g e

Discussion

Maulana Azad Library, Aligarh Muslim University

Chapter -1 Results and discussion

DISCUSSION

Phytocystatins regulate various metabolic and physiological activities of plants owing to their cysteine proteinase inhibitory activity (Irene et al., 2012). They are also involved in various other roles such as protein degradation under normal and diseased conditions, seed development, and programmed cell death. They also inhibit exogenous cysteine proteinases secreted by arthropods and pathogens. Thus, it is important to explore the structure and function of phytocystatins from different sources to have a better understanding of their function as well as the physiological relationship with target proteases. Oryzacystatin was the first phytocystatin isolated from rice. It is structurally and functionally related to egg white lysozyme (Oliveira et al., 2003). Afterwards, phytocystatins have been isolated from numerous plant sources including dicots and monocots species viz. cowpea (Fernandes et al., 1993), potato (Waldron et al., 1993), carrot (Ojima et al., 1997), pineapple (Shyu et al., 2004), strawberry (Martinez et al., 2005), cacao (Pirovani et al., 2010), latex tree (Bangrak and Chotigeat, 2011). Recently, phytocystatins have been purified from other plant sources such as almond (Siddiqui et al., 2016), chickpea (Bhat et al., 2016), Brassica alba (Ahmed et al., 2016), and Brassica juncea (Khan et al., 2016), Recent studies have proven garlic to be a notable anti-oxidative, anti-thrombotic, and anti-hypertensive agent (Petrovska and Cekovska, 2010). Consequently, by virtue of these properties’ garlic forms the fundamental basis of the present study as well as various other research project Maulana Azad Library, Aligarh Muslim University being conducted worldwide.

In the present study, phytocystatin has been isolated from garlic cloves. The homogenate was initially prepared in the extraction buffer, which was then followed by ammonium sulfate fractionation (30-60%) and gel filtration chromatography on Sephacryl S 100 HR. The ammonium sulfate fractionation removed unwanted proteins, and the precipitate obtained after 30-60% fractionation was then dissolved in a minimum volume of 50 mM sodium phosphate buffer (pH 7.5), and dialysis was done. After then, the precipitate 131 | P a g e

Chapter -1 Results and discussion was loaded on to the gel filtration chromatography Sephacryl S 100 HR column. The anti-papain inhibitory activity was checked at each step, in order to confirm the efficiency of the procedure. It was observed that the specific activity increases at each step of purification, thereby confirming the efficiency of the procedure. Table -3 shows step-wise purification of GPC along with different parameters. The percent yield was found to be 48.9 % with a fold purification of 152.6. The fold purification obtained was comparable to the previous reports for purification of phytocystatins isolated from other sources (Ahmed et al., 2016; Khan et al., 2016; Siddiqui et al., 2016). The pooled fractions of purified GPC showed specific inhibition against cysteine proteinases but did not show inhibition against serine proteinases (Fig.19). The homogeneity of GPC was further confirmed by native gel electrophoresis (Fig. 10) which showed the presence of a single band, thereby suggesting the presence of a single protein in the homogenous fractions. The purified GPC also migrated as a single band on SDS-PAGE under reducing and non-reducing conditions, which suggest the absence of any subunit in the purified GPC (Fig. 11).

The molecular weight of GPC was determined under native conditions through gel filtration chromatography on Sephacryl S-100HR column and was found to be 12 kDa. The molecular weight of the purified GPC was found to be 12.5 kDa as determined by SDS-PAGE, which lies in the range of Stefins (Type I cystatins) (Chu et al., 2011). The molecular weight of GPC is comparable tMaulanao that of phytocystatin Azad Library, isolated Aligarh from Brassica Muslim juncea University (Khan et al., 2016). The Stoke’s radius is often used to predict the shape of the protein molecule (Schürmann et al. 2001). The Stokes radius of GPC (17.8 Å) was comparable to that of cytochrome C, thus suggesting that GPC is also a globular protein. The purified GPC was found to be devoid of free thiol groups and carbohydrate moieties. Thus, the above observations provide evidence which affirmed that the purified GPC is similar to type I cystatin (Stefins family) which are low molecular weight cysteine proteinase inhibitors lacking glycan moiety and disulfide bonds. 132 | P a g e

Chapter -1 Results and discussion

The stability of the purified inhibitor was also assessed at different pH and temperature. The pH profile (Fig.16) showed that GPC is stable in the pH range of 6-8 and showed maximum inhibitory activity at pH 7.5. Furthermore, the thermal stability profile of GPC (Fig.17) showed that GPC could withstand heat denaturation up to 60°C and showed maximum inhibitory activity at 37°C. These results are in accordance with other cysteine proteinase inhibitors such as phytocystatin purified from Phaseolus mungo as well as Brassica alba (Ahmed et al., 2016; Sharma et al., 2006). Thermal stability profile of GPC is in close agreement with that of pineapple phytocystatin (Shyu et al., 2004). The antibodies were also raised against GPC, which gave a reaction of identity with the purified inhibitor as exhibited by a single precipitin line on immunodiffusion suggesting that the wells contain immunologically pure preparation of GPC (Fig. 19). The antibody titer value 19952.6 was determined by direct binding ELISA, which suggest a good immune response and is in agreement with the previous report of phytocystatin (Khan et al., 2016). The stoichiometry of GPC-papain binding was found to be 1:1 stoichiometry. Similar results have been reported for the binding of papain to other phytocystatins purified from Brassica juncea and Brassica alba (Ahmed et al., 2016; Khan et al., 2016).

Purified GPC is a non-competitive inhibitor against cysteine proteases viz. papain, ficin, and bromelain. Phytocystatins from other sources like soybean (Zhao et al., 1996), barley seeds (Abe et al., 1994), tomato (Wu and Haard,Maulana 2000) and Azad corn (FernandesLibrary, Aligarhet al., 1991) Muslim were alsoUniversity reported to be non- competitive inhibitors against papain. Kinetic analysis shows that the GPC has the highest affinity for papain as compared to ficin and bromelain. Similar findings have been stated for other phytocystatins (Priyadarshini and Bano, -8 2010). The Ki value of GPC against papain was found to be 8.5x10 M which is comparable with the Ki value of phytocystatins from sesame phytocystatin -8 (Ki=7.9x10 M) (Cheng et al., 2014), and soybean cystatin induced by methyl -8 jasmonate (Ki= 5.7x10 M) (Zhao et al., 1996). The GPC showed stronger binding against papain than phytocystatin purified from Colocasia esculenta 133 | P a g e

Chapter -1 Results and discussion

-8 having Ki value 9.8x10 M (Wang et al., 2008). The dissociation rate constant was also analysed for the GPC – proteinase complex and the value of the dissociation constant of GPC for papain, ficin, and bromelain was 3.2x10-4 s-1, 4.9x10-4 s-1 and 5.8x10-4 s-1, respectively. The result obtained is comparable to that of dissociation constant determined in case of chickpea phytocystatin and phytocystatin isolated from Brassica juncea seeds (Bhat et al., 2016; Khan et al., 2016). The dissociation rate constant determined in case of chickpea phytocystatin for papain, bromelain, and ficin were 3.6x10-4 s-1, 11.3x10-4 s-1 and 7.02x10-4 s-1, respectively (Bhat et al., 2016). The association rate constant was also determined for GPC-proteinase complex, and the value of association rate constant of GPC for papain, ficin, and bromelain was 3.76 x 103 s-1, 4.08 x 103 s-1 and 4.20 x 103 s-1, respectively. The values obtained corroborate with the previous results reported for the phytocystatin isolated from Indian mustard seeds and Brassica juncea seeds (Ahmed et al., 2016; Khan et al., 2016).

The ultra-violet absorption and fluorescence spectroscopy were employed to study the binding of GPC to papain. The ultraviolet absorption spectrum of GPC showed a characteristic protein peak at around 280 nm, which is due to the cumulative absorption of aromatic amino acid residues (Fig. 27). The absorption intensity increases upon the interaction of GPC with papain. The increment in the absorption intensity suggests the exposure of aromatic amino acid residues upon perturbation caused due to GPC-papain interactions. The results are comparable with the earlier reports which exhibited similarMaulana binding Azad pattern Library, for phytocystatin Aligarh – Muslim papain interaction University such as the interaction of phytocystatins purified from almond, Brassica alba and Brassica juncea with papain (Ahmed et al., 2016; Khan et al., 2016). The fluorescence emission spectra of GPC were recorded at an excitation wavelength of 280 nm and showed emission maxima at 337 nm owing to the non-polar side chains of protein (Burstein et al., 1973) (Fig.28). The increment in the fluorescence intensity of GPC-papain spectrum suggests the formation of GPC-papain complex along with the conformational change in either one or both of the proteins. The far-UV circular dichroism study was carried out for 134 | P a g e

Chapter -1 Results and discussion analyzing the secondary structure of purified GPC. The circular dichroism spectra of GPC showed two strong negative peaks at 208 nm and 222 nm, which are characteristic peaks for alpha helix, thereby confirming the presence of alpha-helix in the purified GPC. GPC possesses 33.9 % alpha-helical content, which is comparable to the alpha-helical content present in almond phytocystatin (Siddiqui et al., 2015). FTIR spectroscopy was also employed to study the secondary structure of GPC, which revealed a strong peak at 1656 cm-1 in the amide I region of the IR spectra. The peak at 1656 cm-1 is a characteristic peak for alpha- helix. The far-UV CD and FTIR analysis showed a reduction in the alpha-helical content upon GPC – papain complex formation. Similar findings have been reported for the phytocystatin isolated from Brassica alba seeds (Ahmed et al., 2016).

Maulana Azad Library, Aligarh Muslim University

135 | P a g e

Chapter – 2

Unfolding study of garlic phytocystatin in the presence of denaturants Maulana Azad Library, Aligarh Muslim University

Chapter -2 Results and discussion

RESULTS

UNFOLDING STUDIES OF GARLIC PHYTOCYSTATIN (GPC) IN THE PRESENCE OF UREA AND GUANIDINE HYDROCHLORIDE

1. FUNCTIONAL STUDY

1.1 Effect of urea on the cysteine proteinase inhibitory activity of GPC

The cysteine proteinase inhibitory activity of GPC was monitored in the presence of an increasing concentration of urea after 2 h of incubation. It is evident in figure 30 that the inhibitory activity of GPC decreases with the increase in urea concentration. The loss in inhibitory activity was not significant up to 1 M urea. However, there was a steady decrease in the inhibitory activity of GPC after incubation with 2 M urea (Fig. 30). The GPC loses its 50% inhibitory activity at around 3 M urea. The complete loss of cysteine proteinase inhibitory activity of GPC was observed at 8 M urea.

1.2 Effect of guanidine hydrochloride (GdnHCl) on the cysteine proteinase inhibitory activity of GPC

The cysteineMaulana proteinase Azad inhibitory Library, activity Aligarh of GPC Muslim was University monitored with increasing concentration of GdnHCl after 2 h of incubation. It is evident in figure 31 that the inhibitory activity of GPC decreases with increasing concentration of GdnHCl. The plot of inhibitory activity of GPC vs. GdnHCl concentration exhibits that GPC loses its 50% inhibitory activity at around 2 M GdnHCl. The complete loss of cysteine proteinase inhibitory activity of GPC was observed at 4 M GdnHCl.

136 | P a g e

Chapter -2 Results and discussion

100

ivity ct

a 80

ry ry o

60

inhibit ining ining

a 40

Rem

% 20

0 0 2 4 6 8 Urea [M]

Figure 30. Cysteine proteinase inhibitory assay of GPC in the presence of urea. Maulana Azad Library, Aligarh Muslim University Native GPC (4 µM) was incubated with increasing concentration of urea [0 - 8 M] for 2 h at 37ºC. Inhibitory activity assay was performed using casein as a substrate by the method of Kunitz (1947) as described in methods. Values are mean of three independent determinations.

137 | P a g e

Chapter -2 Results and discussion

100

ty i

v 80

cti

a

ry to

bi 60

nhi

i

ng i

a 40

em R

% 20

0 0 1 2 3 4 GdnHCl [M]

Figure 31. Cysteine proteinase inhibitory assay of GPC in the presence of GdnHCl.

Native GPC (4 µM) was incubated with increasing concentration of guanidine hydrochlorideMaulana Azad [0 -Library, 4 M] for Aligarh 2 h at Muslim 37ºC. Inhibitory University activity assay was performed using casein as a substrate by the method of Kunitz (1947) as described in methods. Values are mean of three independent determinations.

138 | P a g e

Chapter -2 Results and discussion

2. STRUCTURAL STUDIES

2.1 INTRINSIC FLUORESCENCE STUDIES OF GPC IN THE PRESENCE OF DENATURANTS

2.1.1 Effect of urea on the intrinsic fluorescence of GPC

Intrinsic fluorescence studies were performed to investigate the denaturation effect of denaturants on GPC. The fluorescence studies have been extensively used to probe the structural changes in the protein based on emission spectra which is dependent on the electronic and dynamic properties of the fluorophore environment (Prajapati et al., 1998). The fluorescence emission spectra were recorded at an excitation of 280 nm to study the conformational changes and modifications in the tertiary structure GPC (Priyadarshini et al., 2010). Denaturation of GPC was studied on the basis of changes in the microenvironment around aromatic amino acid residues which get perturbed due to denaturant and lead to alteration in the emission intensity as well as emission maxima. Figure 32 shows that GPC exhibits emission maxima at 337 nm, which gets shifted to 350 nm upon an increasing concentration of urea. The red shift in the emission maxima of GPC suggests the unfolding of GPC in the presence of urea. At a lower concentration of urea (0.5 M - 1.0 M), there was a gradual increase in emission intensity with no change in emission maxima. On increasing urea concentration from 2.0 M to 5.0 M, there was a gradualMaulana increase inAzad the fluorescence Library, Aligarh intensity Muslimalong with University a redshift of 6 nm. At 3 M and 4 M urea, GPC shows emission maxima at 342 nm which suggests the tryptophan residues have limited contact with water (Burstein et al., 1973), however, above 5 M urea there was marked increase in the emission intensity and red shift of 13 nm was observed. In the presence of 8 M urea, GPC showed emission maxima at 350 nm, which corresponds to the complete exposure of aromatic residues in an aqueous environment (Burstein et al., 1973). The significant increase in the emission intensity and red shift at 350 nm suggest complete denaturation of GPC.

139 | P a g e

Chapter -2 Results and discussion

180 355 Emission intenstity Emission maxima

160

350 )

) 140

.u.

(nm

(a

120 a

ty

m

i i

345 x a

100 m

ntens

i

n n

o

n n

i

o

s

i s

s 80

i s

i 340 Em

Em 60

40 335

20

0 330 0 0.5 1 2 3 4 5 6 7 8 Urea [M]

Figure 32. Intrinsic fluorescence analysis of GPC in the presence of urea.

Native GPC (4 µM) was incubated with increasing concentration of urea Maulana Azad Library, Aligarh Muslim University [0 - 8 M] for 2 h at 37ºC in 50 mM sodium phosphate buffer. Fluorescence was measured at an excitation wavelength of 280 nm and the emission range was fixed at 300-400 nm. The black dotted circles connected with line shows emission maxima of GPC in the presence of an increasing concentration of urea. Values are mean of three independent determinations.

140 | P a g e

Chapter -2 Results and discussion

2.1.2 Effect of GdnHCl on the intrinsic fluorescence of GPC

The intrinsic fluorescence of GPC was recorded in the presence of increasing concentration (0 - 4 M) of GdnHCl. Figure 33 shows the intrinsic fluorescence of native GPC and GPC in the presence of increasing concentration (0 - 4 M) of GdnHCl. The gradual increase in the emission intensity of GPC was observed with increasing concentration (0 - 4 M) of GdnHCl. The increase in the emission intensity was also accompanied by the increase in the emission maxima. It was observed that GPC in the presence of an increasing concentration of GdnHCl showed a red shift of 8 nm, which indicates the unfolding of GPC.

3. ANS FLUORESCENCE STUDIES OF GPC IN THE PRESENCE OF DENATURANTS

3.1 Effect of urea on the ANS fluorescence of GPC

ANS fluorescence has been extensively used to investigate the non-native conformations of a protein (Semisotnov et al., 1991). ANS binds non- covalently to the hydrophobic residues present within the hydrophobic core (Povarova et al., 2010). Native GPC exhibits low ANS fluorescence intensity, which may be due to the presence of few hydrophobic patches present at the surface of the protein. Figure 34 shows the ANS fluorescence of GPC in the presence of urea. The ANS fluorescence intensity of GPC decreases with the increasingMaulana concentration Azad Library,of urea (0 Aligarh- 8 M). T Muslimhe GPC also University exhibited a red shift of about 10 nm in the presence of an increasing concentration of urea (Fig. 32).

3.2 Effect of GdnHCl on the ANS fluorescence of GPC

The ANS fluorescence of GPC was studied in the presence of an increasing concentration of GdnHCl (0 - 4 M) to detect the presence of non-native conformations. Figure 35 shows the ANS fluorescence of GPC in the presence of GdnHCl.

141 | P a g e

Chapter -2 Results and discussion

300 350 Emission intensity Emision maxima

250

345

)

nm

200 (

a.u.)

a a

(

ity

axim

ns

e m

t 150 340

in

on on

on on

issi issi

100 Em Em 335

50

0 330 0 0.5 1 1.5 2 2.5 3 3.5 4 GdnHCl [M]

Figure 33. Intrinsic fluorescence analysis of GPC in the presence of GdnHCl.

Native GPC (4 µM) was incubated with increasing concentration of GdnHCl [0 Maulana- 4 M] for Azad 2 h at Library, 37ºC in Aligarh50 mM sodiumMuslim phosphate University buffer. Fluorescence was measured at an excitation wavelength of 280 nm, and the emission range was fixed at 300-400 nm. The black dotted circles connected with line shows emission maxima of GPC in the presence of an increasing concentration of urea. Values are mean of three independent determinations.

142 | P a g e

Chapter -2 Results and discussion

46 530 Emission intensity Emission maxima

44

42 )

.u) 525

(a

(nm

40 a

ty

i

m

i

x a

38 m

ntens

i

n n

n n

o

i

o

i

s

s

s i s 36

i 520

Em Em 34

32

30 515 0 1 2 3 4 5 6 7 8 Urea [M]

Figure 34. ANS fluorescence analysis of GPC in the presence of urea.

Native GPC (4 µM) was incubated with increasing concentration of urea [0 - 8 M] for 2 h at 37ºC in 50 mM sodium phosphate buffer. ANS FluorescenceMaulana was Azad measured Library, post Aligarh incubation Muslim at an Universityexcitation wavelength of 380 nm, and the emission range was fixed at 400 - 600 nm. The black dotted circles connected with line shows emission maxima of GPC in the presence of an increasing concentration of urea. Values are mean of three independent determinations.

143 | P a g e

Chapter -2 Results and discussion

70 528 Emission intensity Emission maxima

60

.u)

50 ) (a

524

(nm

ty

a a i

40

axim

ntens

m

i

n n

ion o

i 30

s

iss

s

i m

520 E Em 20

10

0 516 0 0.5 1 1.5 2 2.5 3 3.5 4 GdnHCl [M]

Figure 35. ANS fluorescence analysis of GPC in the presence of GdnHCl.

Native GPC (4 µM) was incubated with increasing concentration of GdnHCl [0 Maulana- 4 M] for Azad 2 h at Library, 37ºC in Aligarh50 mM sodiumMuslim phosphate University buffer. ANS Fluorescence was measured post incubation at an excitation wavelength of 380 nm, and the emission range was fixed at 400 - 600 nm. The black dotted circles connected with line shows emission maxima of GPC in the presence of an increasing concentration of urea. Values are mean of three independent determinations.

144 | P a g e

Chapter -2 Results and discussion

During the unfolding process of GPC, the ANS fluorescence intensity of GPC was found to decrease with the increasing concentration of GdnHCl (Fig. 35). The GPC showed a red shift of 5 nm with the increasing concentration (0 - 4 M) of GdnHCl.

4. ACRYLAMIDE QUENCHING STUDIES OF GPC IN THE PRESENCE OF DENATURANTS

4.1 Acrylamide quenching analysis of GPC in the presence of urea

Acrylamide quenching experiment allows us to probe the solvent accessibility of the tryptophan residues (Pawar and Deshpande, 2000). The increased intrinsic emission and the red shift in GPC upon denaturation was further investigated by acrylamide quenching experiments. Figure 36 shows the Stern- Volmer plot for acrylamide quenching of GPC with increasing concentration of urea. It is evident from the plot that quenching was more pronounced as the concentration of urea increases. An increase in the slope for GPC in the presence of urea as compared to the control (GPC in the absence of urea) indicates the extent of exposure of the tryptophan residues. The maximum quenching was observed at 8 M urea. The Ksv of GPC incubated with urea was found to be more as compared to native GPC (Table 5). The results of the Stern–Volmer plot thus indicate that the tryptophan residues of GPC are more exposedMaulana to solvent Azad during Library, the unfolding Aligarh process, Muslim induced University by urea. Stern- Volmer results are consistent with the intrinsic fluorescence emission results. The red shift in the fluorescence emission maxima and increased quenching of tryptophan fluorescence in the presence of acrylamide suggests the unfolding of GPC.

145 | P a g e

Chapter -2 Results and discussion

4.2 Acrylamide quenching analysis of GPC in the presence of GdnHCl

The acrylamide quenching study of GPC was also done in the presence of GdnHCl. Figure 37 shows the Stern-Volmer plot for acrylamide quenching of GPC with increasing concentration of GdnHCl. It is evident from the plot that quenching was more pronounced as the concentration of GdnHCl increases. An increase in the slope for GPC in the presence of GdnHCl as compared to the control (GPC in the absence of GdnHCl) indicates the extent of exposure of the tryptophan residues. Maximum quenching was observed at 4 M GdnHCl. The

Ksv of GPC incubated with GdnHCl was found to be more as compared to native GPC (Table 5). The results thus indicate that the tryptophan residues of GPC are more exposed to solvent during the GdnHCl induced unfolding process. Furthermore, the red shift in the fluorescence emission maxima and increased quenching of tryptophan fluorescence in the presence of acrylamide suggests the unfolding of GPC.

5. SECONDARY STRUCTURE ANALYSIS OF GPC IN THE PRESENCE OF UREA AND GUANIDINE HYDROCHLORIDE

5.1 Effect of urea on the secondary structure of GPC

Far-UV circular dichroism (CD) studies were performed to investigate the changes in the secondary structure of GPC upon denaturation by urea. Figure Maulana Azad Library, Aligarh Muslim University 38 shows the far-UV CD spectra of native GPC and GPC incubated with increasing concentration (0-8 M) of urea. Native GPC shows the signature peak at 222 nm and 208 nm, which corresponds to the alpha helix. Native GPC contains 33.9% alpha helix as reported by Siddiqui et al. (2017).

146 | P a g e

Chapter -2 Results and discussion

Control 1 M urea 2 M urea 3 M urea 4 M urea 5 M urea 6 M urea 7 M urea 8 M urea 3

2.5

8 M Urea

F

Fo/ 2

1.5

1 0 0.1 0.2 0.3 0.4 0.5 Acrylamide [M]

Figure 36. Acrylamide quenching analysis of GPC in the presence of urea. Maulana Azad Library, Aligarh Muslim University Stern–Volmer plot for the acrylamide quenching of GPC (4 µM) in the presence of an increasing concentration of urea [1 M – 8 M]. The excitation wavelength was set at 295 nm in order to probe tryptophan fluorescence only, and the emission spectrum was recorded in the range of 300 - 400 nm. The control represents GPC without urea.

147 | P a g e

Chapter -2 Results and discussion

Control 0.5 M GdnHCl 1 M GdnHCl 1.5 MGdnHCl 2 M GdnHCl 2.5 M GdnHCl 3 M GdnHCl 3.5 M GdnHCl 4 M GdnHCl 6

5 4 M GdnHCl

4

F Fo/ 3

2

1 0 0.1 0.2 0.3 0.4 0.5 Acrylamide [M]

Figure 37. Acrylamide quenching analysis of GPC in the presence of GdnHCl. Maulana Azad Library, Aligarh Muslim University

Stern–Volmer plot for the acrylamide quenching of GPC (4 µM) in the presence of an increasing concentration of GdnHCl [0.5 M – 4 M]. The excitation wavelength was set at 295 nm in order to probe tryptophan fluorescence, and the emission spectrum was recorded in the range of 300 - 400 nm. The control represents GPC without GdnHCl.

148 | P a g e

Chapter -2 Results and discussion

Table 5: Stern-Volmer quenching constant (Ksv) of GPC in the presence of an increasing concentration of urea and guanidine hydrochloride

-1 -1 Urea [M] Ksv [M ] GdnHCl [M] Ksv [M ]

0 1.46 0 1.46

1 1.53 0.5 2.83

2 1.62 1.0 3.06

3 1.74 1.5 3.40

4 2.26 2.0 3.55

5 2.57 2.5 3.80

6 2.81 3.0 7.40

7 3.10 3.5 8.10

8 3.38 4.0 9.40

Maulana Azad Library, Aligarh Muslim University

Stern-Volmer quenching constant (Ksv) was determined by the standard equation given in the methodology section. Ksv defines the quenching efficiency of the ligand.

149 | P a g e

Chapter -2 Results and discussion

The increment in the concentration of denaturant disrupts the secondary structural elements of the GPC. Figure 38 shows gradual and stepwise disruption in the secondary structure of GPC up to 6 M urea, and almost complete loss of secondary structure was observed at 8 M urea. The loss in % alpha- helix at various stages of urea-induced denaturation is summarized in Table 6.

5.2 Effect of GdnHCl on the secondary structure of GPC

Far-UV circular dichroism (CD) studies were performed to investigate the secondary structural changes in GPC upon denaturation by GdnHCl. Figure 37 shows the far-UV CD spectra of native GPC and GPC incubated with increasing concentration (0 - 4 M) of GdnHCl. Native GPC showed the signature peak at 222 nm and 208 nm, which corresponds to the alpha helix. Native GPC contains 33.9% alpha helix as reported by Siddiqui et al. (2017). It is evident from figure 39 that decrease in the ellipticity values at 222 nm was gradual up to 1.5 M GdnHCl. However, an increase in the GdnHCl concentration above 1.5 M showed a sharp decrease in the ellipticity values, which suggests prominent disruption of secondary structural elements above 1.5 M GdnHCl. The result suggested that the increment in the GdnHCl concentration disrupts the secondary structural elements of the GPC. The complete loss of secondary structure in GPC was observed at 4 M GdnHCl. The loss in % alpha- helix at various stages of denaturation is summarized in Maulana Azad Library, Aligarh Muslim University Table 6.

150 | P a g e

Chapter -2 Results and discussion

0

-2000

GPC -4000

1 M urea

-6000 2 M urea

RE 3 M urea M -8000 4 M urea 5 M urea -10000 6 M urea 7 M urea -12000 8 M urea

-14000 200 210 220 230 240 250 260 Wavelength (nm)

Figure 38. Circular dichroism analysis of GPC in the presence of urea. Maulana Azad Library, Aligarh Muslim University Far-UV circular dichroism spectra of GPC (4 µM) in the presence of an increasing concentration of urea [1 M - 8 M]. The spectra were recorded after incubation of GPC with urea for 2 h at 37ºC in 50 mM sodium phosphate buffer. The spectra were recorded in the range of 200-260 nm, and each spectrum is an average of three scans.

151 | P a g e

Chapter -2 Results and discussion

0

-2000

-4000 GPC

-6000 0.5 M GdnHCl

E E 1 M GdnHCl

R -8000

M 1.5 M GdnHCl -10000 2 M GdnHCl

-12000 3 M GdnHCl 4 M GdnHCl -14000 200 210 220 230 240 250 260 Wavelength (nm)

Figure 39. Circular dichroism analysis of GPC in the presence of GdnHCl. Maulana Azad Library, Aligarh Muslim University Far-UV circular dichroism spectra of GPC (4 µM) in the presence of an increasing concentration of GdnHCl [0.5 M - 4 M]. The spectra were recorded after incubation of GPC with GdnHCl for 2 h at 37ºC in 50 mM sodium phosphate buffer. The spectra recorded in the range of 200-260 nm and each spectrum is an average of three scans.

152 | P a g e

Chapter -2 Results and discussion

Table 6: Secondary structure analysis of GPC under denaturing conditions using urea and guanidine hydrochloride.

% Remain Urea [M] % Alpha-helix alpha-helix 0 33.9 100 1 30.1 88.7 2 26.1 76.9 3 23.9 70.5 4 19.7 58.1 5 16.8 49.5 6 12.7 37.6 7 2.3 6.7 8 0 0 GdnHCl [M] 0 33.9 100 0.5 32.6 96.1 1 30.8 90.1 1.5 27.3 80.5 Maulana2 Azad Library,19.6 Aligarh Muslim University57.8 3 12.7 37.4 4 0 0

Alpha-helical content was determined by the method of Chen et al. (1972) equation given in the methodology section.

153 | P a g e

Chapter -2 Results and discussion

6. FOURIER TRANSFORM INFRARED MEASUREMENTS OF GPC IN THE PRESENCE OF DENATURANTS

6.1 Fourier transform infrared analysis of GPC in the presence of urea

FTIR experiments were also performed to investigate the secondary structural alteration within GPC upon treatment with denaturants. FTIR spectra in the amide I range were recorded to investigate the secondary structural elements of GPC. Figure 40 shows the FTIR spectra of GPC under the denaturing effects of urea. Native GPC showed a signature peak at 1655 cm-1, which corresponds to the alpha helix. Figure 40 showed that the increase in urea concentration alters the secondary structure of GPC in the amide I region. The gradual loss of peak at 1655 cm-1 was observed with increase in urea concentration, and complete loss of signature peak of the alpha helix was observed at 8 M urea.

6.2 Fourier transform infrared analysis of GPC in the presence of GdnHCl

FTIR spectra of native GPC were recorded in the presence of an increasing concentration of GdnHCl. It is evident from figure 41 that native GPC exhibited a characteristic peak at 1655 cm-1, which corresponds to the alpha- helical structure. The slight changes were observed in the characteristic alpha Maulana Azad Library, Aligarh Muslim University helix peak up to 1 M GdnHCl. However, upon increasing the concentration above 1 M GdnHCl, the peak starts getting shifted towards a non-native form of GPC. The complete loss of alpha helix peak (1655 cm-1) at 4 M GdnHCl suggests complete disruption of the secondary structure of native GPC.

154 | P a g e

Chapter -2 Results and discussion

0.9 GPC 2 M urea 4 M urea

6 M urea 8 M urea 0.8

0.7

nce

rba o

0.6 Abs

0.5

0.4 1600 1620 1640 1660 1680 1700 Wavenumber (cm-1)

Figure 40. FTIR analysis of GPC in the presence of urea.

FTIR Maulanaspectra ofAzad GPC Library, (4 µM) Aligarh in the Muslim presence University of an increasing concentration of urea [2 M, 4 M, 6 M, and 8 M]. The spectra were recorded after incubation of GPC with urea for 2 h at 37ºC in 50 mM sodium phosphate buffer. The spectra were recorded in the range of 1600- 1700 cm-1, and each spectrum is an average of three scans.

155 | P a g e

Chapter -2 Results and discussion

0.9

GPC 1 M GdnHCl 2 M GdnHCl

3 M GdnHCl 4 M GdnHCl

0.8

0.7

nce rba

Abso 0.6

0.5

0.4 1600 1620 1640 1660 1680 1700 Wavenumber (cm-1)

Figure 41. FTIR analysis of GPC in the presence of GdnHCl. Maulana Azad Library, Aligarh Muslim University FTIR spectra of GPC (4 µM) in the presence of an increasing concentration of GdnHCl [1 M, 2 M, 3 M, and 4 M]. The spectra were recorded after incubation of GPC with GdnHCl for 2 h at 37ºC in 50 mM sodium phosphate buffer. The spectra were recorded in the range of 1600- 1700 cm-1, and each spectrum is an average of three scans.

156 | P a g e

Chapter -2 Results and discussion

7. EQUILIBRIUM DENATURATION STUDY OF GPC IN THE PRESENCE OF DENATURANTS

7.1 Equilibrium denaturation study of GPC in the presence of urea

The equilibrium transition curve of GPC was plotted as a function of changes in the fluorescence emission with the increasing denaturant concentration. Figure 42 shows the denaturation curve of GPC in the presence of an increasing concentration of urea. It was observed that unfolding of GPC is gradual from 0-8 M urea. The equilibrium constants calculated from the fraction of native GPC in the transition region of the normalized curved were used to determine the free energy change during unfolding. ΔG reflected the protein stability and was calculated by using the linear extrapolation method in which ΔGnative-unfolded shows linear dependence to denaturant concentration (Fig.

43) (Creighton, 1997; Pace, 1986). The conformational stability ΔG(H2O) of GPC determined under denaturing condition using urea was 1.5±0.2 kcal/mol. The transition midpoint of the urea-induced unfolding of GPC obtained by fluorescence emission is summarized in Table 7, along with their respective thermodynamic parameters.

7.2 Equilibrium denaturation study of GPC in the presence of GdnHCl Maulana Azad Library, Aligarh Muslim University Figure 44 shows the denaturation curve of GPC in the presence of an increasing concentration of GdnHCl. Figure 45 shows the conformational stability plot of GPC determined using GdnHCl as a denaturant. The transition midpoint of GdnHCl induced unfolding was observed at around 2.32 M. The

ΔG(H2O) value for GPC determined using GdnHCl induced unfolding was found to be 1.9±0.1 kcal/mol. The transition midpoint of GdnHCl-induced unfolding of GPC obtained by fluorescence emission is summarized in Table 7, along with their respective thermodynamic parameters.

157 | P a g e

Chapter -2 Results and discussion

1

0.9

0.8

0.7 PC

G 0.6 e e

0.5 nativ 0.4

0.3 Fraction 0.2

0.1

0 0 1 2 3 4 5 6 7 8 Urea [M]

Figure 42. Unfolding of GPC in the presence of urea.

Urea-induced equilibrium unfolding transition curve of GPC as measured by changes inMaulana fluorescence Azad emission. Library, The Aligarh fluorescence Muslim spectra University of GPC (4 µM) were recorded in the presence of increasing concentration (0 – 8 M) urea for the denaturation curve. The spectra were recorded post incubation with urea for 2 h at 37ºC in 50 mM sodium phosphate buffer. The data are expressed in terms of the fraction unfolded (Fu) calculated from the standard equation given in the methodology section.

158 | P a g e

Chapter -2 Results and discussion

2

1.5

l)

o 1 l/m

ca 0.5

(k

G Δ 0 -1 0 1 2 3 4 5 6 7 8 -0.5

-1

-1.5 Urea [M]

Figure 43. Conformational Stability plot of GPC in the presence of urea. Maulana Azad Library, Aligarh Muslim University The plot of ΔG vs. [urea] for determining the conformational stability of GPC. The ΔG was calculated according to the standard equation given in the methodology section.

159 | P a g e

Chapter -2 Results and discussion

1

0.9

0.8

0.7 C

GP 0.6 e e

0.5 nativ 0.4

0.3 Fraction 0.2

0.1

0 0 0.5 1 1.5 2 2.5 3 3.5 4 GdnHCl [M]

Figure 44. Unfolding of GPC in the presence of GdnHCl.

GdnHCl-induced equilibrium unfolding transition curve of GPC as Maulana Azad Library, Aligarh Muslim University measured by changes in fluorescence emission. The fluorescence spectra of GPC (4 µM) were recorded in the presence of increasing concentration (0 – 4 M) GdnHCl for the denaturation curve. The spectra were recorded post incubation with GdnHCl for 2 h at 37ºC in 50 mM sodium phosphate buffer. The data are expressed in terms of the fraction unfolded (Fu) calculated from the standard equation given in the methodology section.

160 | P a g e

Chapter -2 Results and discussion

2.5

2

1.5

l)

o 1

l/m

ca (k

0.5

G Δ 0 -1 0 1 2 3 4 -0.5

-1

-1.5 GdnHCl [M]

Figure 45. Conformational stability plot of GPC in the presence of GdnHCl. Maulana Azad Library, Aligarh Muslim University The plot of ΔG vs. [GdnHCl] for determining the conformational stability of GPC. The ΔG was calculated according to the standard equation given in the methodology section.

161 | P a g e

Chapter -2 Results and discussion

Table 7: Urea and guanidine hydrochloride induced unfolding parameters of GPC

Transition ΔG (H20) m Denaturant midpoint [M] (kcal/mol) (kcal/mol/M)

1. Urea 4.7±0.1 1.5±0.2 0.345±0.0008

2. GdnHCl 2.32±0.1 1.9±0.1 0.816±0.05

Values are determined on the basis of the equation given in the methodology section.

Maulana Azad Library, Aligarh Muslim University

162 | P a g e

Discussion

Maulana Azad Library, Aligarh Muslim University

Chapter -2 Results and discussion

DISCUSSION

Protein folding is a crucial process that ascertains the achievement of a specific three-dimensional conformation of protein so that it can perform its destined functions efficiently. Protein folding-unfolding is a complex process which involves a number of stages during the transition from native to unfolded form and vice-versa. Small globular proteins exhibit a two-state model which does not involve any basic intermediates during the transition from native to denatured form (Aune and Tanford, 1969). It is well documented in the literature that protein unfolding may proceeds through intermediate states during the transition from native to unfolded form (Brems et al., 1990; Mitchinson and Pain, 1985). The intermediates formed during the transition from native to unfolded state are referred to as molten globule. Urea and guanidine hydrochloride (GdnHCl) are the two well-known denaturants that are exploited in research labs to decipher the mechanism of protein folding and unfolding. Urea and guanidine hydrochloride (GdnHCl) unfold proteins by solubilizing the non-polar residues of the protein along with the peptide backbone and the polar groups present in the side chain of the proteins (Nandi and Robinson, 1984; Roseman and Jencks, 1975). In the present study, a number of biophysical techniques are employed to investigate the denaturing effects of urea and guanidine hydrochloride on the structure, function, and stability profile of garlic phytocystatin (GPC). It was found that cysteine proteinase inhibitory activity decreases with the increase in Maulana Azad Library, Aligarh Muslim University denaturant concentration, which reflects the alteration in the native structure of GPC. The 50% loss of inhibitory activity of GPC was observed at 3 M urea and 2.5 M GdnHCl (Fig. 30 and 31). The GPC was found to be completely inactivated at 8 M urea and 4 M GdnHCl. The results are comparable to the previous reports, which suggest the reduction of cysteine proteinase inhibitory activity in the presence of urea and guanidine hydrochloride (Aatif et al., 2011; Rashid et al., 2005). The intrinsic fluorescence studies also suggest that GPC gets denatured upon incubation with urea and guanidine hydrochloride. A

163 | P a g e

Chapter -2 Results and discussion gradual increment in fluorescence intensity along with no change in emission maxima was observed up to 1 M urea, suggesting minimum changes in the microenvironment around aromatic residues. However, incubation of GPC with urea above 1 M showed a marked increase in the fluorescence intensity along with a red shift of around 13 nm (Fig. 32). This indicates that incubation of GPC with urea above 1 M results in more exposure of aromatic residues of the protein to the solvent environment. The increase in fluorescence intensity and the red shift is a hallmark of unfolding (Devaraj et al., 2011). The increase in fluorescence intensity may be the after effect of increased separation between the aromatic residues and specific quenching groups of the protein (Halfman and Nishida, 1971; Sommers and Kronman, 1980). It is evident from the results that the denaturing effect of guanidine hydrochloride is more pronounced as there was a marked increase in fluorescence intensity within a narrow range of GdnHCl concentration (Fig. 33). GPC incubated with GdnHCl showed prominent increment in fluorescence intensity up to 1 M suggesting the exposure of aromatic residues which confirms the conformational changes in the protein. A distinct red shift of 8 nm along with increased fluorescence intensity, suggests the complete unfolding of GPC upon incubation at 4 M GdnHCl (Fig. 33). ANS fluorescence studies were carried out to investigate the presence of intermediate species upon denaturation of GPC with urea and GdnHCl. ANS refers to 1, 8 anilino-naphthalene sulfonate that is used to monitor the exposure of hydrophobic regions which are buried inside the protein. The presence of Maulana Azad Library, Aligarh Muslim University intermediate species is confirmed by the level of ANS binding to denatured protein as compared to the native protein. Native GPC showed minimal ANS fluorescence intensity, which suggests the presence of a few hydrophobic patches at the surface of GPC. Upon incubation of GPC with (0 – 8 M) urea, it was observed that increase in urea concentration decreases the ANS fluorescence intensity which indicates the disruption of hydrophobic patches and the absence of any intermediate species during the unfolding of GPC (Fig. 34). The fluorescent probe ANS binds to GPC under the denaturing effects of

164 | P a g e

Chapter -2 Results and discussion

GdnHCl more competently as compared to GPC under denaturing effects of urea. ANS fluorescence intensity was found to be increased up to 1 M GdnHCl that suggests the binding of ANS to the hydrophobic clusters of the protein (Fig. 35). The increase in fluorescence could be attributed to the exposure of more hydrophobic patches of GPC upon incubation with GdnHCl. The biphasic curve of ANS fluorescence may be inferred as the presence of intermediate species at 1 M GdnHCl. Decrease in ANS fluorescence beyond 1M GdnHCl and increase in emission maxima suggests the complete unfolding of GPC at 4 M GdnHCl (Fig. 35). Previous study also showed that human placental cystatin when incubated with GdnHCl lead to the formation of intermediate state during the unfolding of the cystatin (Rashid et al., 2005), however, no intermediate was formed in the presence of urea (Sharma et al., 2006). Acrylamide quenching studies were performed in order to analyze the relative solvent exposure of fluorophores (Eftink and Ghiron, 1981). Urea up to 3 M did not exhibit any noticeable increase in the tryptophan fluorescence quenching while a marked increase in tryptophan quenching was observed above 3 M urea (Fig. 36). Fluorescence quenching was maximum at 8 M urea. The acrylamide quenching results are analyzed by the Stern-Volmer plot, which illustrates the quenching efficiency of a ligand upon binding to fluorophores. The Stern-Volmer plot showed an increased slope for the GPC in the presence of GdnHCl as compared to native GPC reflecting the exposure the tryptophan residues. It is evident from the results that fluorescence quenching was more effective by acrylamide as GdnHCl concentration increases. The Maulana Azad Library, Aligarh Muslim University uneven increase in fluorescence quenching of tryptophan could be attributed to the uneven increased interaction of the buried aromatic residues with the solvent. It is evident from the Stern-Volmer plot that fluorescence quenching was more effective above 2.5 M GdnHCl, and maximum quenching was observed at 4 M GdnHCl (Fig. 37). The Stern-Volmer quenching constants

(Ksv) were determined at each step of denaturation by the standard equation. The Stern-Volmer quenching constant defines the quenching efficiency of the ligand. The high Ksv value of a ligand will quench the fluorophores more

165 | P a g e

Chapter -2 Results and discussion efficiently. The Ksv value of GPC in the absence of denaturant was found to be -1 1.46 M . However, Ksv showed an increment with an increase in denaturant concentration. The Ksv value of GPC incubated with 8 M urea, and 4 M GdnHCl was found to be 3.38 M-1 and 9.40 M-1, respectively (Table 5). These outcomes demonstrate the structural changes in the protein leading to the expanded quencher accessibility. Far – UV circular dichroism results reflected the disruption of the secondary structure of GPC at various stages of denaturation. Native GPC showed 33.9% alpha-helix, which declines with an increase in urea and GdnHCl concentration. It was observed that alpha-helical content reduces to 50% at 5 M urea; however, in the case of GdnHCl, it was observed around 2.5 M (Fig. 38 and 39). A marked decrease in secondary structure between 4 M and 7 M urea indicated that the bulk of secondary structures are disrupted in this region. In the case of GdnHCl, abrupt increase in loss of secondary structure was observed after 3 M and complete loss of alpha-helical content was observed at 4 M. Priyadarshini et al. (2010) showed mid-point transition of goat pancreas cystatin at 3.6 M urea and 3.2 M GdnHCl as deduced from circular dichroism study. FTIR experiments were undertaken to confirm the consistency of circular dichroism results and analyze the changes in the secondary structure of GPC denatured by urea and GdnHCl. The secondary structure of proteins is determined by the amide I and amide II bands of FTIR spectra, the amide I absorb in the range of 1600 – 1690 cm-1 that mainly represents C=0 stretching (Kong and Yu, 2007). Amide II bands absorb in the Maulana Azad Library, Aligarh Muslim University range of 1480 – 1575 cm-1, which reflects the presence of CN stretching and NH bending (Kong and Yu, 2007). In order to explore the changes in the secondary structure of GPC, amide I region was plotted against absorbance. The mean frequency of 1656±2.0 cm-1 has been assigned to the alpha - helix of the protein (Kong and Yu, 2007). Native GPC showed an absorbance peak at 1655 cm-1, which confirmed the presence of alpha - helix. Increase in urea concentration disrupts the peak at 1655 cm-1, which indicates the changes in the secondary structure of GPC. The complete loss of peak was observed at 8 M

166 | P a g e

Chapter -2 Results and discussion urea, suggesting the absence of alpha helix at high concentration (Fig. 40). Similarly, as the concentration of GdnHCl increases, it disrupts the signature peak of the alpha helix at 1655 cm-1. It was observed that the peak at 1655 cm-1 disappeared at 4 M GdnHCl, which indicates the complete disruption of secondary structure (Fig. 41). Denaturation of D-glyceraldehyde-3-phosphate dehydrogenase has been previously monitored in the presence of GdnHCl by FTIR spectroscopy (Li and Zhou, 1996) in which the results showed the alteration in the secondary structure of the enzyme. Similar kind of study using FTIR spectroscopy suggested that glucose oxidase, when incubated with arabinose for 12 days, showed peak loss at 1530 cm-1 confirming the loss of alpha-helical structure (Khan et al., 2012). The equilibrium denaturation curve of GPC in the presence of urea and guanidine hydrochloride was also plotted as a function of change in the fluorescence intensity (Fig. 42 and 44). It was observed that the transition mid-point for the urea and GdnHCl induced unfolding was 4.7±0.1 M and 2.32±0.1 M, respectively. The value of ΔG(H2O) was found to be 1.5±0.2 kcal/mol in the case of urea and 1.9±0.1 kcal/mol in the case of GdnHCl (Fig. 43 and 45). The ΔG(H2O) represents the conformational stability of GPC determined under the denaturing conditions using urea and GdnHCl. It was observed that the value ΔG(H2O) determined using urea-induced unfolding is lower than the stability determined using GdnHCl which might be attributed to an increased level of protonation of the acidic groups and disruption of salt bridges. A number of theories have been put forward in order to understand the Maulana Azad Library, Aligarh Muslim University mechanism of denaturation by urea and GdnHCl. However, the exact mechanism by which urea and GdnHCl denature protein is still the subject of core research. The probable answer to the chaotropes mediated denaturation is the combination of various theories which explains the denaturation process. Urea being an uncharged chaotropic molecule denatures protein either by direct interaction or indirect interaction. Direct interaction involves hydrogen bonding between urea and the peptide bond of a protein. The newly formed hydrogen bond weakens the intermolecular bonds and hydrophobic interactions which

167 | P a g e

Chapter -2 Results and discussion disrupts the overall conformation of a protein. Thus, the hydrophobic inner core of a protein becomes more susceptible for water and urea molecules, thereby, accelerating up the denaturation process. Indirect interaction of urea influences the property of the solvent in which the protein is present. The changes in the attributes of the solvent affect the hydrodynamic property of the solvent itself, which ultimately encourages urea to denature the protein by destabilizing internal forces. The indirect interaction ultimately proceeds towards direct interaction by making an environment which is suitable for urea to interact with the internal bonds. Hydrogen bonds formed between urea and exposed amide bonds are stronger than the amide-amide hydrogen bonds which are necessary for proper folding of the protein (Bennion and Daggett 2003; Hua et al. 2008). On the other side, GdnHCl contains monovalent salt, which shows both chaotropic and ionic impacts. The pKa of GdnHCl is 11, which implies that it would be present as fully protonated form as Gdn+, which contains a delocalized positive charge on its planar structure if the pH is below 11. The conformational stability of proteins depends on the availability of cations (Gdn+) and anions (Cl-1) present in the vicinity of proteins. Proteins confer a number of affinity binding sites with differential specificity for the cations and anions that leads to the emergence of different ways of stabilization of proteins by denaturants. Hence, it is obvious to have a different mode of denaturing action by various denaturants. The cations bind to the negatively charged groups of proteins which result in compaction of native proteins. Hence, it can be said that the ionic association of Gdn+ with the charged groups of protein is Maulana Azad Library, Aligarh Muslim University liable for disrupting electrostatic interactions and denaturation of the proteins. Similarly, in the present study, the GPC may be stabilized at a lower concentration of cation binding to the negatively charged moieties of the GPC. Gdn+ at lower concentration binds to the opposite charged groups and acts as a stabilizer for protein while at higher concentration it acts as a denaturant, which leads to complete unfolding of the protein. Therefore, it can be said that the difference in the nature of both the chaotropes lead to the emergence of different states during the denaturation of GPC by urea and GdnHCl.

168 | P a g e

Chapter – 3

A. Interaction study of fungicide with garlic phytocystatin

B. Interaction study of

Maulanaherbicide Azad Library, Aligarhwith Muslim garlicUniversity phytocystatin

Chapter – 3 A

Interaction of carbendazim (fungicide) with garlic phytocystatin Maulana Azad Library, Aligarh Muslim University

Chapter -3 (A) Results and discussion

RESULTS

(A) INTERACTION OF CARBENDAZIM (FUNGICIDE) WITH GARLIC PHYTOCYSTATIN (GPC)

1. FUNCTIONAL STUDY

1.1 Effect of carbendazim on the cysteine proteinase inhibitory activity of GPC

Cysteine proteinase inhibitory activity assay of GPC was done in order to probe the functional inactivity induced by carbendazim. GPC was incubated with different concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. It was observed that increasing concentration of carbendazim affects the inhibitory activity of GPC. Furthermore, figure 46 suggests that treatment of GPC with a high concentration of carbendazim and longer incubation showed a more pronounced effect on the inhibitory activity of GPC. It is evident from the result that GPC showed minimum inhibitory activity when incubated at 100 µM carbendazim for 12 h.

2. STRUCTURAL STUDY

2.1 ULTRAVIOLETMaulana Azad Library, ABSORPTION Aligarh Muslim STUDY University OF GPC IN THE PRESENCE OF CARBENDAZIM

2.1.1 Effect of carbendazim on UV-absorption of GPC

UV-vis spectroscopy of protein sample is based on the cumulative absorption near 280 nm due to the presence of three aromatic amino acid residues, namely tryptophan, tyrosine, and phenylalanine (Brennan, 1999).

169 | P a g e

Chapter -3 (A) Results and discussion

120

4 hours 12 hours

100

ty i

v *

80 cti

a #

ry

to *

bi 60 #

nhi

i

ng 40 * ni

i #

a * em # * R 20

% #

0 GPC alone 20 40 60 80 100 Carbendazim (µM)

Figure 46. Cysteine proteinase inhibitory assay of GPC in the presence of carbendazim.

Native GPC (4 µM) was incubated with increasing concentration of carbendazimMaulana (20 µM, 40Azad µM, Library, 60 µM, 80Aligarh µM, and Muslim 100 µM) University at 37°C for 4 h and 12 h in 50 mM sodium phosphate buffer (pH 7.5). Inhibitory activity assay was performed using casein as a substrate by the method of Kunitz (1947) as described in methods. Values expressed as mean ± S.E.M. of three independent experiments. *P < 0.05 with respect to GPC alone at 4 h and #P < 0.05 with respect to GPC alone at 12 h.

170 | P a g e

Chapter -3 (A) Results and discussion

The GPC was incubated with different concentrations of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. Figure 47 (A) shows that the increase in carbendazim concentration decreases the absorption intensity of native GPC. Decreased absorption intensity could be attributed to the burial of aromatic residues upon the complex formation of GPC-carbendazim. Figure 47 (B) suggests that incubation of GPC for 12 h showed a noticeable decrease in absorption intensity as compared to the GPC-carbendazim incubated for 4 h. Hence, it can be concluded that carbendazim showed concentration as well as time-dependent effects on GPC.

2.2 INTRINSIC FLUORESCENCE STUDY OF GPC IN THE PRESENCE OF CARBENDAZIM

2.2.1 Effect of carbendazim on the intrinsic fluorescence of GPC

Tryptophan has the highest fluorescence among the three intrinsic fluorophores, namely tryptophan, tyrosine, and phenylalanine. The intrinsic fluorescence of GPC was recorded after incubation with different concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. Figure 48 (A) shows the fluorescence spectra of native GPC and GPC-carbendazim incubated for 4 Maulana Azad Library, Aligarh Muslim University h. It was observed that increasing concentration of carbendazim decreases the fluorescence intensity of native GPC, hence confirmed the interaction of GPC with carbendazim. The decreased fluorescence intensity also reflects changes in the microenvironment around aromatic residues of GPC in the presence of carbendazim. Figure 48 (B) shows the relative fluorescence of GPC incubated with carbendazim, which suggests a prominent reduction in fluorescence intensity at longer incubation time.

171 | P a g e

Chapter -3 (A) Results and discussion

0.5 GPC alone 20 µM CAR 40 µM CAR 60 µM CAR 80 µM CAR 100 µM CAR

0.4

) .u.

a 0.3

nce ( nce rba

0.2 Abso

0.1

0 250 260 270 280 290 300 Wavelength (nm)

Figure 47 (A). UV-absorption analysis of GPC in the presence of carbendazim (CAR) Maulana Azad Library, Aligarh Muslim University Native GPC (4 µM) was incubated with different concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) for 4 h at 37ºC in 50 mM sodium phosphate buffer (pH 7.5). Absorption spectra were recorded in the range of 250-300 nm on a UV-1800 Shimadzu spectrophotometer.

172 | P a g e

Chapter -3 (A) Results and discussion

4 hours 12 hours

0.4

nm

0 * 8

2 *

* @ @ 0.3

*

ensity *

0.2

nce int nce rba

0.1 Abso

0 GPC 20 40 60 80 100 alone Carbendazim (µM)

Figure 47 (B). Relative absorption plot of GPC in the presence of carbendazim

RelativeMaulana absorption Azad of Library, GPC incubated Aligarh withMuslim different University concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) for 4 h and 12 h at 37°Cin 50 mM sodium phosphate buffer (pH 7.5). The values are plotted for absorbance at 280 nm against the different concentration of carbendazim. Values expressed as mean ± S.E.M. of three independent experiments. *P < 0.05 with respect to control at 4 h and #P < 0.05 with respect to control at 12 h.

173 | P a g e

Chapter -3 (A) Results and discussion

500 GPC alone 450 20 µM CAR 400 40 µM CAR

350 60 µM CAR

(a.u.)

300 80 µM CAR

ensiry 100 µM CAR 250

200

escence int escence 150

Fluor 100

50

0 300 320 340 360 380 400 Wavelength (nm)

Figure 48 (A). Intrinsic fluorescence spectra of GPC in the presence of carbendazim (CAR) Maulana Azad Library, Aligarh Muslim University Native GPC (4 µM) was incubated with different concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) for 4 h at 37ºC in 50 mM sodium phosphate buffer (pH 7.5). Fluorescence spectra were measured at an excitation wavelength of 295 nm, and the emission range was fixed at 300-400 nm.

174 | P a g e

Chapter -3 (A) Results and discussion

4 hours 12 hours

500 (a.u.) 400

intensity intensity 300

escence escence 200

100

tive fluor tive Rela 0 GPC 20 40 60 80 100 alone Carbendazim (µM)

Figure 48 (B). Relative fluorescence plot of GPC in the presence of carbendazim.

RelativeMaulana fluorescence Azad Library,of GPC incubated Aligarh Muslimwith different University concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) for 4 h and 12 h at 37°C 37ºC in 50 mM sodium phosphate buffer (pH 7.5). The values are plotted for fluorescence intensity against the different concentration of carbendazim. Values expressed as mean ± S.E.M. of three independent experiments. *P < 0.05 with respect to control at 4 h and #P < 0.05 with respect to control at 12 h.

175 | P a g e

Chapter -3 (A) Results and discussion

2.2.2 Stern-Volmer quenching analysis of GPC in the presence of carbendazim

Quenching studies were performed to assess the relative exposure of fluorophores and its accessibility towards water molecule. Non - ionic acrylamide molecules bind to tryptophan residues, hence becomes a distinguishing tool to study the conformational changes in the protein. Figure 49 (A) shows the Stern-Vomer plot of GPC incubated with different concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. It is apparent from the plot that quenching is concentration dependent and the strong quenching at 100 µM carbendazim suggests that the fluorophores of GPC are maximally buried to the solvent. The results obtained from the quenching experiment are consistent with the intrinsic fluorescence results. Figure 49 (B) shows a modified Stern-Volmer plot for calculating the binding constant for the GPC-carbendazim complex at 4 h and 12 h. The Stern–Volmer quenching constant (Ksv) and binding constant (Kb) for the GPC incubated with carbendazim are listed in Table 8.

3. SYNCHRONOUS FLUORESCENCE STUDY OF GPC IN THE PRESENCE OF CARBENDAZIM

3.1 Effect of carbendazim on the synchronous fluorescence of GPC Maulana Azad Library, Aligarh Muslim University

Synchronous fluorescence reveals information about micro-environment around tyrosine and tryptophan residues of the protein. Therefore, synchronous fluorescence spectra were recorded to elucidate conformational changes mediated by carbendazim. Excitation and emission spectra were scanned simultaneously at a fixed difference of wavelength (Δλ= λemission-λexcitation). Δλ =15 nm and Δλ=60 nm discloses characteristic information about surrounding of tyrosine and tryptophan residues respectively.

176 | P a g e

Chapter -3 (A) Results and discussion

1.5

4 hours 12 hours

1.4

1.3

Fo/F

1.2

1.1

1 0 20 40 60 80 100 Carbendazim [10-6 M]

Figure 49 (A). Stern-Volmer analysis of GPC in the presence of carbendazim Maulana Azad Library, Aligarh Muslim University Stern-Volmer plot of GPC (4 µM) in the presence of different concentrations of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) incubated for 4 h and 12 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The plot was analyzed by the Stern-Volmer quenching equation described in the methodology section.

177 | P a g e

Chapter -3 (A) Results and discussion

0 4 hours 12 hours

-0.2

/f] -0.4

f)

-

fo

[(

-0.6 log

-0.8

-1

-1.2 -4.8 -4.6 -4.4 -4.2 -4 -3.8 log carbendazim [M]

Figure 49 (B). Modified Stern-Volmer analysis of GPC in the presence of carbendazim Maulana Azad Library, Aligarh Muslim University Modified Stern-Volmer plot of GPC in the presence of different concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) incubated for 4 h and 12 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The plot was analyzed by the modified Stern-Volmer equation described in the methodology section.

178 | P a g e

Chapter -3 (A) Results and discussion

Table 8: Stern-Volmer quenching constant (Ksv) and binding constant (Kb) for the interaction of GPC with carbendazim.

-1 -1 Carbendazim Ksv [M ] Kb [M ]

4 h incubation 2.597 x 103 48.9

12 h incubation 3.865 x 103 5.01 x 102

Stern-Vomer quenching constant (Ksv) and binding constant

(Kb) is determined by the Stern-Volmer equation and modified Stern-Volmer equation described in the methodology section.

Maulana Azad Library, Aligarh Muslim University

179 | P a g e

Chapter -3 (A) Results and discussion

The shift in emission maxima at both wavelength difference could be attributed to changes in the polar environment near tyrosine and tryptophan. Henceforth, synchronous fluorescence demonstrated as an unmistakable tool that could be utilized to investigate the shift in polarity around fluorophores. The synchronous fluorescence spectra of GPC in the presence of an increasing concentration of carbendazim (0-100 µM) are shown in figure 50 (A) and (B). Carbendazim was added in successive increments in GPC (4 µM) till it reaches up to 100 µM carbendazim. It is evident from figure 50 (A) and (B) that upon addition of carbendazim to GPC, the synchronous fluorescence decreases gradually for both the fluorophores thus, signifying the interaction between GPC and carbendazim (He et al., 2008). Figure 50 (A) showed that there was no change in emission maxima in the synchronous fluorescence of tyrosine. However, figure 50 (B) exhibited a significant blue shift in the case of tryptophan residues.

4. ISOTHERMAL TITRATION CALORIMETRIC STUDY OF GPC IN THE PRESENCE OF CARBENDAZIM

Isothermal titration calorimetry is a useful tool for delineation of the thermodynamic parameters of ligand-macromolecule interaction. It helps to predict the nature of reaction as well as other thermodynamic parameters. Figure 51 shows the ITC profile of carbendazim (20 µM) with GPC (10 µM). The upper panel represents the raw ITC profile as a result of the injection of Maulana Azad Library, Aligarh Muslim University carbendazim into GPC at 37° C in 50 mM sodium phosphate buffer (pH 7.5). Each heat burst in the curve corresponds to a single ligand injection. The area under these heat bursts was determined by integration to obtain the associated injection heats. The isotherm was fitted to a single-site sequential model for acquiring the best fits. The lower panel represents the heat released per injection plotted as a function of the molar ratio of carbendazim to GPC. It is evident from the lower panel of figure 51 that the interaction of GPC with carbendazim is an exothermic process.

180 | P a g e

Chapter -3 (A) Results and discussion

700 GPC alone 10 µM CAR 20 µM CAR 600 30 µM CAR 40 µM CAR 50 µM CAR

500 60 µM CAR (a.u.) 70 µM CAR 80 µM CAR 90 µM CAR ensity 400 100 µM CAR

300 escence int escence

Fluor 200

100 285 295 305 315 Wavelength (nm)

Figure 50 (A). Synchronous fluorescence spectra of GPC in the presence of carbendazim (CAR) Maulana Azad Library, Aligarh Muslim University Synchronous fluorescence spectra of GPC for tyrosine residues (Δλ= 15 nm). Native GPC (4 µM) was titrated with increasing concentration of carbendazim (10 – 100 µM) in 50 mM sodium phosphate buffer (pH 7.5). The synchronous fluorescence for tyrosine residues was recorded at an excitation wavelength of 240 nm, and the emission spectra were recorded in the range of 255 – 400 nm.

181 | P a g e

Chapter -3 (A) Results and discussion

1100 GPC alone 10 µM CAR

20 µM CAR

30 µM CAR 900

40 µM CAR

(a.u.)

50 µM CAR 60 µM CAR ensity 700 70 µM CAR 80 µM CAR 90 µM CAR 100 µM CAR

escence int escence 500 Fluor

300

100 300 310 320 330 340 350 360 Wavelength (nm)

Figure 50 (B). Synchronous fluorescence spectra of GPC in the presence of carbendazim (CAR) Maulana Azad Library, Aligarh Muslim University Synchronous fluorescence spectra of GPC for tryptophan residues (Δλ= 60 nm). Native GPC (4 µM) was titrated with increasing concentration of carbendazim (10 – 100 µM) in 50 mM sodium phosphate buffer (pH 7.5). The synchronous fluorescence for tryptophan residues was recorded at an excitation wavelength of 240 nm, and the emission spectra were recorded in the range of 300 – 400 nm.

182 | P a g e

Chapter -3 (A) Results and discussion

Figure 51. Isothermal titration calorimetry plot of GPC in the presence of carbendazim Maulana Azad Library, Aligarh Muslim University Isothermal titration calorimetry plot of GPC (10 µM) with carbendazim (20 µM) in 50 mM sodium phosphate buffer (pH 7.5). Titration of GPC shows calorimetric response as successive injections of the ligand is added to the sample cell.

183 | P a g e

Chapter -3 (A) Results and discussion

5. SECONDARY STRUCTURE ANALYSIS OF GPC IN THE PRESENCE OF CARBENDAZIM

5.1 CD ANALYSIS

5.1.1 Effect of carbendazim on the secondary structure of GPC

Circular dichroism reveals the secondary structures of protein and subsequently could be used to examine the changes in the secondary structure of proteins. Far UV circular dichroism spectra were recorded in the range of 190 nm – 260 nm. Figure 52 shows far UV CD spectra of GPC incubated with different concentrations of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. The far UV CD spectrum of GPC exhibits two negative peaks at 208 nm and 222 nm that is a characteristic peak of the alpha-helical structure of proteins (Ghalandari et al., 2014). The incubation of GPC with carbendazim gradually reduces the negative ellipticity, thereby decreasing the alpha-helical content of GPC. The decrease in alpha-helical content of GPC upon incubation with carbendazim suggests destabilization of intermolecular forces which are responsible for maintaining secondary structures ultimately leads to changes in the conformation of the protein. The alpha-helical content was determined by the equation givenMaulana below (Chen Azad et al.,Library, 1972) and Aligarh is listed Muslim in Table University 9.

% alpha-helix = (MRE222 nm – 2340/30300) x 100

184 | P a g e

Chapter -3 (A) Results and discussion

GPC alone 13000 20 µM GPC

9000 40 µM CAR 60 µM CAR 5000 80 µM CAR 100 µM CAR

1000

MRE -3000

-7000

-11000

-15000 195 205 215 225 235 245 255 Wavelength (nm)

Figure 52. Circular dichroism analysis of GPC in the presence of carbendazim (CAR) Maulana Azad Library, Aligarh Muslim University Far-UV circular dichroism spectra of GPC (4 µM) incubated with different concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) incubated for 4 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The spectra were recorded in the range of 200-260 nm, and each spectrum is an average of three scans.

185 | P a g e

Chapter -3 (A) Results and discussion

Table 9: Secondary structure analysis of GPC incubated with carbendazim for 4 hours at 37ºc in 50 millimolar sodium phosphate buffer (pH 7.5).

Carbendazim (µM) % Lost alpha-helix

0 0

20 10.29

40 20.5

60 33.58

80 46.6

100 59.8

Alpha-helicalMaulana content Azad was determinedLibrary, Aligarh by the Muslimmethod University of Chen et al. (1972) equation given in the methodology section.

186 | P a g e

Discussion

Maulana Azad Library, Aligarh Muslim University

Chapter -3 (A) Results and discussion

DISCUSSION

Continuous efforts are being made in a way to increase crop yield and production to serve the increasing mass. Various biological and chemical methods are employed in order to achieve the estimated target of crop yield and protection to plants. Application of pesticide is one of the potent chemical methods that is widely used in this era to eliminate the disastrous effects of pest and pathogens (Damalas and Eleftherohorinos, 2011). Since pesticides brought a revolution in the crop production so, they are considered as an efficient and remarkable agent of plant defense. However, considering the side effects imparted by these pesticides due to their prolonged exposure, integrated pest management was introduced in 1959 as a concept to minimize the use of harmful and detrimental pesticides (Stern et al., 1959). Still, several harmful pesticides are in use to combat pest and pathogens which are unaffected by relatively less potent pesticides. Hence, the present study aimed at examining the effects of carbendazim (fungicide) that is routinely used on crops (Thiaré et al., 2015). Chemically, carbendazim is a methyl 2-benzimidazole carbamate which is used against various orchard and vegetable diseases (Thiare et al., 2015). It is used as a systemic fungicide and protects plant against a wide range of fungal diseases such as mildew, rot and blight disease, and mould. It targets various crops including vegetables, cereals, vines and fruits etc. It has been also reported that purple blotch disease in garlic crops can be managed by usage of carbendazim at 0.2% thrice at 15 days interval (Chaurasia et al., 2007). Maulana Azad Library, Aligarh Muslim University Phytocystatins are plant proteinase inhibitors of cysteine proteases which are implicated in the regulation of biological and physiological activities of the plant. They are found to be involved in the regulation of endogenous cysteine proteases during development and plant growth, programmed cell death, and senescence (Irene et al., 2012). Phytocystatins have established their roles in response to cell damage, jasmonic acid treatment, fungal infection, and biotic-abiotic stress (Irene et al., 2012). The present study employed various

187 | P a g e

Chapter -3 (A) Results and discussion biophysical techniques to evaluate the effects of carbendazim on garlic phytocystatin.

Purified garlic phytocystatin (4 µM) was incubated with different concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h to evaluate the effect of carbendazim on the inhibitory activity of GPC. Inhibitory activity of GPC showed concentration and time-dependent decrease, meaning thereby that the inhibitory activity declines as the concentration of carbendazim increases along with the incubation time. As it can be observed from the figure 46 that incubation of GPC with 10 µM carbendazim for 4 h reduces the inhibitory activity to 80.2 % while incubation at 100 µM carbendazim for 12 h brings significant conformational change in GPC and it retains only 8.3% inhibitory activity. It suggests that increased concentration and incubation time alters the conformation of GPC, which ultimately reduces the functionality of GPC. Black gram and chickpea cystatin showed similar findings when incubated with sodium diethyl dithiocarbamate and mancozeb, respectively (Bhat et al., 2016; Sharma et al., 2005).

The UV absorption spectroscopy was employed to analyze the complex formation between GPC and carbendazim. The UV spectroscopy is a fine and peculiar technique which reflects the changes in terms of absorption of energy by chromophores. Native GPC showed absorbance at 280 nm that is the characteristic peak for proteins. Carbendazim decreases the absorption of Maulana Azad Library, Aligarh Muslim University native GPC, which suggests the burial of aromatic residues of GPC upon interaction. It is apparent from the absorption spectra of GPC that incubation with 100 µM carbendazim results in a noticeable decrease in absorption intensity of GPC-carbendazim complex (Fig. 47 A). Therefore, the decreased absorption intensity upon incubation with carbendazim suggests the formation of GPC- carbendazim complex. The complex formation results in fine structural change within GPC that leads to the emergence of a new conformation of GPC, which is different from native GPC. The GPC was also

188 | P a g e

Chapter -3 (A) Results and discussion incubated with carbendazim for 12 h, which illustrates that incubation time affects the conformation of GPC and induces more pronounced effect (Fig. 47 B).

Intrinsic fluorescence spectroscopy corresponds to protein conformational transitions, subunit association, substrate binding, or denaturation (Sułkowska et al., 2003). Therefore, alteration in the conformation of GPC was also studied by employing tryptophan fluorescence spectroscopy that reflects the conformational changes in GPC. The fluorescence spectra of GPC were recorded in the presence of different concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) after incubation for 4 h and 12 h. Native GPC was excited at 295 nm to monitor the changes around tryptophan residues. It is evident from the fluorescence spectra that increasing the concentration of carbendazim decreases the emission intensity of native GPC (Fig. 48 A). It is also observed that incubation of GPC with carbendazim did not show any shift in the peak of emission maxima. GPC showed strong fluorescence quenching when incubated with 100 µM carbendazim for 12 h suggesting concentration and time-dependent function (Fig. 48 B). Consequently, it can be concluded that decreased fluorescence may be due to the changes in the microenvironment around the aromatic amino acid, in particular around tryptophan residues leading to the compactness of GPC. Moreover, the decreased fluorescence might be the outcome of the decreased distance betweenMaulana the fluorophores Azad Library, and quenching Aligarh groups Muslim of the protein.University Similar findings have been reported in the case of chickpea cystatin when treated with mancozeb (fungicide) (Bhat et al., 2016). Hence, it can be inferred that carbendazim induces conformational changes upon interaction with GPC, which ultimately results in the transition of native GPC to a non-native form of GPC.

The decreased intrinsic fluorescence was further evaluated by Stern- Volmer quenching plot in order to probe the accessibility of fluorophores

189 | P a g e

Chapter -3 (A) Results and discussion towards solvent. The Stern – Volmer quenching constant (Ksv) obtained for the GPC-carbendazim binding was 2.597 x 103 M-1 and 3.865 x 103 M-1 for 4 h and 12 h incubation, respectively (Table 8). The Stern – Volmer quenching constant describes the fluorescence quenching efficiency of a ligand. The higher quenching constant signifies higher quenching efficiency of a ligand thus, GPC showed strong quenching at 100 µM carbendazim when incubated for 12 h (Fig. 49 A). It can also be inferred that incubation of GPC with carbendazim brought changes in the microenvironment around tryptophan residues. The inference obtained from Stern-Volmer plot and decreased fluorescence intensity indicates a change in the microenvironment around fluorophores at higher concentration and longer incubation, thus confirming the structural change within GPC. The binding constant of GPC-carbendazim interaction obtained at 12 h of incubation is higher than that of obtained at 4 h of incubation (Fig. 46 B). This effect may be due to very slow conformational changes in the protein that allowed the carbendazim to find a higher affinity binding state than the original one. Consequently, the availability of higher affinity binding state, thus quenches the GPC more efficiently, thereby bringing more pronounced structural change.

Synchronous fluorescence was also followed to examine the changes in the microenvironment around fluorophores such as tyrosine and tryptophan residues upon binding of carbendazim to garlic phytocystatin. In synchronous fluorescence spectroscopy, excitation and emission monochromators are scanned simultaneouslyMaulana Azad and theLibrary, difference Aligarh in the Muslim excitation University and emission wavelength is exploited to decipher the alterations around fluorophores. The difference of 15 nm and 60 nm in the excitation and emission wavelength provide details about alteration around tyrosine and tryptophan residues, respectively (Bhogale et al., 2014). Moreover, the shift in the emission maxima corresponds to the changes in the polarity around fluorophores thereby altering the microenvironment around fluorophores (Liu et al., 2017). GPC (4 µM) upon titrating with carbendazim (0-100 µM) showed quenching in tyrosine as well as tryptophan synchronous fluorescence spectra of GPC. However, only 190 | P a g e

Chapter -3 (A) Results and discussion tryptophan spectra showed a blue shift which signifies the changes in the microenvironment around tryptophan residue (Fig. 50 B). The binding of carbendazim to GPC rearranged the hydrophobic environment which leads to the increased hydrophobicity in the vicinity of tryptophan residues as well as exposed them towards a non-polar environment. Hence, it can be inferred that tryptophan residues were exposed towards the hydrophobic environment upon binding of carbendazim to GPC. Isothermal titration calorimetry study showed that the binding of carbendazim with GPC was an exothermic process (Fig. 51). The similar thermodynamic reaction has been previously reported in case of interaction of almond phytocystatin with Cu (II) hydroxide (fungicide) and yellow mustard phytocystatin with iprodione (fungicide) (Ahmed et al., 2018; Siddiqui et al., 2017). The results obtained by above biophysical experiments conclude that carbendazim upon binding to GPC brings conformational and functional change which ultimately leads to the transition of GPC to non-native form.

Circular dichroism analysis was performed in order to monitor the effect of carbendazim on the secondary structural elements of GPC. It is evident from the results that GPC losses secondary structure upon incubation with different concentration of carbendazim (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h (Fig. 52). It is further observed that 20 µM carbendazim decreases the alpha-helical content by 10.29 % while 59.8% reduction was observed at 100 µM carbendazim. Thus, the reductionMaulana in the alphaAzad-helical Library, structure Aligarh of GPC Muslim may be University due to the binding of carbendazim with the amino acid residue present in the chain of GPC, thereby, disrupting the network of hydrogen bonds (Kang et al., 2004). Hence, the study confirms that carbendazim disrupts the secondary structure of GPC and transforms it into non-native conformation.

191 | P a g e

Chapter – 3 B

Interaction of oxyfluorfen (herbicide) with garlic phytocystatin Maulana Azad Library, Aligarh Muslim University

Chapter -3 (B) Results and discussion

RESULTS

INTERACTION STUDY OF OXYFLUORFEN (HERBICIDE) WITH GARLIC PHYTOCYSTATIN (GPC)

1. FUNCTIONAL STUDY

1.1 Effect of oxyfluorfen on the cysteine proteinase inhibitory activity of GPC

The cysteine proteinase inhibitory assay was done in order to monitor the inhibitory activity of garlic phytocystatin in the presence of oxyfluorfen. The garlic phytocystatin was incubated with different concentration of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. Figure 53 shows the cysteine proteinase inhibitory assay of garlic phytocystatin in the presence of oxyfluorfen. It was observed that the cysteine proteinase inhibitory activity of garlic phytocystatin reduced to 80.73% in the presence of 10 μM oxyfluorfen, which further declines with increasing concentration of oxyfluorfen. The inhibitory activity of GPC reduced to 23.5% upon incubation with 100 μM oxyfluorfen for 4 h. It was also observed that incubation time affects the cysteine proteinase inhibitory activity. The cysteine proteinase activity profoundly declinesMaulana upon Azad incubation Library, of GPC Aligarh with oxyfluorfen Muslim Universityfor 12 h. Thus, it confirms that higher concentration and longer time of incubation of oxyfluorfen with GPC adversely affects the cysteine proteinase inhibitory activity of garlic phytocystatin.

192 | P a g e

Chapter -3 (B) Results and discussion

120

4 hours 12 hours

100

ty i

v 80 *

acti

ry 60 * to

bi #

*

nhi

i

l 40 * #

# dua

i *

es 20 #

R % 0 GPC 20 40 60 80 100 alone Oxyfluorfen (µM)

Figure 53. Cysteine proteinase inhibitory assay of GPC in the presence of oxyfluorfen

Native GPC (4 µM) was incubated with increasing concentration of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37°C for 4 h and 12Maulana h in 50 mM Azad sodium Library, phosphate Aligarh buffer Muslim (pH 7.5) University. Inhibitory activity assay was performed using casein as a substrate by the method of Kunitz (1947) as described in methods section. Values expressed as mean ± S.E.M. of three independent experiments. *P < 0.05 with respect to GPC alone at 4 h and #P < 0.05 with respect to GPC alone at 12 h.

193 | P a g e

Chapter -3 (B) Results and discussion

2. STRUCTURAL STUDY

2.1 ULTRAVIOLET ABSORPTION STUDY OF GPC IN THE PRESENCE OF OXYFLUORFEN

2.1.1 Effect of oxyfluorfen on UV-absorption of GPC

Proteins absorb at around 280 nm due to the presence of aromatic amino acid residues. UV absorption spectroscopy is a reliable method to monitor perturbations caused by the binding of the ligand to the protein molecule. The perturbations upon binding of a ligand are indicative of a conformational change. Hence, the GPC was monitored in the presence of different concentrations of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) after incubation for 4 h and 12 h. It is evident from figure 54 (A) that GPC absorbs at 280 nm, while on increasing the concentration of oxyfluorfen, the absorption intensity of GPC decreases. It was further observed that 100 μM oxyfluorfen showed a pronounced reduction in the absorption intensity of GPC. Figure 54 (B) shows that incubation of GPC for 12 h showed a prominent decrease in absorption as compared to the GPC incubated with oxyfluorfen for 4 h. Hence, it can be concluded that oxyfluorfen shows concentration as well as time-dependent effects on GPC

2.2 INTRINSICMaulana FLUORESCENCE Azad Library, Aligarh STUDY Muslim OF GPCUniversity IN THE PRESENCE OF OXYFLUORFEN

2.2.1 Effect of oxyfluorfen on the intrinsic fluorescence of GPC

The total intrinsic fluorescence of a protein is a cumulative fluorescence of individual fluorophores present in the protein. The residues which impart fluorescence are tyrosine, phenylalanine & tryptophan.

194 | P a g e

Chapter -3 (B) Results and discussion

GPC alone 20 µM oxyfluorfen 40 µM oxyfluorfen 60 µM oxyfluorfen 0.8 80 µM oxyfluorfen 100 µM oxyfluorfen

0.7

) 0.6

.u. a

0.5 nce ( nce

0.4 rba

0.3 Abso

0.2

0.1

0 240 250 260 270 280 290 300 Wavelength (nm)

Figure 54 (A). UV-absorption analysis of GPC in the presence of oxyfluorfen Maulana Azad Library, Aligarh Muslim University Native GPC (4 µM) was incubated with different concentration of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) for 4 h at 37ºC in 50 mM sodium phosphate buffer (pH 7.5). Absorption spectra were recorded in the range of 240-300 nm on a UV-1800 Shimadzu spectrophotometer.

195 | P a g e

Chapter -3 (B) Results and discussion

0.7 4 hours 12 hours 0.6 * *

# * 0.5 * #

*

(a.u.)

0.4 # # ensity #

0.3 nce int nce

0.2 rba

Abso 0.1

0 GPC 20 40 60 80 100 alone Oxyfluorfen (µM)

Figure 54 (B). Relative absorption plot of GPC in the presence of oxyfluorfen Maulana Azad Library, Aligarh Muslim University Relative absorption of GPC incubated with different concentration of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) for 4 h and 12 h at 37°Cin 50 mM sodium phosphate buffer (pH 7.5). Values expressed as mean ± S.E.M. of three independent experiments. *P < 0.05 with respect to GPC alone at 4 h and #P < 0.05 with respect to GPC alone at 12 h.

196 | P a g e

Chapter -3 (B) Results and discussion

Among them, tryptophan is the major contributor of fluorescence due to its high quantum yield. Intrinsic fluorescence is used to analyze the conformational alteration within the proteins. It reflects the alteration in the local environment of aromatic residues upon interaction with ligands. The intrinsic fluorescence of GPC was monitored after incubation with different concentrations of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. It is evident from figure 55 (A) that native GPC exhibits a strong fluorescence emission peak at 334 nm and oxyfluorfen quenches the fluorescence of native GPC. The reduction in the fluorescence intensity upon the interaction of oxyfluorfen suggests the binding of oxyfluorfen to GPC. Figure 55 (B) shows relative fluorescence of GPC incubated with different concentration of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. The relative fluorescence plot showed a prominent reduction in fluorescence intensity at longer incubation time.

2.2.2 Stern-Volmer quenching analysis of GPC in the presence of oxyfluorfen

Stern-Volmer quenching studies were carried out to analyze the relative exposure of the tryptophan and its accessibility towards water molecule. This method is utilized to study the conformational alteration within the protein. Figure 56 (A) and (B) shows the Stern-Volmer and modified Stern-Volmer plot forMaulana the GPC Azad-oxyfluorfen Library, complex Aligarh, respectivelyMuslim University. The Stern Volmer quenching constant (Ksv) and the number of binding sites are listed in Table 10. Furthermore, the linearity of the Stern-Volmer plot also indicates the existence of either kind of quenching mechanism. As the GPC-oxyfluorfen absorption spectrum is clearly different from that of GPC or oxyfluorfen alone, which is a kind of obvious evidence that they have formed at least one kind of protein-ligand complex with a certain new structure. Hence, it can be inferred that the mode of quenching belongs to a static quenching mechanism.

197 | P a g e

Chapter -3 (B) Results and discussion

1000 GPC alone

900 20 μM oxyfluorfen

) 800 40 μM oxyfluorfen

(a.u. 60 μM oxyfluorfen

700 y

600 80 μM oxyfluorfen ensit

int 500 100 μM oxyfluorfen e e 400

escenc 300 uor

Fl 200

100

0 310 330 350 370 390 Wavelength (nm)

Figure 55 (A). Intrinsic fluorescence spectra of GPC in the presence of oxyfluorfenMaulana Azad Library, Aligarh Muslim University

Native GPC (4 µM) was incubated with different concentration of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) for 4 h at 37ºC in 50 mM sodium phosphate buffer (pH 7.5). Fluorescence spectra were measured at an excitation wavelength of 295 nm, and the emission range was fixed at 310-400 nm.

198 | P a g e

Chapter -3 (B) Results and discussion

1200

4 hours 12 hours

1000

(a.u.)

* # * 800 ensity # * # * # 600 *

# escence int escence

400 Fluor

200

0 GPC 20 40 60 80 100 alone Oxyfluorfen (µM)

Figure 55 (B). Relative fluorescence plot of GPC in the presence of oxyfluorfen

RelativeMaulana fluorescence Azad ofLibrary, GPC incubated Aligarh Muslim with different University concentration of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) for 4 h and 12 h at 37°C 37ºC in 50 mM sodium phosphate buffer (pH 7.5). The values are plotted for fluorescence intensity against the different concentration of oxyfluorfen. Values expressed as mean ± S.E.M. of three independent experiments. *P < 0.05 with respect to GPC alone at 4 h and #P < 0.05 with respect to GPC alone at 12 h.

199 | P a g e

Chapter -3 (B) Results and discussion

1.9

1.8 4 hours 12 hours

1.7

1.6

1.5

Fo/F 1.4

1.3

1.2

1.1

1 0 50 100 150 Oxyfluorfen [10-6 M]

Figure 56 (A). Stern-Volmer analysis of GPC in the presence of oxyfluorfen Maulana Azad Library, Aligarh Muslim University The data of fluorescence experiment was analyzed by Stern-Volmer equation as described in the methodology section. GPC in the presence of different concentrations of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) was incubated for 4 h and 12 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5).

200 | P a g e

Chapter -3 (B) Results and discussion

0 4 hours 12 hours -0.1

-0.2

-0.3

-0.4

] F

)/ -0.5

F -

o -0.6

[(F

g o

l -0.7

-0.8

-0.9

-1 -4.8 -4.6 -4.4 -4.2 -4 -3.8 log oxyfluorfen [M]

Figure 56 (B). Modified Stern-Volmer analysis of GPC in the presence of oxyfluorfen Maulana Azad Library, Aligarh Muslim University Modified Stern-Volmer plot of GPC in the presence of different concentration of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) incubated for 4 h and 12 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The plot was analyzed by the modified Stern-Volmer equation described in the methodology section.

201 | P a g e

Chapter -3 (B) Results and discussion

Table 10: Stern-Volmer quenching constant (Ksv) and binding constant (Kb) for the interaction of GPC with oxyfluorfen.

-1 -1 Oxyfluorfen Ksv [M ] Kb [M ]

4 h incubation 6.89 x 103 9.72 x 103

12 h incubation 8.20 x 103 10.03 x 103

Stern-Vomer quenching constant (Ksv) and binding constant

(Kb) is determined by the Stern-Volmer equation and modified Stern-Volmer equation described in the methodology section.

Maulana Azad Library, Aligarh Muslim University

202 | P a g e

Chapter -3 (B) Results and discussion

Similar reasoning has been documented for various interaction studies such as irisflorentin & bovine serum albumin (Zhang et al., 2008), fisetin and human serum albumin (Roy et al., 2012), cinnamic acid and 2,3-dihydroquinazoline with lysozyme (Hemalatha et al., 2016; Zhang et al., 2011). The binding constant (Kb) was also determined using modified Stern Volmer equation. The binding constant for the garlic phytocystatin-oxyfluorfen complex was found to be 9.72x103 M-1.

3. SYNCHRONOUS FLUORESCENCE STUDY OF GPC IN THE PRESENCE OF OXYFLUORFEN

3.1 Effect of oxyfluorfen on the synchronous fluorescence of GPC

Synchronous fluorescence spectroscopy was undertaken to study the conformational changes within GPC upon binding of oxyfluorfen. It also provides information about the alteration in the micro-environment around tyrosine and tryptophan residues (Wang et al., 2007). The shift in the emission maxima of a protein can predict the changes in polarity around tryptophan and tyrosine. Figure 57 (A) and (B) shows synchronous fluorescence spectra of GPC in the absence and presence of oxyfluorfen (0-100 µM) in 50 mM sodium phosphate buffer (pH 7.5) with fixed Δλ=60 nm and Δλ=15 nm respectively. The addition of oxyfluorfen decreases the intensity of synchronous fluorescenceMaulana in bothAzad cases Library,. However, Aligarh no shift Muslim in the University emission maxima was observed in case of tryptophan residues, suggesting that there is no perturbation in the microenvironment around the aforesaid residue upon binding of oxyfluorfen. However, a red shift was observed in case of tyrosine residues, which suggests the alteration of microenvironment around tyrosine residues upon binding of oxyfluorfen to GPC.

203 | P a g e

Chapter -3 (B) Results and discussion

1000 GPC alone

900 10 μM oxyfluorfen 20 μM oxyfluorfen 800

30 μM oxyfluorfen (a.u.)

700 40 μM oxyfluorfen 50 μM oxyfluorfen 600

ensity 60 μM oxyfluorfen 500 70 μM oxyfluorfen 400 80 μM oxyfluorfen

escence int escence 90 μM oxyfluorfen 300 100 μM oxyfluorfen

Fluor 200

100

0 300 320 340 360 380 400 Wavelength (nm)

Figure 57 (A). Synchronous fluorescence spectra of GPC in the presence of oxyfluorfen

SynchronousMaulana fluorescence Azad spectra Library, of GPC Aligarh for tryptophanMuslim University residues (Δλ=

60 nm). Native GPC (4 µM) was titrated with increasing concentration of oxyfluorfen (10 – 100 µM) in 50 mM sodium phosphate buffer (pH 7.5). The synchronous fluorescence for tryptophan residues were recorded at an excitation wavelength of 240 nm, and the emission spectra were recorded in the range of 300 – 400 nm.

204 | P a g e

Chapter -3 (B) Results and discussion

600 GPC alone 10 μM oxyfluorfen 20 μM oxyfluorfen

500 30 μM oxyfluorfen

) 40 μM oxyfluorfen

50 μM oxyfluorfen (a.u.

400 60 μM oxyfluorfen y 70 μM oxyfluorfen

ensit 80 μM oxyfluorfen 300

int 90 μM oxyfluorfen e e 100 μM oxyfluorfen

200

escenc uor

Fl 100

0 255 275 295 315 335 355 Wavelength (nm)

Figure 57 (B). Synchronous fluorescence spectra of GPC in the presence of oxyfluorfen

SynchronousMaulana fluorescence Azad Library, spectra Aligarh of GPC Muslim for tyrosine University residues (Δλ= 15 nm). Native GPC (4 µM) was titrated with increasing concentration of oxyfluorfen (10 – 100 µM) in 50 mM sodium phosphate buffer (pH 7.5). The synchronous fluorescence for tyrosine residues were recorded at an excitation wavelength of 240 nm, and the emission spectra were recorded in the range of 255 – 400 nm.

205 | P a g e

Chapter -3 (B) Results and discussion

4. ISOTHERMAL TITRATION CALORIMETRIC STUDY OF GPC IN THE PRESENCE OF OXYFLUORFEN

Isothermal titration calorimetry (ITC) is a reliable technique employed to analyze thermodynamic parameters upon binding of small molecules to biological macromolecules. The ITC can also be employed to determine the energetics associated with the binding of ligands (Wang et al., 2014). Therefore, the nature of thermodynamic reaction associated with the binding of oxyfluorfen to GPC was determined by ITC. The upper panel in figure 58 shows the raw ITC profile of the binding of oxyfluorfen with GPC. Each peak in the isotherm corresponds to a single injection of oxyfluorfen into the GPC solution. The lower panel shows the heat released per injection plotted as a function of the molar ratio of oxyfluorfen to GPC solution. The best fit was obtained by following one site model. It is evident from figure 58 that the binding of oxyfluorfen to GPC is an exothermic reaction.

5. SECONDARY STRUCTURE ANALYSIS OF GPC IN THE PRESENCE OF OXYFLUORFEN

5.1 CD ANALYSIS

5.1.1 Effect of oxyfluorfen on the secondary structure of GPC Maulana Azad Library, Aligarh Muslim University

Circular dichroism (CD) is primarily employed in the elucidation of the secondary structure of proteins. The changes in secondary structural elements of proteins upon interaction with small molecules can be easily monitored by using this technique. CD is also used to determine the protein conformations by unraveling the backbone (peptide bonds) conformation of proteins.

206 | P a g e

Chapter -3 (B) Results and discussion

Maulana Azad Library, Aligarh Muslim University

Figure 58. Isothermal titration calorimetry plot of GPC in the presence of oxyfluorfen

Isothermal titration calorimetry plot of GPC (10 µM) with oxyfluorfen (20 µM) in 50 mM sodium phosphate buffer (pH 7.5). Titration of GPC shows calorimetric response as successive injections of the ligand is added to the sample cell.

207 | P a g e

Chapter -3 (B) Results and discussion

Figure 59 shows the CD spectra of GPC incubated with different concentration of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. The circular dichroism spectrum of native GPC shows two sharp negative minima peaks at 222 nm and 208 nm which correspond to the alpha-helix structure. The spectrum showed the presence of a high percentage of alpha-helical content which represents the transition of π-π* and n-π* of the α-helix structure. However, when GPC incubated with increasing concentration of oxyfluorfen, the GPC-oxyfluorfen spectrum showed a reduction in the negative ellipticity without any significant shift of the peaks.

The reduction in the negative ellipticity suggests the reduction in the alpha-helical content of GPC. Oxyfluorfen (100 μM) significantly reduces the alpha-helical content of GPC, thereby suggesting the alteration in the secondary structure of GPC. The alpha-helical content was determined by the equation given below (Chen et al., 1972) and is listed in Table 11.

% alpha-helix = (MRE222 nm – 2340/30300) x 100

Previous reports also suggested an alteration in secondary structure upon interaction of pesticides with chickpea phytocystatin and almond phytocystatin (Bhat et al., 2016; Siddiqui et al., 2017; Siddiqui and Bano, 2018; Sohail et al., 2017).

Maulana Azad Library, Aligarh Muslim University

208 | P a g e

Chapter -3 (B) Results and discussion

25000 GPC alone 20 µM oxyfluorfen 20000 40 µM oxyfluorfen 15000 60 µM oxyfluorfen

10000 80 µM oxyfluorfen 100 µM oxyfluorfen

5000 MRE 0

-5000

-10000

-15000 190 210 230 250 Wavelenth (nm)

Figure 59. Circular dichroism analysis of GPC in the presence of oxyfluorfen Maulana Azad Library, Aligarh Muslim University Far-UV circular dichroism spectra of GPC (4 µM) in the presence different concentration of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) incubated for 4 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The spectra were recorded in the range of 190-260 nm, and each spectrum is an average of three scans.

209 | P a g e

Chapter -3 (B) Results and discussion

Table 11: Secondary structure analysis of GPC after 4 hours incubation with oxyfluorfen.

Oxyfluorfen (µM) % Loss alpha-helix 0 0 20 11.30 40 20.80 60 28.73 80 35.64 100 41.64

Alpha-helical content was determined by the method of Chen et al. (1972) equation given in the methodology section. Maulana Azad Library, Aligarh Muslim University

210 | P a g e

Discussion

Maulana Azad Library, Aligarh Muslim University

Chapter -3 (B) Results and discussion

DISCUSSION

Herbicides are used for disruption and hampering the growth of weeds. They share about 48% of the total usage of pesticides but are used in lower quantities in developing countries because they prefer controlling of weed by hand weeding (Gupta, 2011). Earlier used herbicides were difficult to handle because of highly toxic nature and show non-specificity to the crop. The application of herbicides has exponentially increased and showed major health concern due to its exposure. Oxyfluorfen (diphenyl ether herbicide) inhibits protoporp- hyrinogen oxidase enzyme which is essential for chlorophyll biosynthesis and heme synthesis (Birchfield and Casida, 1997)(Birchfield and Casida, 1997)(Birchfield and Casida, 1997). It can contaminate the surface water thereby accumulates in living system. The accumulation and exposure of oxyfluorfen can exert toxic effects in the plant and animals. Therefore, it is recommended to evaluate the toxic potential of herbicides before usage in the fields.

Phytocystatins are cysteine proteinase inhibitors implicated in the regulation of biological and physiological activities of the plant. They are found to be involved in the regulation of endogenous cysteine proteases during development and plant growth, programmed cell death, and senescence (Irene et al., 2012). They have also shown their roles in response to cell damage, jasmonic acid treatment, fungal infection, and biotic-abiotic stress (Irene et al., 2012).Maulana In the present Azad study,Library, garlic Aligarh phytocystatin Muslim has University been incubated with oxyfluorfen in order to study its structural and functional transition. The garlic phytocystatin was incubated with 20 µM, 40 µM, 60 µM, 80 µM, and 100 µM oxyfluorfen for 4 h and 12 h at 37°C in sodium phosphate buffer (pH 7.5). The functional activity of GPC was monitored with the help of cysteine proteinase inhibitory assay which revealed that inhibitory activity of GPC declines with increasing concentration of oxyfluorfen. The cysteine proteinase inhibitory activity of GPC was also affected by the incubation time and showed that longer incubation of GPC with oxyfluorfen reduces the inhibitory activity to a 211 | P a g e

Chapter -3 (B) Results and discussion greater extent. The cysteine proteinase inhibitory activity of GPC reduces to 23.8% and 13.8% when incubated with 100 µM oxyfluorfen for 4 h and 12 h, respectively (Fig. 53). The decreased inhibitory activity could be due to the conformational alteration within garlic phytocystatin which ultimately leads to the transition of native garlic phytocystatin to a non-native form. The transition of native garlic phytocystatin (inhibitor) to non-native form reduces the probability of the inhibitor to bind to the enzyme, thereby affecting the efficiency of the inhibitor. Various pesticide-protein interaction studies have shown that pesticide can disrupt the normal functioning of proteins viz. inhibition of acetylcholine esterase by chlorpyrifos (Das and Barone, 1999); inhibition of enzyme activity by mancozeb; oxidative stress induced by glyphosate damages hemoglobin (Kwiatkowska et al., 2014). Similar results have been reported for the interaction of phytocystatin with sodium diethyldithiocarbamate (Sharma et al., 2005), mancozeb, glyphosate, and chlorpyrifos (Bhat et al., 2016). The previous report illustrates similar findings for the interaction of almond phytocystatin with pendimethalin, methoxyfenozide, and Cu (II) hydroxide (Siddiqui et al., 2017). Ultraviolet absorption spectroscopy was employed to study the interaction of garlic phytocystatin with oxyfluorfen. Figure 54 (A) showed that the absorption intensity reduces in the presence of an increasing concentrations of oxyfluorfen, thereby suggesting the interaction of oxyfluorfen with GPC. The decrease in absorption intensity was more pronounced when incubated for 12 h as compared to 4 h incubation (Fig. 54 B). The hypochromic shift suggests a Maulana Azad Library, Aligarh Muslim University structural change in garlic phytocystatin upon interaction with oxyfluorfen, thereby further confirming the formation of garlic phytocystatin-oxyfluorfen complex. Thus, the reduction in the absorption intensity of GPC upon complex formation with oxyfluorfen might be due to the burial of earlier exposed aromatic residues. The previous reports also suggested that incubation of yellow mustard phytocystatin with the oxadiargyl reduces the absorption intensity of native phytocystatin and inferred as the formation of the phytocystatin-oxadiargyl complex formation (Ahmed et al., 2017).

212 | P a g e

Chapter -3 (B) Results and discussion

Intrinsic fluorescence spectroscopy was undertaken to study the conformational alteration within GPC upon incubation with different concentrations of oxyfluorfen (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h and 12 h. The fluorescence spectra were recorded at an excitation wavelength of 295 nm for native GPC and GPC-oxyfluorfen complex (Fig. 55 A). The fluorescence spectrum of GPC exhibited a strong emission peak at 334 nm. The fluorescence spectra showed quenching of native GPC in the presence of oxyfluorfen. The incubation of GPC with 100 μM oxyfluorfen showed strong quenching as compared to the quenching at a lower concentration of oxyfluorfen. Hence, the observations confirmed that oxyfluorfen has concentration-dependent effects on garlic phytocystatin. Moreover, the decreased intensity could be attributed to the changes in the microenvironment around fluorophores. It also suggests the burial of aromatic residues which were earlier exposed. The fluorescence of GPC in the presence of oxyfluorfen was also monitored for different incubation time (Fig. 55 B). It was observed that longer incubation quenches the fluorescence of native GPC more effectively and strongly, thereby reflecting that higher concentration and longer incubation adversely alters the conformation of native GPC. Similar fluorescence quenching results have been reported in case of interaction of pesticides with black gram phytocystatin (Sharma et al., 2005), chickpea cystatin (Bhat et al., 2016) and almond cystatin (Siddiqui et al., 2017). Previous reports have also shown the quenching of human serum albumin by glyphosate and chlorpyrifos, respectively (Han et al., Maulana Azad Library, Aligarh Muslim University 2012; Yue et al., 2008).

The reduction in the fluorescence was further analyzed by Stern-Volmer quenching method in order to investigate the accessibility of fluorophores towards solvent. The Stern-Volmer quenching constant (Ksv) was also determined for the binding of GPC to oxyfluorfen (Fig. 56). The Stern – Volmer quenching constant describes the fluorescence quenching efficiency of 3 -1 a ligand. The Ksv obtained for the GPC-oxyfluorfen binding was 6.89 x 10 M and 8.20 x 103 M-1 for 4 h and 12 h of incubation, respectively (Table 10). It 213 | P a g e

Chapter -3 (B) Results and discussion was observed that GPC incubated at 100 µM oxyfluorfen for 12 h showed strong quenching as compared to the incubation for 4 h. The higher quenching constant signifies higher quenching efficiency of a ligand thus, GPC showed strong quenching at 100 µM oxyfluorfen when incubated for 12 h. The binding 3 -1 constant (Kb) obtained for the GPC-oxyfluorfen interaction was 9.72 x 10 M and 10.03x 103 M-1 for 4 h and 12 h incubation (Table 10). The higher binding constant value at 12 h of incubation might be due to very slow conformational changes in the protein that allowed the oxyfluorfen to find a higher affinity binding state than the original one. Consequently, the availability of higher affinity binding state, thus quenches the GPC more efficiently, thereby bringing more noticeable structural change. The inference obtained from Stern-Volmer plot and decreased fluorescence intensity indicates changes in the micro- environment around fluorophores at higher concentration and longer incubation, thus confirming the structural change within GPC.

Synchronous fluorescence spectroscopy was also performed to examine the alteration in the microenvironment around the tyrosine and tryptophan residues of GPC. GPC upon titrating with oxyfluorfen (0-100 µM) in 50 mM sodium phosphate buffer (pH 7.5) showed quenching of synchronous fluorescence in case of tyrosine as well as tryptophan residues (Fig. 57 A and B). However, only tyrosine synchronous fluorescence spectra showed a redshift which signifies the changes in the microenvironment around tyrosine residue. The red shift further suggests that the addition of oxyfluorfen drives the tyrosine residuesMaulana of GPC Azad from Library, the non -Aligarhpolar micro Muslim-environment University to a more polar environment thereby reducing the hydrophobicity (Naveenraj et al., 2012; Varlan and Hillebrand, 2010; Zhang et al., 2014). The similar conclusion has been drawn by the previous findings which signify the alteration of micro- environment around tyrosine residues upon binding of nordihydro-guaiaretic acid to human serum albumin and bovine serum albumin (Nusrat et al., 2016). Isothermal titration calorimetry study showed that the binding of oxyfluorfen with GPC was an exothermic process (Fig. 58). The similar thermodynamic reaction has been previously reported in case of interaction of yellow mustard 214 | P a g e

Chapter -3 (B) Results and discussion phytocystatin with oxadiargyl (herbicide) (Ahmed et al., 2017). The results obtained by above biophysical experiments conclude that oxyfluorfen upon binding to GPC brings conformational change, which ultimately leads to the transition of GPC to non-native form. Circular dichroism analysis was performed to study the effect of oxyfluorfen on the secondary structure of GPC. Far-UV circular dichroism spectra of GPC showed two negative peaks at 208 nm and 222 nm, which are characteristic peaks for alpha-helix. It is evident from figure 59 that the negative ellipticity of native GPC reduces with the increasing concentration of oxyfluorfen. The reduction in the negative ellipticity of native GPC suggests the alteration in the secondary structure of GPC and reduction in the alpha-helical content of GPC upon binding of oxyfluorfen. The reduction in the alpha-helical content could be due to the destabilization of the polypeptide backbone. Furthermore, it can also be inferred that the various interactive forces viz. hydrogen bonding, van der Waals forces and hydrophobic interactions involved in maintaining the secondary of protein get disrupted upon binding of oxyfluorfen to garlic phytocystatin. Therefore, it can be concluded that oxyfluorfen disrupts the secondary structure as well as affects the physiological functions of GPC. It has been previously reported that the alpha-helical content of chickpea phytocystatin, almond phytocystatin, and yellow mustard phytocystatin reduces in the presence of glyphosate (herbicide), pendimethalin (herbicide), and oxadiargyl (herbicide) (Ahmed et al., 2017; Bhat et al., 2016; Siddiqui et al., 2017). The study confirmed the functional and structural alteration within GPC Maulana Azad Library, Aligarh Muslim University upon interaction with oxyfluorfen.

215 | P a g e

Chapter – 4 Aggregation study of garlic phytocystatin assisted by Maulanatrifluoroethanol Azad Library, Aligarh Muslim University (TFE)

Chapter -4 Results and discussion

RESULTS

AGGREGATION OF GARLIC PHYTOCYSTATIN (GPC) ASSISTED BY TRIFLUOROETHANOL (TFE)

1. FUNCTIONAL STUDY

1.1 Effect of TFE on the cysteine proteinase inhibitory activity of GPC

Cysteine proteinase inhibitory assay of GPC was performed to analyze the effects of TFE on the functionality of the inhibitor. Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90%) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 hours. It is evident from figure 60 that native GPC in the absence of TFE showed maximum inhibitory activity and was taken as a reference (100%). A sharp decrease in inhibitory activity was observed during the treatment of GPC with increasing concentration of TFE. The GPC showed 37.31% to 94.27% decrement in the inhibitory activity of GPC using 10% to 50 % TFE. The decreased inhibitory activity might be due to the structural alteration, which reduces the probability of the inhibitor to bind on the surface of the enzyme. Hence, the increment in the TFE concentration disrupts the activity of the inhibitor, thereby confirming the negative impact on the functional status of the GPC. Maulana Azad Library, Aligarh Muslim University 2. STRUCTURAL STUDY

2.1 Effect of TFE on the intrinsic fluorescence of GPC

Aromatic amino acid residues absorb at a wavelength of 280 nm and emit in the range of 300 to 400 nm. Conformational and structural alterations in a protein can be easily monitored by probing the aromatic residues as a reporter since their emission spectra depends on their position in the protein. The intrinsic fluorescence of GPC (4 µM) was recorded after incubation with

216 | P a g e

Chapter -4 Results and discussion

120

100

ctivity a

80 ry

60

inhibito l l

dua 40 Resi

% 20

0 0 10 20 30 40 50 60 70 80 90 %TFE

Figure 60. Cysteine proteinase inhibitory assay of GPC in the presence of TFE

Native GPCMaulana (4 µM) was Azad incubated Library, with Aligarh increasing Muslim concentration University of TFE (0-90%) at 37°C for 4 h in 50 mM sodium phosphate buffer (pH 7.5). Inhibitory activity assay was performed using casein as a substrate by the method of Kunitz (1947) as described in methods. Each value represents the average of three independent experiments performed in duplicates.

217 | P a g e

Chapter -4 Results and discussion increasing concentration of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 61 (A) shows the fluorescence spectra of GPC alone and GPC incubated with TFE (0-90% v/v). Figure 61 (B) shows relative fluorescence spectra of GPC against increasing concentration of TFE (0-90%). GPC showed emission maxima at 337 nm, and the fluorescence intensity initially increases and then decreases with increasing TFE concentration. An insignificant increase in the fluorescence intensity was observed at 10% TFE concentration, and no change was observed in the emission maxima. A gradual increase in the fluorescence intensity was observed up to 20% TFE. The maximum fluorescence intensity was observed at 30% TFE, which is two times of the native GPC along with the redshift. The enhanced fluorescence at 30% TFE along with redshift could be attributed to the exposure of aromatic residues, which suggests partial unfolding and loose packaging of GPC (Shamsi et al., 2016). The partially unfolded state with loose packaging can be regarded as a molten globule state. A sharp decline was observed in fluorescence intensity from 30% TFE to 60% TFE and gradual decrease beyond 60% TFE. The reduction in the fluorescence intensity along with redshift could be attributed to the aggregation of the protein as the aromatic residues of the protein may get exposed to more polar solvent (Rehman et al., 2015).

3. ANS FLUORESCENCE STUDIES OF GPC

3.1 EffectMaulana of Azad TFE Library, on the ANSAligarh fluorescence Muslim University of GPC in the presence of TFE

ANS dye does not fluoresce, but it gives fluorescence upon binding to the hydrophobic patches of the protein. Hence, it can be used as an essential tool to decipher the intermediates formed during the transition stages of proteins. In figure 62 (A), GPC showed insignificant fluorescence due to the less availability of hydrophobic patches at the surface of the GPC. However, GPC treated with 10% TFE showed marginal increase in the ANS fluorescence.

218 | P a g e

Chapter -4 Results and discussion

160 GPC alone 10% TFE 20% TFE 30% TFE 40% TFE 50% TFE 60% TFE 70% TFE 80% TFE

140

120

(a.u.)

100 ensity 80

60 escence int escence

40 Fluor

20

0 300 320 340 360 380 400 Wavelength (nm)

Figure 61 (A). Intrinsic fluorescence emission spectra of GPC in the absence and presence of TFE Maulana Azad Library, Aligarh Muslim University Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90%) at 37°C for 4 h in 50 mM sodium phosphate buffer (pH 7.5). Fluorescence spectra were measured at an excitation wavelength of 280 nm, and the emission range was fixed at 300-400 nm.

219 | P a g e

Chapter -4 Results and discussion

160

140 (a.u.) 120

intensity intensity 100

80 escence escence

60

tive Fluor tive 40

Rela 20

0 0 10 20 30 40 50 60 70 80 90 % TFE (v/v)

Figure 61 (B). Relative fluorescence plot of GPC in the presence of TFE

RelativeMaulana fluorescence Azad Library,of GPC Aligarh (4 µM) Muslim incubated University with increasing concentration of TFE (0-90%) for 4 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The samples were excited at 280 nm, and the emission spectra were recorded in the range of 300 – 400 nm. The values are plotted for fluorescence intensity against the different concentration of TFE.

220 | P a g e

Chapter -4 Results and discussion

At 20% TFE, the increase in the fluorescence intensity was about 4-fold as compared to untreated GPC, and the maximum increase was observed at 30% TFE, which is 8-fold. The partially unfolded state at 20% TFE suggests the initialization of formation of molten globule that is a step in the formation of a molten globule. The enhancement of fluorescence at 30% TFE could be attributed to the maximum exposure of the hydrophobic patches upon unfolding of GPC and can be regarded as the molten globule state of GPC (Bhat and Bano, 2014). Further increment in TFE concentration showed a sharp decline in the fluorescence intensity suggesting burial of earlier exposed hydrophobic patches due to cross-linking of protein molecules (Amani and Naeem, 2013). The cross-linking of protein molecules reduces the accessibility of ANS dye to bind hydrophobic patches, thereby reducing fluorescence intensity. A sharp decline in fluorescence intensity was observed at 40% TFE, and stabilization effect was observed beyond 40% TFE. The gradual change in fluorescence intensity was observed at and beyond 40% TFE, suggesting stabilizing effects of TFE at higher concentration. It was found that TFE at higher concentration strengthens the intra-protein electrostatic interaction and hydrogen bonds, bringing about highly rigid protein structure (Bhat and Bano, 2014). Figure 62 (B) shows relative ANS fluorescence emission plot of GPC incubated with increasing concentration of TFE (0-90% v/v) at 37ºC in 50 mM sodium phosphate buffer for 4 h. Maximum ANS fluorescence intensity was plotted against respective TFE concentration.

4. SECONDARYMaulana Azad STRUCTURE Library, Aligarh ANALYSIS Muslim OFUniversity GPC IN THE PRESENCE OF TFE

4.1 Circular dichroism measurements of GPC in the presence of TFE

Circular dichroism (CD) spectroscopy was performed to analyze the secondary structural changes within GPC upon incubation with TFE. Far UV circular dichroism spectra were recorded in the range of 195 nm – 255 nm.

221 | P a g e

Chapter -4 Results and discussion

350 ANS alone GPC 10 % TFE 20% TFE 30% TFE 40% TFE 50% TFE 60% TFE 70% TFE 80% TFE 90% TFE 300

250

cence 200

es

r

uo l

150

S F S

N A 100

50

0 400 450 500 550 600 Wavelength (nm)

Figure 62 (A). ANS fluorescence emission spectra of GPC in the presence of TFE

NativeMaulana GPC (4 µM)Azad was Library, incubated Aligarh with increasing Muslim Universityconcentration of TFE (0-90%) at 37°C for 4 h in 50 mM sodium phosphate buffer (pH 7.5). ANS fluorescence spectra were measured at an excitation wavelength of 380 nm, and the emission range was fixed at 400 - 600 nm. ANS alone represents the spectrum of ANS molecules in buffer only. GPC in the absence of TFE has been taken as a reference.

222 | P a g e

Chapter -4 Results and discussion

350

300

250 escence escence

200 Fluor

150 ANS ANS

tive tive 100 Rela 50

0 0 10 20 30 40 50 60 70 80 90 % TFE

Figure 62 (B). Relative ANS fluorescence plot of GPC in the presence of TFE

Relative fluorescenceMaulana Azadin which Library, 4 µM Aligarh of GPC Muslim was Universityincubated with increasing concentration of TFE (0-90%) for 4 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The samples were excited at 380 nm, and the emission spectra were recorded in the range of 400 – 600 nm. The values are plotted for fluorescence intensity against different concentrations of TFE.

223 | P a g e

Chapter -4 Results and discussion

Figure 63 shows far UV CD spectra of GPC (4 µM) incubated with increasing concentration of TFE (0-90%) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. It can be seen from figure 63 that native GPC showed two negative minima at 208 nm and 222 nm, which are the characteristic peak of the alpha helix. The decrease in the negative ellipticity was observed up to 20% TFE without any change in peak. GPC treated with 30% TFE showed an increase in negative ellipticity, thereby restoring the native-like structure. Hence, this stage can be regarded as a molten globule state. Increasing TFE concentration beyond 30% induces a noticeable structural change and is accompanied by a peak shift. GPC incubated with 60-90% TFE showed a change in peak from 222 nm to 218 nm along with a gradual increase in negative ellipticity. A gradual rise in negative ellipticity suggests the increase in sheet content in GPC (Fig. 63), thus signifying the presence of a more aggregated structure. GPC upon incubation with 90% TFE showed a negative peak at 218 nm along with maximum negative ellipticity. Hence, GPC incubated with increasing concentration of TFE induces structural changes which are accompanied by a shift in characteristic peak along with a change in secondary structure.

5. AGGREGATION SPECIFIC ASSAYS OF GPC IN THE PRESENCE OF TFE

5.1 Turbidity assay of GPC in the presence of TFE

TurbidityMaulana assay wasAzad performed Library, to Aligarhanalyze the Muslim formation University of aggregated GPC. Native GPC (4 µM) was incubated with increasing concentration of TFE (0- 90%) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 64 shows turbidity measurements of GPC in the presence of increasing concentration of TFE. The native GPC showed minimum absorbance at 350 nm, thereby confirming the absence of any aggregated structure. However, GPC incubated with increasing concentration of TFE showed gradual increment in absorbance, thus suggesting the formation of GPC aggregates. It is evident from figure 64 that maximum absorbance was observed at 90% TFE.

224 | P a g e

Chapter -4 Results and discussion

GPC alone 10% TFE 20% TFE 30% TFE 40% TFE 50% TFE 30 60% TFE 70% TFE 80% TFE 90% TFE 20

10

0

deg -10 m -20

-30

-40

-50

-60 195 205 215 225 235 245 255 Wavelength (nm)

Figure 63. Circular dichroism spectra of GPC in the absence and presence of TFE Maulana Azad Library, Aligarh Muslim University Far-UV circular dichroism spectra of GPC (4 µM) was taken in the presence of an increasing concentration of TFE (0-90%) for 4 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The spectra were recorded in the range of 195-255 nm, and each spectrum is an average of three scans. GPC alone represents 0% TFE and taken as reference for the experiment.

225 | P a g e

Chapter -4 Results and discussion

1.4

1.2

nm 1

0 5

0.8 nce @3 nce

0.6 rba

Abso 0.4

0.2

0 0 10 20 30 40 50 60 70 80 90 % TFE

Figure 64. Turbidity assay of GPC in the presence of TFE

Native GPC (4 µM) was incubated with increasing concentration of TFE Maulana Azad Library, Aligarh Muslim University (0-90%) at 37°C for 4 h in 50 mM sodium phosphate buffer (pH 7.5). Absorbance was recorded at 350 nm on a UV-1800 Shimadzu spectrophotometer. 0% TFE represent GPC in the absence of TFE, and each bar represents mean±SEM of three experiments.

226 | P a g e

Chapter -4 Results and discussion

5.2 Rayleigh scattering assay of GPC in the presence of TFE

Rayleigh scattering assay was performed to monitor the initiation and formation of aggregation. Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90%) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 65 shows Rayleigh scattering measurements of GPC in the presence of an increasing concentration of TFE. The native GPC showed minimum fluorescence; however, the fluorescence intensity increases as the concentration of TFE increases. The significant increase in fluorescence intensity confirms the formation of the aggregated structure of GPC.

5.3 Thioflavin-T fluorescence measurements of GPC in the presence of TFE

ThT is a benzothiazole dye which primarily binds to the intermolecular beta- sheets structure of aggregated protein hence used to confirm the amyloid structure of the protein. The fluorescence is due to the immobilization of ThT molecule to the binding sites which are present on the fibrils. The ThT dye conformers get bound to the sites and give enhanced fluorescence. Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90%) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 66 shows ThT fluorescence measurements of GPC in the presence of increasing concentration of TFE. It is evident from figure 66 that the ThT fluorescence of GPC increases upon the increasing concentration of TFE, thereby confirming the transition ofMaulana native GP AzadC towards Library, non- Aligarhnative form. Muslim There University was a gradual increment in ThT fluorescence up to 60% TFE. It was observed that 70% of TFE induces pronounced effects and fluorescence intensity increases up to 12 times, indicating the formation of aggregates fibrils. The ThT fluorescence was maximum at 90% TFE that is 28 times as compared to native GPC. The pronounced enhancement in fluorescence at 90% TFE confirmed the transition of native GPC and presence of an aggregated form of GPC (Naeem et al., 2015).

227 | P a g e

Chapter -4 Results and discussion

800

700

nm

0 600

5

3 at at

500

ty i

400

ntens i

300

cence cence

es r

uo 200

l F

100

0 0 10 20 30 40 50 60 70 80 90

% TFE

Figure 65. Rayleigh scattering measurement of GPC in the presence of TFE Maulana Azad Library, Aligarh Muslim University Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90%) at 37°C for 4 h in 50 mM sodium phosphate buffer (pH 7.5). Fluorescence was recorded at an excitation wavelength of 350, and the emission was recorded in the range of 300 – 400 nm. The fluorescence intensity was plotted against increasing concentration of TFE. 0% TFE represent GPC in the absence of TFE, and each bar represents mean±SEM of three experiments.

228 | P a g e

Chapter -4 Results and discussion

600 GPC alone 10% TFE 500 20% TFE

30% TFE

400 40% TFE

50% TFE escence

300 60% TFE fluor

70% TFE 80% TFE

ThT 200 90% TFE

100

0 460 480 500 520 540 560 580 600 Wavelength (nm)

Figure 66. ThT fluorescence spectra of GPC in the presence of TFE

Native GPCMaulana (4 µM) was Azad incubated Library, with Aligarh increasing Muslim concentration University of TFE (0-90%) at 37°C for 4 h in 50 mM sodium phosphate buffer (pH 7.5). Fluorescence was recorded at an excitation wavelength of 440, and the emission was recorded in the range of 460 – 600 nm. GPC alone represents 0% TFE and taken as reference for the experiment.

229 | P a g e

Chapter -4 Results and discussion

The sharp increase in fluorescence from 60% TFE to 90% TFE suggest the progressive increase of aggregated structure that is the formation of a more beta-sheet structure on subsequent addition of TFE.

5.4 Congo red assay of GPC in the presence of TFE

Congo red assay was employed to confirm the aggregation mediated by TFE. Congo red dye is structurally similar to Thioflavin T and shows specificity towards beta-pleated sheet structure. Binding of Congo red to the sheet structure results in increased absorption along with characteristic redshift, which corresponds to the formation of aggregated structure. Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90%) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 67 shows Congo red absorbance measurements of GPC in the presence of increasing concentration of TFE. Figure 67 shows that native GPC upon binding to Congo red exhibited slight absorption at 490 nm. The marginal increase in the absorption was observed at 10% TFE. The increment in absorption was gradual from 20% TFE to 50% TFE. However, a sharp increase was observed above 60% TFE. The sharp increase in absorption is accompanied by redshift, which could be attributed to the lateral attachment of a non-polar molecule of Congo red dye along the length of fibrillar aggregates (Naeem et al., 2015). The gradual increase in absorption suggests the transition of native GPC towards non-native form and the sharp absorption along with the redshift confirmed the presence of aggregated structure at a higher concentration of TFE. 6. SCANNINGMaulana Azad ELECTRON Library, Aligarh MICROSCOPY Muslim University OF GPC IN THE PRESENCE OF TFE

Scanning electron microscopy was done to analyze the morphology of the aggregates of GPC in the presence of TFE. Figure 68 (A) shows the morphology of native GPC (4 µM), which is devoid of any aggregated structure. However, figure 68 (B) shows the presence of a sheath-like structure at 60% TFE, which looks similar to the aggregates. The sheath-like structures are the protofibrils formed in the initial stages of amyloid aggregates.

230 | P a g e

Chapter -4 Results and discussion

4 GPC alone 10% TFE 20% TFE 30% TFE 40% TFE 50% TFE 60% TFE 70% TFE

80% TFE 90% TFE 90% TFE

) 3

.u.

a nce ( nce

rba 2 Abso

1

0 400 450 500 550 600 Wavelength (nm)

Figure 67. Congo red absorbance spectra of GPC in the presence of TFE Maulana Azad Library, Aligarh Muslim University Native GPC (4 µM) was incubated with increasing concentration of TFE (0-90%) at 37°C for 4 h in 50 mM sodium phosphate buffer (pH 7.5). Absorbance spectra were recorded in the range of 400 – 600 nm. GPC alone represents 0% TFE and taken as reference for the experiment.

231 | P a g e

Chapter -4 Results and discussion

(A) Maulana Azad Library, Aligarh Muslim University Figure 68. Scanning electron microscopy of GPC in the presence of TFE

Scanning electron microscopy images of GPC incubated with TFE. (A) Native GPC as a negative control showing no aggregation. (B) GPC incubated with 60% TFE for 4 h at 37ºC in 50 mM sodium phosphate buffer (pH 7.5).

232 | P a g e

Chapter -4 Results and discussion

Maulana Azad Library,(B) Aligarh Muslim University

233 | P a g e

Discussion

Maulana Azad Library, Aligarh Muslim University

Chapter -4 Results and discussion

DISCUSSION

Protein aggregation along with amyloid fibrillation is a complex mechanism and yet to be revealed. It was found that protein aggregates and amyloid fibrils share a common core structure which does not depend on the sequence of amino acids (Morshedi et al., 2010). Protein aggregation involves the generation of the partially unfolded state which exposes the hydrophobic residues. Exposure of hydrophobic residues lead to strong intermolecular hydrophobic interaction hence facilitates the formation of cross-linked structures. An organic solvent like TFE can induce protein aggregation thus used to decipher the in vitro protein aggregation mechanism. TFE weakens the hydrophobic forces which are required to maintain the tertiary structure of a protein, thereby disrupting the native structure of a protein (Naeem et al., 2016). In the present study, TFE is used to induce aggregation state in garlic phytocystatin (GPC) to study the structural and conformational changes. Several biophysical techniques are employed to investigate the structural and functional changes within GPC upon treatment with TFE.

Purified garlic phytocystatin (4 µM) was incubated with increasing concentrations of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h to evaluate the effect of TFE on the inhibitory activity of GPC. Cysteine proteinase inhibitory assay was followed to analyze the functional status of the garlic phytocystatin (Fig. 60). It was observed that the inhibitory activityMaulana of GPC Azad reduces Library, at the subsequent Aligarh Muslim addition Universityof TFE. GPC showed 37.31% to 98.28% reduction in the inhibitory activity upon increasing concentration of TFE from 10% to 70%. The decreased inhibitory activity could be attributed to the conformational changes brought about by TFE upon interaction with the inhibitor, thereby weakening the hydrophobic or hydrogen bonding within the inhibitor. In a previous report, chickpea phytocystatin also showed a reduction in the inhibitory activity upon incubation with increasing concentration of TFE (Bhat and Bano, 2014). Intrinsic fluorescence spectroscopy was carried out to analyze the changes in the environment of 234 | P a g e

Chapter -4 Results and discussion aromatic residues in the presence of TFE. The fluorescence spectra of GPC were recorded in the presence of an increasing concentration of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) after incubation for 4 h (Fig. 61 A). Native GPC was excited at 280 nm to monitor the changes around aromatic residues. It was observed that TFE has concentration- dependent effects on the fluorescence of GPC (Fig. 61 B). It is evident from results that GPC showed maximum fluorescence intensity at 30% TFE which can be attributed to the partial unfolding of GPC. The partially unfolded the state of GPC is regarded as molten globule state. The partial unfolding of GPC facilitates the interaction of residues to form a compact and aggregated form of protein. The decrease in fluorescence intensity has been previously reported when cellulase was treated with TFE and buffalo kidney cystatin was incubated with a high concentration of glyoxal (Iram and Naeem, 2012; Shamsi et al., 2016). Hence, increasing concentration of TFE induces structural changes within GPC, thereby showing the transition of native GPC to non-native GPC.

ANS fluorescence was employed to investigate the presence of molten globule structure during the transition of native GPC to the aggregated structure. The ANS fluorescence spectra of GPC (4 µM) were recorded in the presence of an increasing concentration of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) after incubation for 4 h (Fig. 62 A). It was observed that ANS does not show any fluorescence which may be due to the absence of hydrophobic patches on the surface, however on increasing the TFE concentrationMaulana ANS fluorescence Azad Library, increases Aligarh which Muslimconfirm edUniversity the structural transition of native GPC and exposure of hydrophobic residues at the surface of GPC. The maximum ANS fluorescence was noted at 30% TFE, suggesting the presence of molten globule at this stage (Fig. 62 B). Further increase in TFE concentration reduces the ANS fluorescence intensity, which could be attributed to the interlocking of exposed residues or adherence of partially unfolded state and does not allow ANS dye to bound and show fluorescence. The decrease in ANS fluorescence suggests the formation of an aggregated state of GPC upon an increase in TFE concentration. A similar inference has 235 | P a g e

Chapter -4 Results and discussion been reported when cellulase was incubated with TFE and monitored for ANS fluorescence (Iram and Naeem, 2012).

Circular dichroism was also done to analyze the changes in the secondary structure of GPC. The CD spectra of GPC (4 µM) were recorded in the presence of an increasing concentration of TFE (0-90% v/v) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) after incubation for 4 h (Fig. 63). It was observed that native GPC showed two negative peaks at 208 and 222 nm, which are characteristic peaks for the alpha helix. It can be seen in figure 63 that negative ellipticity reduces up to 20% TFE treatment. However, at 30% TFE, GPC showed increased negative ellipticity suggesting the presence of native-like structure at this concentration. Therefore, this stage can be regarded as a molten globule state during the transition of GPC upon treatment with TFE. Further increase in TFE concentration showed a decline in negative peak, which may be due to the conformational changes within GPC upon interaction with TFE. It was observed that GPC in the presence of 60% TFE showed a shift in the negative peak towards 218 nm and did not exhibit the characteristic peak at 208 or 222 nm. The negative peak at 218 nm corresponds to the beta- sheet structure of the protein, thereby confirming the transition of GPC towards non-native form. It is also evident from figure 63 that there is a continuous increase in negative maxima at 218 nm, which suggest a gradual increase in beta-sheet content at higher TFE concentration. It can be concluded that the presence of beta-sheet structure at a higher level of TFE could have triggered the formationMaulana of theAzad aggregated Library, structure Aligarh of MuslimGPC. It has University been reported earlier that caprine brain cystatin incubated with a high concentration of methylglyoxal showed a shift towards 218 nm thereby confirming the transition of the alpha helix to β-sheet structure (Bhat et al., 2015).

Turbidity assay and Rayleigh scattering assay were performed to analyze the formation of aggregates. The assays were performed after incubation of GPC (4 µM) with increasing concentration of TFE (0-90%) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h (Fig. 64 & 65). Both

236 | P a g e

Chapter -4 Results and discussion the analyses showed that the higher concentration of TFE induces aggregation within GPC. ThT and Congo red assays were also performed to confirm the presence of aggregates. ThT assay showed increased fluorescence intensity at 480 nm as compared to native GPC hence, confirmed the gradual formation of aggregates upon the increasing concentration of TFE. ThT fluorescence was observed 28 times higher than native GPC at 90% TFE (Fig. 66). The high ThT fluorescence confirmed the presence of aggregates. Congo red assay showed an increase in absorbance along with peak shift at higher concentration of TFE, which is indicative of the formation of aggregates (Fig. 67). Similar inferences have been reported for the chickpea cystatin incubated with TFE (Bhat and Bano, 2014).

In a way to visualize high-resolution images of aggregates scanning electron microscopy was done. SEM analysis revealed the presence of dense sheath-like structure at 60% TFE that can be regarded as the early stage of aggregation (Fig. 68 A & B). The sheath-like structure looks similar to the protofibrils formed in the initial stages of amyloid aggregates. Hydrophobic interactions are involved in stabilization of protofibrils and play an essential role. Further, increase in TFE concentration can induce significant structural change resulting in the emergence of massive protein aggregates. Iram and Naeem had shown similar kind of morphological changes and the formation of fibrillar aggregates upon treatment of hemoglobin with TFE (Iram and Naeem, 2013). A similar result has been reported in case of formation of human serum albumin aggregatesMaulana in the Azad presence Library, of acetonitrile Aligarh (NaeemMuslim and University Amani, 2013). Hence, the present study confirmed the functional and structural alteration in the native garlic phytocystatin in the presence of trifluoroethanol.

237 | P a g e

Chapter – 5

Effect of heavy metals (Zn+2 & Cd+2) on structure and function of Maulana Azad Library, Aligarh Muslim University garlic phytocystatin

Chapter -5 Results and discussion

RESULTS

EFFECT OF HEAVY METALS (Zn+2 AND Cd+2) ON FUNCTION AND STRUCTURE OF GARLIC PHYTOCYSTATIN (GPC)

1. FUNCTIONAL STUDY

1.1 Effect of heavy metals (Zn+2 and Cd+2) on the cysteine proteinase inhibitory activity of GPC

The cysteine proteinase inhibitory assay was done to analyze the functional activity of GPC in the absence and presence of zinc and cadmium. Native GPC (4 µM) was incubated with different concentrations of Zn+2 and Cd+2 (20 µM to 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 69 shows the cysteine proteinase inhibitory assay of GPC in the presence of zinc and cadmium. It is evident from the fig. 69 that native GPC (control) shows maximum inhibitory activity and taken as a reference. The result showed that the inhibitory activity of GPC gradually decreases with increasing concentration of zinc and cadmium. It was observed that an increase in the concentration of zinc and cadmium is directly related to the reduction in the cysteine proteinase inhibitory activity. The decrease in cysteine proteinase inhibitory activity at a higher concentration of zinc and cadmium suggests a concentration-dependent effect. Moreover, it was also observed that cadmium reduces cysteine proteinase inhibitory activity of GPC more profoundly Maulana Azad Library, Aligarh Muslim University compared to zinc.

2. STRUCTURAL STUDY

2.1 Ultraviolet absorption study of GPC in the presence of heavy metals (Zn+2 and Cd+2)

The UV-vis absorption spectroscopy was performed to monitor the conformational changes and complex formation of GPC upon treatment with

238 | P a g e

Chapter -5 Results and discussion

120 Zinc Cadmium

100

ctivity a

80

60 l inhibitory l

40

Residua

% 20

0 GPC 20 µM 40 µM 60 µM 80 µM 100 µM alone

Figure 69. Cysteine proteinase inhibitory assay of GPC in the presence of heavy metals (Zn+2 and Cd+2)

Native GPC (4 µM) was incubated with 20 µM, 40 µM, 60 µM, 80 µM and 100 µMMaulana zinc and Azad cadmium Library, at 37Aligarh°C for Muslim 4 h in 5University0 mM sodium phosphate buffer (pH 7.5). Control refers to GPC in the absence of any metal. Values expressed as mean ± S.E.M. of three independent experiments. *P < 0.05 with respect to GPC alone and #P < 0.05 with respect to GPC alone.

239 | P a g e

Chapter -5 Results and discussion metals (Chi and Liu, 2011; Guo et al., 2009; Busenlehner et al., 2001; Valeur and Berberan-Santos, 2012). The UV spectroscopy provides an insight into the conformational alteration within biomolecules upon binding of a small molecule or ligand (Chi and Liu, 2011; Busenlehner et al., 2001). The protein solution exhibits an absorption peak at 280 nm which is caused mainly due to the presence of aromatic amino acid residues such as Tyr, Trp and Phe residues (Herman S. Mansur et al., 2000; Liu et al., 2013). Native GPC (4 µM) was incubated with different concentration of Zn+2 and Cd+2 (20 µM to 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 70 (A) and (B) shows the UV absorption spectra of native GPC and GPC treated with zinc and cadmium, respectively. It was observed from fig. 70 (A) that the native GPC shows absorption maxima at 280 nm, which is a characteristic peak for aromatic amino acid residues. On treatment with zinc and cadmium, the absorption intensity of GPC declines but no change was observed in the absorption maxima. It can be inferred that zinc and cadmium form complex upon interaction with GPC and might alter the conformation of native GPC.

2.2 Intrinsic fluorescence study of GPC in the presence of heavy metals (Zn+2 and Cd+2)

The fluorescence spectroscopy was employed to study the interaction between GPC and metals. It can also be used to investigate the mode of fluorescence quenching, binding constant, and the number of binding sites (Yue et al. 2018). Maulana Azad Library, Aligarh Muslim University The protein molecules have three major fluorophores viz. phenylalanine, tyrosine, and tryptophan, which contributes to the fluorescence of protein molecules (Papadopoulou et al., 2004). Phenylalanine has low quantum yield, and tyrosine gets totally quenched, therefore tryptophan imparts maximum fluorescence (Sułkowska, 2002). The protein molecules show cumulative fluorescence when excited at 280 nm. Therefore, GPC was excited at 280 nm in order to study the cumulative fluorescence of all the three major fluorophores present within GPC.

240 | P a g e

Chapter -5 Results and discussion

0.3 GPC alone 20 µM zinc 40 µM zinc 60 µM zinc 80 µM zinc 100 µM zinc

0.25

)

.u. 0.2

(a nce nce

rba 0.15

o

bs A 0.1

0.05

0 240 250 260 270 280 290 300

Wavelength (nm)

Figure 70 (A). UV-absorption spectra of GPC treated with zinc

Native GPCMaulana (4 µM) was Azad incubated Library, with Aligarh 20 µM, Muslim 40 µM, University60 µM, 80 µM, and 100 µM zinc at 37°C for 4 h in 50 mM sodium phosphate buffer (pH 7.5). Absorption spectra were recorded in the range of 240-300 nm on a UV-1800 Shimadzu spectrophotometer.

241 | P a g e

Chapter -5 Results and discussion

0.4 GPC alone 20 µM cadmium 40 µM cadmium 60 µM cadmium 0.35 80 µM cadmium 100 µM cadmium

0.3

)

.u. 0.25 (a

nce nce 0.2

rba o

bs 0.15 A

0.1

0.05

0 245 255 265 275 285 295 Wavelength (nm)

Figure 70 (B). UV-absorption spectra of GPC treated with cadmium Maulana Azad Library, Aligarh Muslim University Native GPC (4 µM) was incubated with 20 µM, 40 µM, 60 µM, 80 µM and 100 µM cadmium at 37°C for 4 h in 50 mM sodium phosphate buffer (pH 7.5). Absorption spectra were recorded in the range of 240-300 nm on a UV-1800 Shimadzu spectrophotometer.

242 | P a g e

Chapter -5 Results and discussion

Native GPC (4 µM) was incubated with different concentration of Zn+2 and Cd+2 (20 µM to 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 71 (A) shows the fluorescence spectra of native GPC and GPC treated with zinc at different concentrations. The GPC exhibited a strong emission peak at 332 nm, which quenches in the presence of zinc. The fluorescence quenching of GPC increases at higher concentration of zinc along with the blue shift in the emission maxima. Figure 71 (B) shows the fluorescence spectra of native GPC treated with different concentrations of cadmium. It was observed that cadmium also quenches the fluorescence of GPC and a slight red-shift was observed in the native GPC when treated with 100 µM cadmium. The observations suggest the formation of GPC-cadmium complex.

2.3 Stern-Volmer quenching analysis of GPC in the presence of heavy metals (Zn+2 and Cd+2)

The mode of fluorescence quenching was analyzed for the complex formation. The mode of fluorescence quenching depends on the basis of interaction or binding of a fluorophore with quencher viz. static quenching, dynamic quenching and both (Papadopoulou et al., 2004; Bi et al., 2011; Silva et al., 2004). Static mode of fluorescence quenching refers to the formation of non- fluorescent ground state fluorophore-quencher complex, while the dynamic quenching refers to the interaction between fluorophore and quencher through Maulana Azad Library, Aligarh Muslim University the collisional method. GPC was incubated with different concentration of Zn+2 and Cd+2 (20 µM to 100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 72 (A) and (B) shows the Stern-Volmer plot for the GPC- zinc and GPC-cadmium complex, respectively. The Stern-Volmer quenching 3 constant (Ksv) obtained for GPC-zinc and GPC-cadmium complex is 2.7 x 10 M-1 and 2.5 x 103 M-1, respectively. The modified Stern-Volmer determines the binding parameters and the number of binding sites.

243 | P a g e

Chapter -5 Results and discussion

10000 GPC alone

9000 20 μM zinc 40 μM zinc 8000 60 μM zinc

(a.u.) 7000 80 μM zinc

6000 100 μM zinc ensity 5000

4000

escence int escence 3000

2000 Fluor

1000

0 300 320 340 360 380 400 Wavelength (nm)

Figure 71 (A). Intrinsic fluorescence spectra of GPC treated with zinc

Native GPC (4 µM) was incubated with 20 µM, 40 µM, 60 µM, 80 µM, and 100Maulana µM zinc Azad at 37 °CLibrary, for 4 h Aligarh in 50 mM Muslim sodium University phosphate buffer (pH 7.5). Fluorescence spectra were measured at an excitation wavelength of 280 nm, and the emission range was fixed at 300-400 nm.

244 | P a g e

Chapter -5 Results and discussion

1200 GPC alone

20 µM cadmium

) 1000 40 µM cadmium (a.u.

60 µM cadmium y

800 80 µM cadmium enist

100 µM cadmium int

e e 600 escenc

400

uor Fl

200

0 300 320 340 360 380 400 Wavelength (nm)

Figure 71 (B). Intrinsic fluorescence spectra of GPC treated with cadmium

Native GPCMaulana (4 µM) was Azad inc ubatedLibrary, with Aligarh 20 µM, Muslim 40 µM, University60 µM, 80 µM and 100 µM cadmium at 37°C for 4 h in 50 mM sodium phosphate buffer (pH 7.5). Fluorescence spectra were measured at an excitation wavelength of 280 nm, and the emission range was fixed at 300-400 nm.

245 | P a g e

Chapter -5 Results and discussion

Figure 72 (C) and (D) shows the modified Stern-Volmer plot for the GPC-zinc and GPC-cadmium complex, respectively. The binding parameters and number of binding sites are listed in Table 12 along with the Stern-Volmer quenching parameters.

3. SYNCHRONOUS FLUORESCENCE STUDY OF GPC IN THE PRESENCE OF HEAVY METALS (Zn+2 and Cd+2)

Synchronous fluorescence spectroscopy is a useful technique to analyze the changes in the microenvironment of biomolecules upon binding of ligands, thereby reflecting the conformational changes in the biomolecules. Hence, the synchronous fluorescence spectroscopy was used to unravel the insights of protein conformation upon binding of ligands. It is possible by scanning the excitation and emission monochromators simultaneously at a fixed difference of wavelength between excitation and emission (Yue et al., 2016a). The detailed information about the changes in the microenvironment around fluorophores can be easily monitored by using synchronous fluorescence spectroscopy. The ligand was added successively at a fixed difference between excitation wavelength and emission wavelength. The difference in the excitation wavelength and emission wavelength is used to study the changes around different fluorophore. Thereby, adjusting Δλ =15 nm reflects the changes around tyrosine residues while Δλ = 60 nm shows the changes around tryptophan residues. GPC was titrated with increasing concentration of Zn+2 Maulana Azad Library, Aligarh Muslim University and Cd+2 (10 - 100 µM) in 50 mM sodium phosphate buffer (pH 7.5).

Figure 73 (A) and (B) shows the synchronous fluorescence spectra of GPC-zinc at Δλ =15 nm and Δλ = 60 nm, respectively. It was observed from fig. 73 (A) and (B) that the fluorescence intensity of GPC reduced gradually upon binding of zinc in both the cases thereby supporting the fluorescence quenching results and confirmed the binding of zinc with GPC (He et al., 2008).

246 | P a g e

Chapter -5 Results and discussion

1.35

1.3

1.25

1.2 Fo/F 1.15

1.1

1.05

1 0 20 40 60 80 100 120 Zinc [µM]

Figure 72 (A). Stern-Volmer analysis of GPC in the presence of zinc

Stern-VolmerMaulana plot of GPCAzad in Library, the presence Aligarh of different Muslim concentrations University of zinc (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) incubated for 4 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The plot was analyzed by the Stern-Volmer quenching equation described in the methodology section.

247 | P a g e

Chapter -5 Results and discussion

1.3

1.25

1.2

Fo/F 1.15

1.1

1.05

1 0 20 40 60 80 100 120 Cadmium [µM]

Figure 72 (B). Stern-Volmer analysis of GPC in the presence of cadmiumMaulana Azad Library, Aligarh Muslim University

Stern-Volmer plot of GPC in the presence of different concentrations of cadmium (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) incubated for 4 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The plot was analyzed by the Stern-Volmer quenching equation described in the methodology section.

248 | P a g e

Chapter -5 Results and discussion

-0.4

-0.5

-0.6

-0.7

/F] F)

- -0.8

Fo [(

-0.9

g lo -1

-1.1

-1.2

-1.3 -4.8 -4.6 -4.4 -4.2 -4 -3.8 log zinc [M]

Figure 72 (C). Modified Stern-Volmer analysis of GPC in the presence ofMaulana zinc Azad Library, Aligarh Muslim University

Modified Stern-Volmer plot of GPC in the presence of different concentrations of zinc (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) incubated for 4 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The plot was analyzed by the modified Stern-Volmer equation described in the methodology section.

249 | P a g e

Chapter -5 Results and discussion

-0.4

-0.6

-0.8

/F]

F) -

Fo -1

[(

g lo

-1.2

-1.4

-1.6 -4.8 -4.6 -4.4 -4.2 -4 -3.8 log cadmium [M]

Figure 72 (D). Modified Stern-Volmer analysis of GPC in the presenceMaulana of cadmium Azad Library, Aligarh Muslim University

Modified Stern-Volmer plot of GPC in the presence of different concentrations of cadmium (20 µM, 40 µM, 60 µM, 80 µM, and 100 µM) incubated for 4 h at 37°C in 50 mM sodium phosphate buffer (pH 7.5). The plot was analyzed by the modified Stern-Volmer equation described in the methodology section.

250 | P a g e

Chapter -5 Results and discussion

Table 12: Stern-Volmer quenching constant (Ksv) and +2 binding constant (Kb) for the interaction of GPC with Zn and Cd+2

Metal-GPC -1 -1 Ksv (M ) n Kb (M ) complex

Zinc-GPC 2.78x103 0.92 1.3 x103

Cadmium-GPC 2.53 x 103 1.2 1.5 x 104

Stern-Vomer quenching constant (Ksv) and binding constant

(Kb) is determined by the Stern-Volmer equation and modified Stern-Volmer equation as described in the methodology section. TheMaulana value ofAzad n represents Library, Aligarh the number Muslim of bindingUniversity sites.

251 | P a g e

Chapter -5 Results and discussion

The fluorescence quenching of GPC by zinc was accompanied by blue-shift in the emission maxima for Δλ = 60 nm, thereby reflecting the alteration in the microenvironment around tryptophan residues. Figure 73 (C) and (D) shows the synchronous fluorescence spectra of GPC-cadmium at Δλ =15 nm and Δλ = 60 nm, respectively. It was observed that the fluorescence intensity of GPC decreases with the successive addition of cadmium (10 µM- 100 µM) along with the red shift in the emission maxima for Δλ = 60 nm. The decrement in the fluorescence intensity and change in emission maxima of GPC could be attributed to the complex formation of GPC-cadmium along with the perturbation in the microenvironment around tryptophan residues. However, no change was observed in the emission maxima of GPC in case of tyrosine residues. The observations confirmed that the binding of zinc and cadmium to GPC altered the microenvironment around aromatic residues and increases the hydrophobicity around fluorophores (Chi et al., 2010)

4. THREE-DIMENSIONAL FLUORESCENCE STUDY OF GPC IN THE PRESENCE OF HEAVY METALS (Zn+2 and Cd+2)

Three-dimensional (3D) fluorescence spectroscopy is used to study the structural and conformational alteration upon binding of small molecules (Zhang et al., 2008a). This technique can be employed to analyze the conformational perturbation in the protein molecule upon binding of ligands by Maulana Azad Library, Aligarh Muslim University changing excitation and emission wavelength simultaneously (Yue et al., 2016b). Native GPC (4 µM) was incubated with of Zn+2 (100 µM) and Cd+2 (100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 74 (A) and (C) shows the 3D the fluorescence spectra in the absence and presence of zinc (100 µM), respectively. Figure 74 (B) and (D) shows the contour map of GPC in the absence and presence of zinc (100 µM).

252 | P a g e

Chapter -5 Results and discussion

9000 GPC alone 10 μM zinc 8000 20 μM zinc

7000 30 μM zinc

6000 40 μM zinc (a.u.) 50 μM zinc 5000

60 μM zinc ensity 4000 70 μM zinc 80 μM zinc 3000 90 μM zinc

escence int escence 2000 100 μM zinc

Fluor 1000

0 255 275 295 315 335 355 Wavelength (nm)

Figure 73 (A). Synchronous fluorescence spectra of GPC in the presence of zinc Maulana Azad Library, Aligarh Muslim University Synchronous fluorescence spectra of GPC for tyrosine residues (Δλ= 15 nm). Native GPC (4 µM) was titrated with increasing concentration of zinc (10 – 100 µM) in 50 mM sodium phosphate buffer (pH 7.5). The synchronous fluorescence for tyrosine residues was recorded at an excitation wavelength of 240 nm, and the emission spectra were recorded in the range of 255 – 400 nm.

253 | P a g e

Chapter -5 Results and discussion

12000 GPC alone 10 μM zinc

10000 20 μM zinc

30 μM zinc

(a.u.)

8000 40 μM zinc

ensity 50 μM zinc 6000 60 μM zinc 70 μM zinc

escence int escence 4000 80 μM zinc

90 μM zinc Fluor 2000 100 μM zinc

0 300 320 340 360 380 400 Wavelength (nm)

Figure 73 (B). Synchronous fluorescence spectra of GPC in the presence of zinc

SynchronousMaulana fluorescence Azad Library, spectra Aligarh of GPC Muslim for tryptophan University residues (Δλ= 60 nm). Native GPC (4 µM) was titrated with increasing concentration of zinc (10 – 100 µM) in 50 mM sodium phosphate buffer (pH 7.5). The synchronous fluorescence for tryptophan residues was recorded at an excitation wavelength of 240 nm, and the emission spectra were recorded in the range of 300 – 400 nm.

254 | P a g e

Chapter -5 Results and discussion

10000 GPC alone

9000 10 µM cadmium

20 µM cadmium

8000 (a.u.)

30 µM cadmium 7000 40 µM cadmium

ensity 6000 50 µM cadmium 5000 60 µM cadmium

4000 70 µM cadmium escence int escence

3000 80 µM cadmium Fluor 2000 90 µM cadmium 100 µM cadmium 1000

0 255 275 295 315 335 355 Wavelength (nm)

Figure 73 (C). Synchronous fluorescence spectra of GPC in the presence of cadmium Maulana Azad Library, Aligarh Muslim University Synchronous fluorescence spectra of GPC for tyrosine residues (Δλ= 15 nm). Native GPC (4 µM) was titrated with increasing concentration of cadmium (10 – 100 µM) in 50 mM sodium phosphate buffer (pH 7.5). The synchronous fluorescence for tyrosine residues was recorded at an excitation wavelength of 240 nm, and the emission spectra were recorded in the range of 255 – 400 nm.

255 | P a g e

Chapter -5 Results and discussion

12000 GPC alone

10 µM cadmium

10000 20 µM cadmium (a.u.)

30 µM cadmium 40 µM cadmium 8000 ensity 50 µM cadmium 60 µM cadmium 6000 70 µM cadmium

escence int escence 80 µM cadmium 4000

90 µM cadmium Fluor 100 µM cadmium 2000

0 300 320 340 360 380 400 Wavelength (nm)

Figure 73 (D). Synchronous fluorescence spectra of GPC in the presence of cadmium

SynchronousMaulana fluorescence Azad Library, spectra Aligarh of GPC Muslim for tryptophan University residues (Δλ= 60 nm). Native GPC (4 µM) was titrated with increasing concentration of cadmium (10 – 100 µM) in 50 mM sodium phosphate buffer (pH 7.5). The synchronous fluorescence for tryptophan residues was recorded at an excitation wavelength of 240 nm, and the emission spectra were recorded in the range of 300 – 400 nm.

256 | P a g e

Chapter -5 Results and discussion

The 3D fluorescence spectra of zinc show three characteristic peaks, and each peak corresponds to different attributes. The three peaks are peak a, peak b and peak c. The peak ‘a’ corresponds to the Rayleigh scattering peak (λex = λem), peak ‘b’ is mainly caused by the tyrosine and tryptophan residues, and peak c is second order scattering peak (λem)= 2(λex) (Hu et al., 2006). The fluorescence intensity of peak ‘a’ increases in the presence of zinc, which suggests the formation of the GPC-zinc complex. Furthermore, it also suggests the increase in the diameter of macromolecule, thereby enhancing the scattering effect (Tian et al., 2010). Figure 74 (C) shows that the fluorescence intensity of peak ‘b’ decreases in the presence of zinc as compared to the peak ‘b’ of native GPC. The decreased fluorescence intensity suggests the behavioral changes in tryptophan residues, thereby confirming the conformational alteration in the microenvironment around tryptophan residues within GPC (Juarez et al., 2009; Tian et al., 2005).

Figure 75 (A) and (C) show the 3D dimensional fluorescence spectra of GPC in the absence and presence of cadmium, respectively. Figure 75 (B) and (D) shows the contour map of GPC in the absence and presence of cadmium, respectively. Figure 75 (A) shows that the fluorescence intensity of peak ‘a’ increases in the case of the cadmium-GPC complex as compared to native GPC, which confirms the binding of cadmium. The increase in the fluorescence intensity may be inferred as the increase in the diameter of the macromolecule and higher scattering effect (Tian et al., 2010). Figure 75 (C) shows that theMaulana fluorescence Azad intensity Library, of peak Aligarh ‘b’ decreases Muslim in University the presence of cadmium as compared to native GPC. This suggests alteration around tryptophan residues upon binding of cadmium, thereby confirming the conformational changes within GPC.

257 | P a g e

Chapter -5 Results and discussion

A B a

b c

C D a

c b

Figure 74. Three-dimensional fluorescence spectra of GPC in the presence of zinc

Three-dimensional fluorescence spectra of GPC (A) and GPC treated Maulana Azad Library, Aligarh Muslim University with zinc (C). Contour map of GPC (B) and GPC treated with zinc (D). Native GPC (4 µM) was incubated with 100 µM zinc at 37°C in 50 mM sodium phosphate buffer for 4 h before obtaining the spectra. The spectra were recorded at λex and λem of 200 - 350 nm and 200 - 600 nm respectively.

258 | P a g e

Chapter -5 Results and discussion

A B a

b c

C D

a

b c

Figure 75. Three-dimensional fluorescence spectra of GPC in the presence of cadmium

Three-dimensional fluorescence spectra of GPC (A) and GPC treated with cadmium (C). Contour map of GPC (B) and GPC treated with cadmium (D). Native GPC (4 µM) was incubated with 100 µM cadmium at Maulana Azad Library, Aligarh Muslim University 37°C in 50 mM sodium phosphate buffer for 4 h before obtaining the spectra.

The spectra were recorded at λex and λem of 200 - 350 nm and 200 - 600 nm respectively.

259 | P a g e

Chapter -5 Results and discussion

5. SECONDARY STRUCTURE ANALYSIS OF GPC IN THE PRESENCE OF HEAVY METALS (Zn+2 and Cd+2)

5.1 CD ANALYSIS

5.1.1 Effect of heavy metals (Zn+2 and Cd+2) on the secondary structure of GPC

Circular dichroism spectroscopy is an important technique to determine the secondary structure of the protein in solution along with with the changes in the secondary structure (Chen et al., 2012). The secondary structure of a protein is comprised of various secondary structural elements such as alpha-helix, beta- sheet, beta-turn, loops, and random coil. The far-UV circular dichroism spectra provide detailed information regarding the secondary structure of the protein, and it also helps to unravel the changes in the secondary structure of protein upon binding of small molecules or ligands. Far-UV circular dichroism spectroscopy to investigate the possible effect of metals on the secondary structure of GPC and the changes in the secondary structure of GPC upon incubation with metals. Native GPC (4 µM) was incubated with Zn+2 (100 µM) and Cd+2 (100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 76 shows the far-UV circular dichroism spectra of GPC and GPC treated with zinc (100 µM). It is evident from the fig. 76 that GPC shows strong negative ellipticity peak at 208 nm (pi→pi) and 222 nm (n→pi), which are theMaulana characteristic Azad peak Library, for alpha Aligarh-helix (Kelly Muslim et al.,University 2005). Thereby, the negative ellipticity at 222 nm confirms the presence of alpha-helical structure within GPC (Siddiqui et al., 2017). The negative ellipticity declines in the presence of zinc (100 µM), which suggests the binding of zinc to GPC and confirms the formation of the GPC-zinc complex. Figure 76 shows the far-UV circular dichroism spectra of GPC incubated with cadmium (100 µM). The negative ellipticity at 222 nm was found to be less for GPC incubated with cadmium as compared to the negative ellipticity in case of GPC alone.

260 | P a g e

Chapter -5 Results and discussion

200 GPC alone

150 100 µM zinc 100 µM cadmium

100

deg 50 m

0

-50

-100 190 210 230 250 Wavelength (nm)

Figure 76. Circular dichroism analysis of GPC in the presence of Zn+2 and Cd+2

Far-UV circularMaulana dichroism Azad spectra Library, of GPCAligarh (4 µM)Muslim incubated University with Zn+2 (100 µM) and Cd+2 (100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. The spectra were recorded in the range of 190-260 nm, and each spectrum is an average of three scans.

261 | P a g e

Chapter -5 Results and discussion

Hence, the difference in the ellipticity suggests the binding of cadmium to GPC and reduction in the alpha-helical content of GPC.

6. AGGREGATION SPECIFIC ASSAYS OF GPC IN THE PRESENCE OF HEAVY METALS (Zn+2 and Cd+2)

Aggregation specific assays were also performed to investigate the presence of any aggregates upon binding of metals (zinc and cadmium) to GPC. The Thioflavin-T and Congo red assay are the specific assays used to confirm the presence of aggregates. ThT dye gives strong fluorescence upon binding to amyloid fibrils, hence confirms the presence of aggregates. Similarly, Congo red dye gives strong absorbance upon binding to beta-pleated sheet.

6.1 Thioflavin-T fluorescence of GPC in the presence of Zn+2 and Cd+2

Native GPC (4 µM) was incubated with different concentration of Zn+2 (100 µM) and Cd+2 (100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 77 shows ThT fluorescence measurements of GPC in the presence of zinc and cadmium. It is evident from fig. 77 that the ThT fluorescence of GPC increases in the presence of Zn+2 (100 µM) and Cd+2 (100 µM). The pronounced enhancement in ThT fluorescence in the presence of Zn+2 (100 µM) and Cd+2 (100 µM) confirmed the transition of native GPC and presenceMaulana of an aggregated Azad Library, form of GPCAligarh (Naeem Muslim et al., 2015) University. 6.2 Congo red measurements of GPC in the presence of Zn+2 and Cd+2

Native GPC (4 µM) was incubated with Zn+2 (100 µM) and Cd+2 (100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Figure 78 shows Congo red absorbance measurements of GPC in the presence of zinc and cadmium.

262 | P a g e

Chapter -5 Results and discussion

250 GPC alone

100 µM zinc

200 100 µM cadmium

escence escence 150

Fluor

ThT 100

50

0 450 470 490 510 530 550 Wavelength (nm)

Figure 77. ThT fluorescence spectra of GPC in the presence of Zn+2 and Cd+2

Native GPCMaulana (4 µM) wasAzad incubated Library, with Aligarh zinc Muslim (100 µM) University and cadmium (100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Fluorescence was recorded at an excitation wavelength of 440, and the emission was recorded in the range of 460 – 600 nm. GPC alone represents the fluorescence in the absence of zinc or cadmium and taken as reference for the experiment.

263 | P a g e

Chapter -5 Results and discussion

Figure 78 shows that native GPC upon binding to Congo red exhibited slight absorption at 490 nm. The Congo red absorbance increases in the presence of zinc and cadmium, which suggest the formation of aggregates. The sharp increase in absorption is accompanied by redshift, which could be attributed to the lateral attachment of a non-polar molecule of Congo red dye along the length of fibrillar aggregates (Naeem et al., 2015). The increased Congo red absorbance and red shift suggest the transition of native GPC towards non- native form as well as confirms the formation of aggregates of GPC in the presence of zinc (100 µM) and cadmium (100 µM).

7. SCANNING ELECTRON MICROSCOPY OF GPC IN THE PRESENCE OF HEAVY METALS (Zn+2 and Cd+2)

Scanning electron microscopy is a useful technique to analyze the morphological changes upon binding of ligands. The present study employed scanning electron microscopy to investigate the morphology of GPC upon addition of metals. The GPC was incubated with 100 µM zinc and 100 µM cadmium, respectively for 4 h in 50 mM sodium phosphate buffer, pH 7.5 before obtaining the scanning electron micrographs. Figure 79 shows the scanning electron micrographs of (A) native GPC (4 µM), (B) GPC incubated with zinc (100 µM) and (c) GPC incubated with cadmium (100 µM). Native GPC shows no morphological appearance while the micrograph of GPC incubated with zinc shows the appearance of a non-fibrillar structure or Maulana Azad Library, Aligarh Muslim University amorphous structure. The presence of non-native structure suggests the interaction of GPC with zinc (Fig. 79 B). The micrograph of GPC upon interaction with cadmium showed the formation of fibrillar structure (Fig. 79 C). The above observations conclude that the binding of zinc and cadmium to GPC alters the morphology and conformation of GPC.

264 | P a g e

Chapter -5 Results and discussion

1 GPC alone

0.9 100 µM zinc 0.8 100 µM cadmium

0.7

)

0.6

.u. a

0.5 nce ( nce

0.4 rba

0.3 Abso 0.2

0.1

0 400 450 500 550 600 Wavelength (nm)

Figure 78. Congo red absorbance of GPC in the presence of Zn+2 and Cd+2 Maulana Azad Library, Aligarh Muslim University Native GPC (4 µM) was incubated with zinc (100 µM) and cadmium (100 µM) at 37°C in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. Absorbance spectra were recorded in the range of 400 – 600 nm. GPC alone represents the absorbance in the absence of zinc or cadmium and taken as reference for the experiment.

265 | P a g e

Chapter -5 Results and discussion

(A) Figure 79 (A-C). Scanning electron microscopy of GPC in the absence and presence of Zn+2 and Cd+2 Maulana Azad Library, Aligarh Muslim University Scanning electron microscopy images of GPC incubated with heavy metals. (A) Native GPC (4 µM) as a negative control showing no aggregation. (B) GPC incubated with Zn+2 (100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h. (C) GPC incubated with Cd+2 (100 µM) at 37ºC in 50 mM sodium phosphate buffer (pH 7.5) for 4 h.

266 | P a g e

Chapter -5 Results and discussion

Maulana Azad Library, Aligarh Muslim University (B)

267 | P a g e

Chapter -5 Results and discussion

Maulana Azad Library, Aligarh Muslim University

(C)

268 | P a g e

Bibliography

BIBLIOGRAPHY

Aastrand, P.O., Wallqvist, A., Karlstroem, G., 1994. Molecular Dynamics Simulations of 2 m Aqueous Urea Solutions. J. Phys. Chem. 98, 8224- 8233. Aatif, M., Rahman, S., Bano, B., 2011. Protein unfolding studies of thiol- proteinase inhibitor from goat (Capra hircus) muscle in the presence of urea and GdnHCl as denaturants. Eur. Biophys. J. 40, 611-617.

Abe, K., Emori, Y., Kondo, H., Arai, S., Suzuki, K., 1988. The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli. J. Biol. Chem. 263, 7655-7659. Abe, M., Abe, K., Iwabuchi, K., Domoto, C., Arai, S., 1994. Corn cystatin I expressed in Escherichia coli: Investigation of its inhibitory profile and occurrence in corn kernels. J. Biochem. 116, 488-492. Abraham, Z., Martinez, M., Carbonero, P., Diaz, I., 2006. Structural and functional diversity within the cystatin gene family of Hordeum vulgare. J. Exp. Bot. 57, 4245-4255. Abrahamson, M., Alvarez-Fernandez, M., Nathanson, C.-M., 2003. Cystatins. Biochem. Soc. Symp. 179-199. Abrahamson, M., Barrett, A.J., Salvesen, G., Grubb, A., 1986. Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J. Biol. Chem. 261, 11282-11289. Abrahamson, M., Jonsdottir, S., Olafsson, I., Jensson, O., Grubb, A., 1992. HereditaryMaulana cystatin Azad Library, C amyloid Aligarh angiopathy: Muslim identification University of the disease- causing mutation and specific diagnosis by polymerase chain reaction based analysis. Hum. Genet. 89, 377-380. Abrahamson, M., Ritonja, A., Brown, M.A., Grubb, A., Machleidt, W., Barrett, A.J., 1987. Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin. J. Biol. Chem. 262, 9688-9694. Adam, A., Albert, A., Calay, G., Closset, J., Damas, J., Franchlmont, P., 1985. Human kininogens of low and high molecular mass: Quantification by radloimmunoassay and determination of reference values, CLIN. CHEM.

280 | P a g e

Bibliography

31. 423-426. Adedara, I.A., Vaithinathan, S., Jubendradass, R., Mathur, P.P., Farombi, E.O., 2013. Kolaviron prevents carbendazim-induced steroidogenic dysfunction and apoptosis in testes of rats. Environ. Toxicol. Pharmacol. 35, 444-453. Adetumbi, M., Javor, G.T., Lau, B.H., 1986. Allium sativum (garlic) inhibits lipid synthesis by Candida albicans. Antimicrob. Agents Chemother. 30, 499-501. Adler, B.B., Beuchat, L.R., 2002. Death of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in garlic butter as affected by storage temperature. J. Food Prot. 65, 1976-1980. Aguiar, J.M., Franco, O.L., Rigden, D.J., Bloch, C., Monteiro, A.C.S., Flores, V.M.Q., Jacinto, T., Xavier-Filho, J., Oliveira, A.E.A., Grossi-de-Sá, M.F., Fernandes, K.V.S., 2006. Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests. Proteins 63, 662-670.

- - - , A., Blanco-Labra, A., 2004. A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis suaveolens L.) inhibits proteases from the larger grain borer Prostephanus truncatus (Coleoptera: Bostrichidae). Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 138, 81-89. Ahmed, A., Shamsi, A., Bano, B., 2018. Deciphering the toxic effects of iprodione, a fungicide and malathion, an insecticide on thiol protease inhibitor isolated from yellow Indian mustard seeds. Environ. Toxicol. Pharmacol. 61, 52-60. Ahmed, A., Shamsi, A., Bano, B., 2017. Oxadiargyl induced conformational transition of cystatin isolated from yellow mustard seeds: Biophysical and biochemical approach. Int. J. Biol. Macromol. 98, 802-809. Ahmed, A., Maulana Shamsi, A.,Azad Bano, Library, B., 2016. Aligarh Purification Muslim andUniversity biochemical characterization of phytocystatin from Brassica alba. J. Mol. Recognit. 29, 223-231. Ahn, J.E., Salzman, R.A., Braunagel, S.C., Koiwa, H., Zhu-Salzman, K., 2004. Functional roles of specific bruchid protease isoforms in adaptation to a soybean protease inhibitor. Insect Mol. Biol. 13, 649-657. Aktar, M.W., Sengupta, D., Chowdhury, A., 2009. Impact of pesticides use in agriculture: their benefits and hazards. Interdiscip. Toxicol. 2, 1-12. Alavaikko, M., Aine, R., Rinne, A., Järvinen, M., Blanco, G., Apaja-Sarkkinen,

281 | P a g e

Bibliography

M., Hopsu-Havu, V.K., 1985. Behaviour of dendritic reticulum cells possessing immunoreactive acid cysteine-proteinase inhibitor in human lymphoid secondary follicles and in follicular-centre cell lymphomas. Int. J. Cancer 35, 319-325. Alavanja, M.C.R., Bonner, M.R., 2012. Occupational pesticide exposures and cancer risk: A review. J. Toxicol. Environ. Heal. Part B 15, 238-263. Allaire, M., Chernaia, M.M., Malcolm, B.A., James, M.N.G., 1994. Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin- like serine proteinases. Nature 369, 72-76. Almeida, P.C., Nantes, I.L., Chagas, J.R., Rizzi, C.C.A., Faljoni-Alario, A., Carmona, E., Juliano, L., Nader, H.B., Tersariol, I.L.S., 2001. Cathepsin B activity regulation. J. Biol. Chem. 276, 944-951. Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salvesen, G., Thornberry, N.A., Wong, W.W., Yuan, J., 1996. Human ICE/CED-3 protease nomenclature. Cell 87, 171. Amagase, H., Milner, J.A., 1993. Impact of various sources of garlic and their constituents on 7,12-dimethylbenz[a]anthracene binding to mammary cell DNA. Carcinogenesis 14, 1627-1631. Amani, S., Naeem, A., 2013. Detection and analysis of amorphous aggregates and fibrils of cytochrome c in the presence of phenolic acids. Int. J. Biol. Macromol. 58, 104-112. Anastasi, A., Brown, M.A., Kembhavi, A.A., Nicklin, M.J., Sayers, C.A., Sunter, D.C., Barrett, A.J., 1983. Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochem. J. 211, 129-138. Andrews, P., 1964. Estimation of the molecular weights of proteins by SephadexMaulana gel Azad-filtration. Library, Bioche Aligarhm. J. 91, 222Muslim-233. University Annadana, S., Schipper, B., Beekwilder, J., Outchkourov, N., Udayakumar, M., Jongsma, M.A., 2003. Cloning, functional expression in Pichia pastoris, and purification of potato cystatin and multicystatin. J. Biosci. Bioeng. 95, 118-123. Antalis, T.M., Shea-Donohue, T., Vogel, S.N., Sears, C., Fasano, A., 2007. Mechanisms of disease: protease functions in intestinal mucosal pathobiology. Nat. Clin. Pract. Gastroenterol. Hepatol. 4, 393-402. Arai, S., Matsumoto, I., Emori, Y., Abe, K., 2002. Plant seed cystatins and their

282 | P a g e

Bibliography

target enzymes of endogenous and exogenous origin. J. Agric. Food Chem. 50, 6612-6617. Armen, R.S., DeMarco, M.L., Alonso, D.O.V, Daggett, V., 2004. Pauling and y’ pha-pleated sheet structure may define the prefibrillar amyloidogenic intermediate in amyloid disease. Proc. Natl. Acad. Sci. U. S. A. 101, 11622-11627. Ashraf, R., Aamir, K., Shaikh, A.R., Ahmed, T., 2005. Effects of garlic on dyslipidemia in patients with type 2 diabetes mellitus. J. Ayub Med. Coll. Abbottabad 17, 60-64. Astrand, P.O., Wallqvist, A., Karlstrom, G., Linse, P., 1991. Properties of urea–water solvation calculated from a new ab initio polarizable intermolecular potential. J. Chem. Phys. 95, 8419-8429. Aune, K.C., Tanford, C., 1969. Thermodynamics of the denaturation of lysozyme by guanidine hydrochloride. I. Dependence on pH at 25°. Biochemistry 8, 4579-4585. Balbin, M., Hall, A., Grubb, A., Masonn, R.W., Lopez-Otin, C., Abrahamson, M., 1994. Structural and functional characterization of two allelic variants of human cystatin D sharing a characteristic inhibition spectrum against mammalian cysteine proteinases. J. Biol. Chem. 269, 23156-23162. Banerjee, S.K., Maulik, S.K., 2002. Effect of garlic on cardiovascular disorders: a review. Nutr. J. 1, 1-14. Bangrak, P., Chotigeat, W., 2011. Molecular cloning and biochemical characterization of a novel cystatin from Hevea rubber latex. Plant Physiol. Biochem. 49, 244-250. Barak, P., Helmke, P.A., 1993. The chemistry of zinc, In"Zinc in Soils and Plants". (Ed: Robson, A.D.), Springer; Netherland: Dordrecht, 1-13. Maulana Azad Library, Aligarh Muslim University Barrett, A.J., 1986a. The cystatins: a diverse superfamily of cysteine peptidase inhibitors. Biomed. Biochim. Acta 45, 1363-1374. Barrett, A.J., 1986b. Cysteine proteinase inhibitors of the cystatin superfamily. Proteinase Inhib. 515-569. Barrett, A.J., 1981. Cystatin, the egg white inhibitor of cysteine proteinases. Methods Enzymol. 80, 771-778. Barrett, A.J., Davies, M.E., Grubb, A., 1984. The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors. Biochem. Biophys. Res. Commun. 120, 631-666.

283 | P a g e

Bibliography

Barrett, A.J v E bb 1984 Th p c f h γ-trace (cystatin C) amongst the cysteine proteinase inhibitors. Biochem. Biophys. Res. Commun. 120, 631-636. Barrett, A.J., Rawlings, N.D., O Brien, E.A., 2001. The MEROPS database as a protease information system. J. Struct. Biol. 134, 95-102. Barrett, A.J., Rawlings, N.D., Woessner, J.F., 1998. Handbook of proteolytic enzymes. Academic Press. Barrett, A.J., Salvesen, G., 1986. Proteinase inhibitors. Elsevier; Amsterdam:New York. Bashir, K., Ishimaru, Y., Nishizawa, N.K., 2012. Molecular mechanisms of zinc uptake and translocation in rice. Plant Soil 361, 189-201. Bateman, A., Birney, E., Cerruti, L., Durbin, R., Etwiller, L., Eddy, S.R., Griffiths-Jones, S., Howe, K.L., Marshall, M., Sonnhammer, E.L.L., 2002. The Pfam protein families database. Nucleic Acids Res. 30, 276-80. Bayan, L., Koulivand, P.H., Gorji, A., 2014. Garlic: a review of potential therapeutic effects. Avicenna J. phytomedicine 4, 1-14. Belenghi, B., Acconcia, F., Trovato, M., Perazzolli, M., Bocedi, A., Polticelli, F., Ascenzi, P., Delledonne, M., 2003. AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death. Eur. J. Biochem. 270, 2593-604. Benavides, M.P., Gallego, S.M., Tomaro, M.L., 2005. Cadmium toxicity in plants. Brazilian J. Plant Physiol. 17, 21-34. Benchabane, M., Schluter, U., Vorster, J., Goulet, M.C., Michaud, D., 2010. Plant cystatins. Biochimie 92, 1657-1666. Bennion, B.J., Daggett, V., 2003. The molecular basis for the chemical denaturationMaulana Azadof proteins Library, by urea. Aligarh Proc. Natl. Muslim Acad. UniversitySci. U. S. A. 100, 5142- 5147. Bergmann, E.M., Mosimann, S.C., Chernaia, M.M., Malcolm, B.A., James, M.N., 1997. The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition. J. Virol. 71, 2436-2448. Betti, C., Vanhoutte, I., Coutuer, S., De Rycke, R., Mishev, K., Vuylsteke, M., Aesaert, S., Rombaut, D., Gallardo, R., De Smet, F., Xu, J., Van Lijsebettens, M., Van Breusegem, F., Inzé, D., Rousseau, F., Schymkowitz, J., Russinova, E., 2016. Sequence-specific protein

284 | P a g e

Bibliography

aggregation generates defined protein knockdowns in plants. Plant Physiol. 171, 773-787. Bhat, S.A., Bano, B., 2014. Conformational behaviour and aggregation of chickpea cystatin in trifluoroethanol: Effects of epicatechin and tannic acid. Arch. Biochem. Biophys. 562, 51-61. Bhat, S.A., Bhat, W.F., Bano, B., 2016. Spectroscopic evaluation of the interaction between pesticides and chickpea cystatin: comparative binding and toxicity analyses. Environ. Sci. Process. Impacts 18, 872-881. Bhat, S.A., Bhat, W.F., Shah, A., Khan, M.S., Khan, R.H., Bano, B., 2016. Purification and biochemical characterization of a cystatin-like thiol proteinase inhibitor from Cicer arietinum (Chickpea). J. Chromatogr. Sep. Tech. 08, 1-13. Bhat, W.F., Bhat, S.A., Khaki, P.S.S., Bano, B., 2015. Employing in vitro analysis to test the potency of methylglyoxal in inducing the formation of amyloid-like aggregates of caprine brain cystatin. Amino Acids 47, 135- 146. Bhattacharyya, A., Mazumdar, S., Leighton, S.M., Babu, C.R., 2006. A Kunitz proteinase inhibitor from Archidendron ellipticum seeds: Purification, characterization, and kinetic properties. Phytochemistry 67, 232-241. Bhogale, A., Patel, N., Mariam, J., Dongre, P.M., Miotello, A., Kothari, D.C., 2014. Comprehensive studies on the interaction of copper nanoparticles with bovine serum albumin using various spectroscopies. Colloids Surf. B. Biointerfaces 113, 276-84. Bi, S., Yan, L., Wang, B., Bian, J., Sun, Y., 2011. Spectroscopic and voltammetric characterizations of the interaction of two local anesthetics with bovine serum albumin. J. Lumin. 131, 866-873. Bignold, L.P.,Maulana Coghlan, AzadB.L.D., Library, Jersmann, Aligarh H.P.A., Muslim2006. Cancer University morphology, carcinogenesis and genetic instability: a background. EXS 96, 1-24. Bijina, B., Chellappan, S., Basheer, S.M., Elyas, K.K., Bahkali, A.H., Chandrasekaran, M., 2011. Protease inhibitor from Moringa oleifera leaves: Isolation, purification, and characterization. Process Biochem. 46, 2291-2300. Birchfield, N.B., Casida, J.E., 1997. Protoporphyrinogen oxidase of mouse and maize: Target site selectivity and thiol effects on peroxidizing herbicide action. Pestic. Biochem. Physiol. 57, 36-43.

285 | P a g e

Bibliography

Bita, C.E., Gerats, T., 2013. Plant tolerance to high temperature in a changing environment: scientific fundamentals and production of heat stress-tolerant crops. Front. Plant Sci. 4, 1-18. Bjorck, L., Grubb, A., Kjellen, L., 1990. Cystatin C, a human proteinase inhibitor, blocks replication of herpes simplex virus. J. Virol. 64, 941-943. Bjork, I., Brieditis, I., Abrahamson, M., 1995. Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases. Biochem. J. 306, 513-518. Black, R.A., Kronheim, S.R., Sleath, P.R., 1989. Activation of interleukin-1 beta by a co-induced protease. FEBS Lett. 247, 386-390. Blakley, B.R., Yole, M.J., Brousseau, P., Boermans, H., Fournier, M., 1999. Effect of chlorpyrifos on immune function in rats. Vet. Hum. Toxicol. 41, 140-144. B ck E 2010 c th : The lore and the science. RSC Pub; Cambridge:U.K. Blow, D.M., Birktoft, J.J., Hartley, B.S., 1969. Role of a buried acid group in the mechanism of action of chymotrypsin. Nature 221, 337-340. Bobek, L.A., Levine, M.J., 1992. Cystatins - inhibitors of cysteine proteinases. Crit. Rev. Oral Biol. Med. 3, 307-332. Bode, W., Engh, R., Musil, D., Thiele, U., Huber, R., Karshikov, A., Brzin, J., Kos, J., Turk, V., 1988. The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J. 7, 2593-2599. Bode, W., Huber, R., 2000. Structural basis of the endoproteinase-protein inhibitorMaulana interaction. Azad Library, Biochim. AligarhBiophys. ActaMuslim. 1477, University 241-252. Brannigan, J.A., Dodson, G., Duggleby, H.J., Moody, P.C.E., Smith, J.L., Tomchick, D.R., Murzin, A.G., 1995. A protein catalytic framework with an N-terminal nucleophile is capable of self-activation. Nature 378, 416- 419. Brems, D.N., Brown, P.L., Heckenlaible, L.A., Frank, B.H., 1990. Equilibrium denaturation of insulin and proinsulin. Biochemistry 29, 9289–9293. Brennan, J.D., 1999. Focal point. Appl. Spectrosc. 53, 106-121. Brik, A., Wong, C.-H., 2003. HIV-1 protease: mechanism and drug discovery.

286 | P a g e

Bibliography

Org. Biomol. Chem. 1, 5-14. Brown, W.M., Dziegielewska, K.M., 1997. Friends and relations of the cystatin superfamily-new members and their evolution. Protein Sci. 6, 5-12. Brzin, J., Kopitar, M., Turk, V., Machleidt, W., 1983. Protein inhibitors of cysteine proteinases. I. Isolation and characterization of stefin, a cytosolic protein inhibitor of cysteine proteinases from human polymorphonuclear granulocytes. Hoppe. Seylers. Z. Physiol. Chem. 364, 1475-1480. Brzin, J., Popovic, T., Turk, V., Borchart, U., Machleidt, W., 1984. Human cystatin, a new protein inhibitor of cysteine proteinases. Biochem. Biophys. Res. Commun. 118, 103-109. Brzin, J., Ritonja, A., Popovic, T., Turk, V., 1990. Low molecular mass protein inhibitor of cysteine proteinases from soybean. Biol. Chem. Hoppe. Seyler. 371, 167-170. Buchet, J.P., Lauwerys, R., Roels, H., Bernard, A., Bruaux, P., Claeys, F., Ducoffre, G., de Plaen, P., Staessen, J., Amery, A., 1990. Renal effects of cadmium body burden of the general population. Lancet. 336, 699-702. Buddanavar, A.T., Nandibewoor, S.T., 2017. Multi-spectroscopic characterization of bovine serum albumin upon interaction with atomoxetine. J. Pharm. Anal. 7, 148-155. Burstein, E.A., Vedenkina, N.S., Ivkova, M.N., 1973. Fluorescence and the location of tryptophan residues in protein molecules. Photochem. Photobiol. 18, 263-279. Busenlehner, L.S., Cosper, N.J., Scott, R.A., Rosen, B.P., Wong, M.D., Giedroc, D.P., 2001. Spectroscopic Properties of the Metalloregulatory Cd(II) and Pb(II) Sites of S. aureus pI258 CadC. Biochemistry 40, 4426- 4436. Maulana Azad Library, Aligarh Muslim University Butler, E.A., Flynn, F. V, 1961. The occurrence of post-gamma protein in urine: a new protein abnormality. J. Clin. Pathol. 14, 172-178. Canchi, D.R., Garci, A.E., 2011. Backbone and side-chain contributions in protein denaturation by urea. Biophys. J. 100, 1526-1533. Canesi, L., Ciacci, C., Piccoli, G., Stocchi, V., Viarengo, A., Gallo, G., 1998. In vitro and in vivo effects of heavy metals on mussel digestive gland hexokinase activity: The role of glutathione. Comp. Biochem. Physiol. Part C Pharmacol. Toxicol. Endocrinol. 120, 261-268. Capasso, A., 2013. Antioxidant action and therapeutic efficacy of Allium

287 | P a g e

Bibliography

sativum L. Molecules 18, 690-700. Cavallito, C.J., Bailey, J.H., 1944. Allicin, the antibacterial principle of Allium sativum. I. Isolation, physical properties and antibacterial action. J. Am. Chem. Soc. 66, 1950-1951. Cazale, A.C., Clemens, S., 2001. Arabidopsis thaliana expresses a second functional phytochelatin synthase. FEBS Lett. 507, 215-219. Cercos, M., Carbonell, J., 1993. Purification and characterization of a thiol- protease induced during senescence of unpollinated ovaries of Pisum sativum. Physiol. Plant. 88, 267-274. Cerretti, D.P., Kozlosky, C.J., Mosley, B., Nelson, N., Van Ness, K., Greenstreet, T.A., March, C.J., Kronheim, S.R., Druck, T., Cannizzaro, L.A., 1992. Molecular cloning of the interleukin-1 beta converting enzyme. Science 256, 97-100. Chakrabortee, S., Kayatekin, C., Newby, G.A., Mendillo, M.L., Lancaster, A., Lindquist, S., 2016. Luminidependens (LD) is an Arabidopsis protein with prion behavior. Proc. Natl. Acad. Sci. 113, 6065–6070. Chan, J.Y.Y., Yuen, A.C.Y., Chan, R.Y.K., Chan, S.W., 2013. A review of the cardiovascular benefits and antioxidant properties of allicin. Phyther. Res. 27, 637-646. Chauhan, L.K., Saxena, P.N., Gupta, S.K., 2001. Evaluation of cytogenetic effects of isoproturon on the root meristem cells of Allium sativum. Biomed. Environ. Sci. 14, 214-219. Chaurasia, P.C.P., Prasad, J.B., Mandal, A., 2007. Management of leaf blight of garlic with fungicides in Central Tarai of Nepal, Nepal Agric. Res. J. 8, 63-66

Chen, Maulana J.M., Dando, Azad P.M., Library, Rawlings, Aligarh N.D., Muslim Brown, University M.A., Young, N.E., Stevens, R.A., Hewitt, E., Watts, C., Barrett, A.J., 1997. Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase. J. Biol. Chem. 272, 8090-8098. Chen, J.M., Dando, P.M., Stevens, R.A., Fortunato, M., Barrett, A.J., 1998. Cloning and expression of mouse legumain, a lysosomal endopeptidase. Biochem. J. 335, 111-117. Chen, M.S., Johnson, B., Wen, L., Muthukrishnan, S., Kramer, K.J., Morgan, T.D., Reeck, G.R., 1992. Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing

288 | P a g e

Bibliography

activity of a truncated form of the protein. Protein Expr. Purif. 3, 41-49. Chen, T., Zhu, S., Shang, Y., Ge, C., Jiang, G., 2012. Binding of dihydromyricetin to human hemoglobin: Fluorescence and circular dichroism studies. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 93, 125-130. Chen, Y.H., Yang, J.T., Martinez, H.M., 1972. Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry 11, 4120-4131. Cheng, M.L., Tzen, J.T.C., Shyu, D.J.H., Chou, W.M., et al., 2014. Functional characterization of the N-terminal and C-terminal domains of a sesame group II phytocystatin. Bot. Stud. 55, 18. Cheng, T., Hitomi, K., van Vlijmen-Willems, I.M.J.J., de Jongh, G.J., Yamamoto, K., Nishi, K., Watts, C., Reinheckel, T., Schalkwijk, J., Zeeuwen, P.L.J.M., 2006. Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-. J. Biol. Chem. 281, 15893-15899. Cheng, X.W., Huang, Z., Kuzuya, M., Okumura, K., Murohara, T., 2011. cathepsins in atherosclerosis-based vascular disease and its complications. Hypertension 58, 978-986. Cheng, X.W., Shi, G.P., Kuzuya, M., Sasaki, T., Okumura, K., Murohara, T., 2012. Role for cysteine protease cathepsins in heart disease. Circulation 125, 1551-1562. Chi, Z., Liu, R., 2011. Phenotypic characterization of the binding of tetracycline to human serum albumin. Biomacromolecules 12, 203-209. Chi, Z., Liu, R., Teng, Y., Fang, X., Gao, C., 2010. Binding of oxytetracycline to bovine serum albumin: Spectroscopic and molecular modeling investigations.Maulana J. Agric. Azad Food Library, Chem. 58,Aligarh 10262 -Muslim10269. University Chiti, F., Dobson, C.M., 2006. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333-366. Choi, S.J., Reddy, S. V, Devlin, R.D., Menaa, C., Chung, H., Boyce, B.F., Roodman, G.D., 1999. Identification of human asparaginyl endopeptidase (legumain) as an inhibitor of osteoclast formation and bone resorption. J. Biol. Chem. 274, 27747-27753. Christeller, J.T., Farley, P.C., Marshall, R.K., Anandan, A., Wright, M.M., Newcomb, R.D., Laing, W.A., 2006. The squash aspartic proteinase

289 | P a g e

Bibliography

inhibitor SQAPI is widely present in the cucurbitales, comprises a small multigene family, and is a member of the phytocystatin family. J. Mol. Evol. 63, 747-757. Christova, P.K., Christov, N.K., Imai, R., 2006. A cold inducible multidomain cystatin from winter wheat inhibits growth of the snow mold fungus, Microdochium nivale. Planta 223, 1207-1218. Chu, M.H., Liu, K.L., Wu, H.Y., Yeh, K.W., Cheng, Y.S., 2011. Crystal structure of tarocystatin-papain complex: implications for the inhibition property of group-2 phytocystatins. Planta. 234, 243-254. Clemens, S., 2006. Evolution and function of phytochelatin synthases. J. Plant Physiol. 163, 319-332. Cobbett, C.S., 2000. Phytochelatin biosynthesis and function in heavy-metal detoxification. Curr. Opin. Plant Biol. 3, 211-216. Cohen, M., 2007. Environmental toxins and health- the health impact of pesticides. Aust. Fam. Physician 36, 1002-1004. Colosio, C., Corsini, E., Barcellini, W., Maroni, M., 1999. Immune parameters in biological monitoring of pesticide exposure: current knowledge and perspectives. Toxicol. Lett. 108, 285-295. Conclusion on the peer review of the pesticide risk assessment of the active substance carbendazim, 2010. EFSA J. 8, 1598-1674. Copeland, R.A., 2005. Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem. Anal. 46, 1-265. Corsini, E., Sokooti, M., Galli, C.L., Moretto, A., Colosio, C., 2013. Pesticide induced immunotoxicity in humans: a comprehensive review of the existingMaulana evidence. Azad Toxicology Library, 307,Aligarh 123- 1Muslim35. University ht T E 1997 P t t ct : p ct c pp ch Second Edition. Oxford University Press; Oxford: U.K. Csoma, C., Polgar, L., 1984. Proteinase from germinating bean cotyledons. Evidence for involvement of a thiol group in catalysis. Biochem. J. 222, 769-776. Cstorer, A., Ménard, R., 1994. Catalytic mechanism in papain family of cysteine peptidases. Methods Enzymol. 244, 486-500. Dall, E., Brandstetter, H., 2016. Structure and function of legumain in health

290 | P a g e

Bibliography

and disease. Biochimie 122, 126-150. Damalas, C.A., Eleftherohorinos, I.G., 2011. Pesticide exposure, safety issues, and risk assessment indicators. Int. J. Environ. Res. Public Health 8, 1402- 1419. Das, K.P., Barone, S., 1999. Neuronal differentiation in PC12 cells is inhibited by chlorpyrifos and its metabolites: Is acetylcholinesterase inhibition the site of Action. Toxicol. Appl. Pharmacol. 160, 217-230. Dave, S., Mahajan, S., Chandra, V., Dkhar, H.K., Sambhavi, Gupta, P., 2010. Specific molten globule conformation of stem bromelain at alkaline pH. Arch. Biochem. Biophys. 499, 26-31. Davis, S.R., Perrie, R., Apitz-Castro, R., 2003. The in vitro susceptibility of Scedosporium prolificans to ajoene, allitridium and a raw extract of garlic (Allium sativum). J. Antimicrob. Chemother. 51, 593-597. De Sousa-Pereira, P., Abrantes, J., Pinheiro, A., Colaço, B., Vitorino, R., Esteves, P.J., 2014. Evolution of C, D and S-type cystatins in mammals: An extensive gene duplication in primates. PLoS One 9, e109050. De Souza, A., Medeiros, A.D.R., de Souza, A.C., Wink, M., Siqueira, I.R., Ferreira, M.B.C., Fernandes, L., Loayza Hidalgo, M.P., Torres, I.L. da S., 2011. Evaluation of the impact of exposure to pesticides on the health of the rural population: Vale do Taquari, State of Rio Grande do Sul (Brazil). Cien. Saude Colet. 16, 3519-3528. De Wit, J.C., Notermans, S., Gorin, N., Kampelmacher, E.H., 1979. Effect of garlic oil or onion oil on toxin production by Clostridium botulinum in meat slurry. J. Food Prot. 42, 222-224. DeLa Cadena, R.A., Colman, R.W., 1991. Structure and functions of human kininogens. Trends Pharmacol. Sci. 12, 272-275. Maulana Azad Library, Aligarh Muslim University Delaha, E.C., Garagusi, V.F., 1985. Inhibition of mycobacteria by garlic extract (Allium sativum). Antimicrob. Agents Chemother. 27, 485-486. Delledonne, M., Allegro, G., Belenghi, B., Balestrazzi, A., Picco, F., Levine, A., Zelasco, S., Calligari, P., Confalonieri, M., 2001. Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance. Mol. Breed. 7, 35-42. Devaraj, K.B., Kumar, P.R., Prakash, V., 2011. Comparison of activity and conformational changes of ficin during denaturation by urea and guanidine

291 | P a g e

Bibliography

hydrochloride. Process Biochem. 46, 458-464. Di Cera, E., 2009. Serine proteases. IUBMB Life 61, 510-515. Diaz-Mendoza, M., Velasco-Arroyo, B., Gonzalez-Melendi, P., Martinez, M., Diaz, I., 2014. C1A cysteine protease-cystatin interactions in leaf senescence. J. Exp. Bot. 65, 3825-3833. Dietert, R.R., 2011. Role of developmental immunotoxicity and immune dysfunction in chronic disease and cancer. Reprod. Toxicol. 31, 319-326. Dill, K.A., 1990. Dominant forces in protein folding. Biochemistry 29, 7133- 7155.

Dixon, M., 1972. The graphical determination of Km and Ki. Biochem. J. 129, 197-202. Dixon, M., 1953. The determination of enzyme inhibitor constants. Biochem. J. 55, 170-171. Drazkiewicz, M., Tukendorf, A., Baszynski, T., 2003. Age-dependent response of maize leaf segments to cadmium treatment: Effect on chlorophyll fluorescence and phytochelatin accumulation. J. Plant Physiol. 160, 247- 254. DuBois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 2002. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350-356. Duncan, E.M., Muratore-Schroeder, T.L., Cook, R.G., Garcia, B.A., Shabanowitz, J., Hunt, D.F., Allis, C.D., 2008. Cathepsin L proteolytically processes histone H3 during mouse embryonic stem cell differentiation. Cell 135, 284-294. Duramad, P., Holland, N.T., 2011. Biomarkers of immunotoxicity for environmentalMaulana Azad and Library, public healt Aligarhh research. Muslim Int. J.University Environ. Res. Public Health 8, 1388-1401. Dutt, S., Singh, V.K., Marla, S.S., Kumar, A., 2010. In silico analysis of sequential, structural and functional diversity of wheat cystatins and its implication in plant defense. Genomics. Proteomics Bioinformatics 8, 42- 56. Duxbury, T., 1985. Ecological aspects of heavy metal responses in microorganisms. Adv. Microb. Ecol. 8, 185-235. Eftink, M.R., Ghiron, C.A., 1981. Fluorescence quenching studies with

292 | P a g e

Bibliography

proteins. Anal. Biochem. 114, 199-227. Ellman, R., 1969. Tissue sulphydryl groups. Biochem. Methods. 28, 446-451. Englund, P.T., King, T.P., Craig, L.C., Walti, A., 1968. Studies on ficin. I. Its Isolation and characterization. Biochemistry 7, 163-175. Erez, E., Fass, D., Bibi, E., 2009. How intramembrane proteases bury hydrolytic reactions in the membrane. Nature 459, 371-378. Ernst, W.H.O., Krauss, G.J., Verkleij, J.A.C., Wesenberg, D., 2007. Interaction of heavy metals with the sulphur metabolism in angiosperms from an ecological point of view. Plant. Cell Environ. 31, 123-143. Fan, T.J., Han, L.H., Cong, R.S., Liang, J., 2005. Caspase family proteases and apoptosis. Acta Biochim. Biophys. Sin. (Shanghai). 37, 719-27. Farinati, S., Dalcorso, G., Varotto, S., Furini, A., 2010. The Brassica juncea BjCdR15, an ortholog of Arabidopsis TGA3, is a regulator of cadmium uptake, transport and accumulation in shoots and confers cadmium tolerance in transgenic plants. New Phytol. 185, 964-978. Fenwick, G.R., Hanley, A.B., 1985. Allium species poisoning. Vet. Rec. 116, 28. Fernandes, K.V.S., Campos, F.A.P., Do Val, R.R., Xavier-Filho, J., 1991. The expression of papain inhibitors during development of cowpea seeds. Plant Sci. 74, 179-184. Fernandes, K.V.S., Sabelli, P.A., Paul Barratt, D.H., Richardson, M., Xavier- Filho, J., Shewry, P.R., 1993. The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors. Plant Mol. Biol. 23, 215-219. Fields, B.N., Knipe, D.M. David M., Howley, P.M., Griffin, D.E., 2001. Fields virology,Maulana Fourth ed Azadition. LippincottLibrary, Aligarh Williams Muslim & Wilkins University; Philadelphia: United States. Finer, E.G., Franks, F., Tait, M.J., 1972. Nuclear magnetic resonance studies of aqueous urea solutions. J. Am. Chem. Soc. 94, 4424-4429. Finkenstaedt, J.T., 1957. Intracellular distribution of proteolytic enzymes in rat liver tissue. Proc. Soc. Exp. Biol. Med. 95, 302-304. Fossum, K., Whitaker, J.R., 1968. Ficin and papain inhibitor from chicken egg white. Arch. Biochem. Biophys. 125, 367-375. Fraki, J.E., 1976. Human skin proteases. Separation and characterization of two

293 | P a g e

Bibliography

acid proteases resembling cathepsin B1 and cathepsin D and of an inhibitor of cathepsin B1. Arch. Derm. Res. 255, 317-330. Frank, H.S., Franks, F., 1968. Structural approach to the solvent power of water for hydrocarbons; Urea as a structure breaker. J. Chem. Phys. 48, 4746- 4757. Freije, J.P., Abrahamson, M., Olafsson, I., Velasco, G., Grubb, A., Lopez-Otin, C., 1991. Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor. J. Biol. Chem. 266, 20538- 20543. Freije, J.P., Balbing, M., Abrahamson, M., Velasco, G., Dalboge, H., Grubb, A., Lopez-Otin, C., 1993. Human cystatin D. cDNA cloning, characterization of the Escherichia coli expressed inhibitor, and identification of the native protein in saliva. J.Biol.Chem. 268, 15737- 15744. Freire, E., Schon, A., Hutchins, B.M., Brown, R.K., 2013. Chemical denaturation as a tool in the formulation optimization of biologics. Drug Discov. Today. 18, 1007-1013. Friedlander, R.M., Chen, M., Ona, V.O., Li, M., Ferrante, R.J., Fink, K.B., Zhu, S., Bian, J., Guo, L., Farrell, L.A., Hersch, S.M., Hobbs, W., Vonsattel, J.P., Cha, J.H.J., 2000. Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat. Med. 6, 797-801. Fromtling, R.A., Bulmer, G.S., 1978. In vitro effect of aqueous extract of garlic (Allium sativum) on the growth and viability of Cryptococcus neoformans. Mycologia 70, 397-405. Fruton, J.S., Irving, G.W., Bergmann, M., 1941. On the proteolytic enzymes of animalMaulana tissues Azad II. The Library, composite Aligarh nature Muslimof beef spleen University cathepsin. J. Biol. Chem. 138, 249-262 Fujinaga, M., Cherney, M.M., Oyama, H., Oda, K., James, M.N.G., 2004. The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum. Proc. Natl. Acad. Sci. 101, 3364-3369. Gaines, T.B., 1969. Acute toxicity of pesticides. Toxicol. Appl. Pharmacol. 14, 515-534. Garcia-Hernandez, M., Murphy, A., Taiz, L., 1998. Metallothioneins 1 and 2 have distinct but overlapping expression patterns in Arabidopsis. Plant Physiol. 118, 387-397. 294 | P a g e

Bibliography

Gast, K., Zirwer, D., Muller-Frohne, M., Damaschun, G., 2008. Trifluoroethanol-induced conformational transitions of proteins: Insights f th ff c b tw α-lactalbumin and ribonuclease A. Protein Sci. 8, 625-634. Gatehouse, A.M.R., Norton, E., Davison, G.M., Babbé, S.M., Newell, C.A., Gatehouse, J.A., 1999. Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo. J. Insect Physiol. 45, 545-558. Gebhardt, R., Beck, H., 1996. Differential inhibitory effects of garlic-derived organosulfur compounds on cholesterol biosynthesis in primary rat hepatocyte cultures. Lipids 31, 1269-76. Geoffroy, L., Teisseire, H., Couderchet, M., Vernet, G., 2002. Effect of oxyfluorfen and diuron alone and in mixture on antioxidative enzymes of Scenedesmus obliquus. Pestic. Biochem. Physiol. 72, 178-185. Ghalandari, B., Divsalar, A., Saboury, A.A., Haertle, T., Parivar, K., Bazl, R., Eslami-Moghadam, M., Amanlou, M., 2014. Spectroscopic and theoretical investigation of oxali–p t ct w th β-lactoglobulin. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 118, 1038-1046. Ghannoum, M.A., 1988. Studies on the anticandidal mode of action of Allium sativum (Garlic). Microbiology 134, 2917-2924. Gilles, A.M., Lecroisey, A., Keil, B., 1984. Primary structure of alpha- clostripain light chain. Eur. J. Biochem. 145, 469-476. Gocheva, V., Joyce, J.A., 2007. Cysteine cathepsins and the cutting edge of cancer invasion. Cell Cycle 6, 60-64. Goldschmidt, L., Teng, P.K., Riek, R., Eisenberg, D., 2010. Identifying the amylome, proteins capable of forming amyloid-like fibrils. Proc. Natl. Acad. Sci.Maulana 107, 3487 Azad-3492. Library, Aligarh Muslim University Goulet, B., Baruch, A., Moon, N.-S., Poirier, M., Sansregret, L.L., Erickson, A., Bogyo, M., Nepveu, A., 2004. A cathepsin L isoform that is devoid of a signal peptide localizes to the nucleus in S phase and processes the CDP/Cux transcription factor. Mol. Cell 14, 207-219. Grandjean, O., Aeschlimann, A., 1973. Contribution to the study of digestion in ticks: histology and fine structure of the midgut ephithelium of Ornithodorus moubata, Murray (Ixodoidea, Argasidae). Acta Trop. 30, 193-212.

295 | P a g e

Bibliography

Green, G.D., Kembhavi, A.A., Davies, M.E., Barrett, A.J., 1984. Cystatin-like cysteine proteinase inhibitors from human liver. Biochem. J. 218, 939-946. Green, T.R., Ryan, C.A., 1972. Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science 175, 776- 777. Grogan, H.M., Jukes, A.A., 2003. Persistence of the fungicides thiabendazole, carbendazim and prochloraz-Mn in mushroom casing soil. Pest Manag. Sci. 59, 1225-1231. Grubb, A., Horio, M., Hansson, L.O., et al., 2014. Generation of a new cystatin C-based estimating equation for glomerular filtration rate by use of 7 assays standardized to the international calibrator. Clin. Chem. 60, 974- 986. Grubb, A.O., 2000. Cystatin C properties and use as diagnostic marker. Adv. Clin. Chem. 35, 63-99. Grubb, A.O., Weiber, H., Lofb 1983 Th γ-trace concentration of normal human seminal plasma is thirty-six times that of normal human blood plasma. Scand. J. Clin. Lab. Investig. 43, 421-425. Gruden, K., Strukelj, B., Ravnikar, M., Poljsak-Prijatelj, M., Mavric, I., Brzin, J., Pungercar, J., Kregar, I., 1997. Potato cysteine proteinase inhibitor gene family: molecular cloning, characterisation and immunocytochemical localisation studies. Plant Mol. Biol. 34, 317-23. Grudkowska, M., Zagdanska, B., 2004. Multifunctional role of plant cysteine proteinases. Acta Biochim Pol. 51, 609-624 Guo, N.L., Lu, D.P., Woods, G.L., Reed, E., Zhou, G.Z., Zhang, L.B., Waldman, R.H., 1993. Demonstration of the anti-viral activity of garlic extract against human cytomegalovirus in vitro. Chin. Med. J. 106, 93-96. Maulana Azad Library, Aligarh Muslim University Guo, X., Zhang, L., Sun, X., Han, X., Guo, C., Kang, P., 2009. Spectroscopic studies on the interaction between sodium ozagrel and bovine serum albumin. J. Mol. Struct. 928, 114-120. Gupta, P.K., 2011. Herbicides and fungicides. Reprod. In "Reproductive and Developmental Toxicology". Academic Press: Elsevier. 503-521. Guroff, G., 1964. A Neutral, calcium-activated proteinase from the soluble fraction of rat brain. J. Biol. Chem. 239, 149-155. Gutierrez-Campos, R., Torres-Acosta, J.A., Saucedo-Arias, L.J., Gomez-Lim, M.A., 1999. The use of cysteine proteinase inhibitors to engineer

296 | P a g e

Bibliography

resistance against potyviruses in transgenic tobacco plants. Nat. Biotechnol. 17, 1223-1226. Halfman, C.J., Nishida, T., 1971. Nature of the alteration of the fluorescence spectrum of bovine serum albumin produced by the binding of dodecyl sulfate. Biochim. Biophys. Acta - Protein Struct. 243, 294-303. Han, X. Le, Tian, F.F., Ge, Y.S., Jiang, F.L., Lai, L., Li, D.W., Yu, Q.L., Wang, J., Lin, C., Liu, Y., 2012. Spectroscopic, structural and thermodynamic properties of chlorpyrifos bound to serum albumin: A comparative study between BSA and HSA. J. Photochem. Photobiol. B Biol. 109, 1-11. Hao, F., Jing, M., Zhao, X., Liu, R., 2015. Spectroscopy, calorimetry and molecular simulation studies on the interaction of catalase with copper ion. J. Photochem. Photobiol. B Biol. 143, 100-106. Harrach, T., Eckert, K., Schulze-Forster, K., Nuck, R., Grunow, D., Maurer, H.R., 1995. Isolation and partial characterization of basic proteinases from stem bromelain. J. Protein Chem. 14, 41-52. Hartley, B.S., 1960. Proteolytic Enzymes. Annu. Rev. Biochem. 29, 45-72. Hashimoto, Y., Kakegawa, H., Narita, Y., Hachiya, Y., Hayakawa, T., Kos, J., Turk, V., Katunuma, N., 2001. Significance of cathepsin B accumulation in synovial fluid of rheumatoid arthritis. Biochem. Biophys. Res. Commun. 283, 334-339. Hass, G.M., Ryan, C.A., 1980. Carboxypeptidase inhibitor from ripened tomatoes; Purification and properties. Phytochemistry 19, 1329-1333. Hassanein, H.M.A., 2002. Toxicological effects of the herbicide oxyfluorfen on acetylcholinesterase in two fish species: Oreochromis niloticus and Gambusia affinis. J. Environ. Sci. Heal. Part A 37, 521-527. Maulana Azad Library, Aligarh Muslim University He, Y., Wang, Y., Tang, L., Liu, H., Chen, W., Zheng, Z., Zou, G., 2008. Binding of puerarin to human serum albumin: A spectroscopic analysis and molecular docking. J. Fluoresc. 18, 433-442. Hedstrom, L., 2002. Serine protease mechanism and specificity. Chem. Rev. 102, 4501-4524. Hemalatha, K., Madhumitha, G., Al-Dhabi, N.A., Arasu, M.V., 2016. Importance of fluorine in 2,3-dihydroquinazolinone and its interaction study with lysozyme. J. Photochem. Photobiol. B Biol. 162, 176-188. Henderson, P.J.F., 1972. A linear equation that describes the steady-state

297 | P a g e

Bibliography

kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors. Biochem. J. 127, 321-333. Herman S. Mansur, Zelia P. Lobato, Rodrigo L. Orefice, Wander L. Vasconcelos, Cintia Oliveira, and, Machado, L.J., 2000. Surface Functionalization of porous glass netw k : Eff ct bovine serum albumin and porcine insulin immobilization. Biomacromolecules 1, 789- 797. Heyno, E., Klose, C., Krieger-Liszkay, A., 2008. Origin of cadmium-induced reactive oxygen species production: mitochondrial electron transfer versus plasma membrane NADPH oxidase. New Phytol. 179, 687-699. Hill, B.C., Hanna, C.A., Adamski, J., Pham, H.P., Marques, M.B., Williams, L.A., 2017. Ficin-treated red cells help identify clinically significant alloantibodies masked as reactions of undetermined specificity in gel microtubes. Lab. Med. 48, 24-28. Hopsu-Havu, V.K., Joronen, I., Rinne, A., Jarvinen, M., 1985. Production of acid and neutral cysteine-proteinase inhibitors by a cultured human skin epithelium cell line. Arch. Dermatol. Res. 277, 452-456. Hsing, A.W., Chokkalingam, A.P., Gao, Y.T., Madigan, M.P., Deng, J., Gridley, G., Fraumeni, J.F., 2002. Allium vegetables and risk of prostate cancer: a population-based study. J. Natl. Cancer Inst. 94, 1648-1651. Hu, Y.J., Liu, Y., Sun, T.Q., Bai, A.M., Lu, J.Q., Pi, Z.B., 2006. Binding of anti-inflammatory drug cromolyn sodium to bovine serum albumin. Int. J. Biol. Macromol. 39, 280-285. Hua, L., Zhou, R., Thirumalai, D., Berne, B.J., 2008. Urea denaturation by stronger dispersion interactions with proteins than water implies a 2-stage unfolding. Proc. Natl. Acad. Sci. 105, 16928-16933. Huang,Maulana Y., Wang, Azad K.K., Library, 2001. The Aligarh calpain family Muslim and University human disease. Trends Mol. Med. 7, 355-62. Husain, S.S., Lowe, G., 1970. The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin. Biochem. J. 117, 333-340. Hwang, J.E., Hong, J.K., Lim, C.J., Chen, H., Je, J., Yang, K.A., Kim, D.Y., Choi, Y.J., Lee, S.Y., Lim, C.O., 2010. Distinct expression patterns of two Arabidopsis phytocystatin genes, AtCYS1 and AtCYS2, during development and abiotic stresses. Plant Cell Rep. 29, 905-915.

298 | P a g e

Bibliography

Imada, C., 2005. Enzyme inhibitors and other bioactive compounds from marine actinomycetes. Antonie Van Leeuwenhoek 87, 59-63. Iram, A., Alam, T., Khan, J.M., Khan, T.A., Khan, R.H., Naeem, A., 2013. Molten globule of hemoglobin proceeds into aggregates and advanced glycated end products. PLoS One 8, e72075. Iram, A., Naeem, A., 2013. Detection and analysis of protofibrils and fibrils of hemoglobin: Implications for the pathogenesis and cure of heme loss related maladies. Arch. Biochem. Biophys. 533, 69-78. Iram, A., Naeem, A., 2012. Trifluoroethanol and acetonitrile induced formation of the molten globule states and aggregates of cellulase. Int. J. Biol. Macromol. 50, 932-938. Irene, D., Chung, T.Y., Chen, B.J., Liu, T.H., Li, F.Y., Tzen, J.T.C., Wang, C.I., Chyan, C.L., 2012. Solution structure of a phytocystatin from Ananas comosus and its molecular interaction with papain. PLoS One 7, e47865. Isemura, S., Saitoh, E., Ito, S., Isemura, M., Sanada, K., 1984. Cystatin S: a cysteine proteinase inhibitor of human saliva. J. Biochem. 96, 1311-1314. Isemura, S., Saitoh, E., Sanada, K., 1987. Characterization and amino acid sequence of a new acidic cysteine proteinase inhibitor (cystatin SA) structurally closely related to cystatin S, from human whole saliva. J. Biochem. 102, 693-704. Isemura, S., Saitoh, E., Sanada, K., 1986. Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S. FEBS Lett. 198, 145-149. Isemura, S., Saitoh, E., Sanada, K., Minakata, K., 1991. Identification of full- sized forms of salivary (S-type) cystatins (cystatin SN, cystatin SA, cystatin S, and two phosphorylated forms of cystatin S) in human whole saliva andMaulana determination Azad Library, of phospho Aligarhrylation Muslim sites of University cystatin S. J. Biochem. 110, 648-54. Jaga, K., Dharmani, C., 2003. Sources of exposure to and public health implications of organophosphate pesticides. Rev. Panam. Salud Publica 14, 171-185. Janowski, R., Kozak, M., Jankowska, E., Grzonka, Z., Grubb, A., Abrahamson, M., Jaskolski, M., 2001. Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping. Nat. Struct. Biol. 8, 316-320.

299 | P a g e

Bibliography

Jarvinen, M., 1978. Purification and some characteristics of the human epidermal SH-protease inhibitor. J. Invest. Dermatol. 71, 114-118. Jarvinen, M., 1976. Healing of a crush injury in rat striated muscle. 4. Effect of early mobilization and immobilization on the tensile properties of gastrocnemius muscle. Acta Chir. Scand. 142, 47-56. Jarvinen, M., Rinne, A., 1982. Human spleen cysteineproteinase inhibitor: Purification, fractionation into isoelectric variants and some properties of the variants. Biochim. Biophys. Acta - Protein Struct. Mol. Enzymol. 708, 210-217. Jensen, K., Oestergaard, P.R., Wilting, R., Lassen, S.F., 2010. Identification and characterization of a bacterial glutamic peptidase. BMC Biochem. 11, 1-12. Johnson, M.G., Vaughn, R.H., 1969. Death of Salmonella typhimurium and Escherichia coli in the presence of freshly reconstituted dehydrated garlic and onion. Appl. Microbiol. 17, 903-905. Johnson, S.L., Pellecchia, M., 2006. Structure- and fragment-based approaches to protease inhibition. Curr. Top. Med. Chem. 6, 317-329. Johri, N., Jacquillet, G., Unwin, R., 2010. Heavy metal poisoning: the effects of cadmium on the kidney. BioMetals 23, 783-792. Joshi, B.N., Sainani, M.N., Bastawade, K.B., Gupta, V.S., Ranjekar, P.K., 1998. Cysteine protease inhibitor from pearl millet: A new class of antifungal protein. Biochem. Biophys. Res. Commun. 246, 382-387. Juaarez, J., Lopez, S.G., Cambon, A., Taboada, P., Mosquera, V., 2009. Influence of electrostatic interactions on the fibrillation process of human serum albumin. J. Phys. Chem. B 113, 10521-10529.

Kahle,Maulana H., 1993. ResponseAzad Library, of roots Aligarhof trees to Muslim heavy metals. University Environ. Exp. Bot. 33, 99-119. Kang, J., Liu, Y., Xie, M.-X., Li, S., Jiang, M., Wang, Y.-D., 2004. Interactions of human serum albumin with chlorogenic acid and ferulic acid. Biochim. Biophys. Acta - Gen. Subj. 1674, 205-214. Karami-Mohajeri, S., Abdollahi, M., 2011. Toxic influence of organophosphate, carbamate, and organochlorine pesticides on cellular metabolism of lipids, proteins, and carbohydrates. Hum. Exp. Toxicol. 30, 1119-1140. Kassi, E., Moutsatsou, P., 2010. Estrogen receptor signaling and its relationship

300 | P a g e

Bibliography

to in systemic lupus erythematosus. J. Biomed. Biotechnol. 2010, 1-14. Katrahalli, U., Jaldappagari, S., Kalanur, S.S., 2010. Probing the binding of fluoxetine hydrochloride to human serum albumin by multispectroscopic techniques. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 75, 314- 319. Keilova, H., Tomasek, V., 1975. Inhibition of cathepsin C by papain inhibitor from chicken egg white and by complex of this inhibitor with cathepsin B1. Collect. Czechoslov. Chem. Commun. 40, 218-224. Kelly, S.M., Jess, T.J., Price, N.C., 2005. How to study proteins by circular dichroism. Biochim. Biophys. Acta - Proteins Proteomics 1751, 119-139. Kepler, D., 2002. Proteases in biology and med c I “E y b ch t y” (E : p N ) Q t v B 78 517 Keppler, D., 2006. Towards novel anti-cancer strategies based on cystatin function. Cancer Lett. 235, 159-176. Khan, S., Ahmad, S., Siddiqi, M.I., Bano, B., 2016. Physico-chemical and In silico analysis of a phytocystatin purified from Brassica juncea cultivar RoAgro 5444. Biochem. Cell Biol. 94, 584-596. Khan, S., Khan, N.A., Bano, B., 2017. In-sights into the effect of heavy metal stress on the endogenous mustard cystatin. Int. J. Biol. Macromol. 105, 1138-1147. Khan, T.A., Amani, S., Naeem, A., 2012. Glycation promotes the formation of genotoxic aggregates in glucose oxidase. Amino Acids 43, 1311-1322. Kirici, M., Atamanalp, M., Beydemir, S., 2017. In vitro effects of some metal ions on glutathione reductase in the gills and liver of Capoeta trutta, RegulatoryMaulana Mechanisms Azad in Library,Biosystems. Aligarh 8, 66-70. Muslim University Kirschke, H., Wiederanders, B., Bromme, D., Rinne, A., 1989. Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins. Biochem. J. 264, 467-473. Klimov, D.K., Straub, J.E., Thirumalai, D., 2004. Aqueous urea solution destabilizes A 16-22 oligomers. Proc. Natl. Acad. Sci. 101, 14760–14765. Knowles, L.M., Milner, J.A., 2003. Diallyl Disulfide Induces ERK Phosphorylation and Alters Gene Expression Profiles in Human Colon Tumor Cells. J. Nutr. 133, 2901–2906. https://doi.org/10.1093/jn/133.9.2901

301 | P a g e

Bibliography

K ch P L w L 1996 c : th c c th p t c application of Allium sativum L. and related species. Second edition. Williams & Wilkins. Kohler, A., Blaudez, D., Chalot, M., Martin, F., 2004. Cloning and expression of multiple metallothioneins from hybrid poplar. New Phytol. 164, 83-93. Koiwa, H., Bressan, R.A., Hasegawa, P.M., 1997. Regulation of protease inhibitors and plant defense. Trends Plant Sci. 2, 379-384. Kondo, H., Emori, Y., Abe, K., Suzuki, K., Arai, S., 1989. Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin. Gene 81, 259-265. Kong, J., Yu, S., 2007. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim. Biophys. Sin. (Shanghai). 39, 549-559. Kopitar, M., Brzin, J., Zvonar, T., Locnikar, P., Kregar, I., Turk, V., 1978. Inhibition studies of an intracellular inhibitor on thiol proteinases. FEBS Lett. 91, 355-359. Kostura, M.J., Tocci, M.J., Limjuco, G., Chin, J., Cameron, P., Hillman, A.G., Chartrain, N.A., Schmidt, J.A., 1989. Identification of a monocyte specific pre-interleukin 1 beta convertase activity. Proc. Natl. Acad. Sci. 86, 5227– 5231. Kramer, U., 2010. Metal Hyperaccumulation in plants. Annu. Rev. Plant Biol. 61, 517-534. Kresheck, G.C., Scheraga, H.A., 1965. The temperature dependence of the enthalpy of formation of the amide hydrogen bond; the urea model. J. Phys. Chem. 69, 1704-1706.

Krishnan,Maulana V.G.M., Azad Murugan, Library, K., Aligarh 2015. Purification, Muslim University characterization and kinetics of protease inhibitor from fruits of Solanum aculeatissimum Jacq. Food Sci. Hum. Wellness 4, 97-107. Kubota, K., Wakabayashi, K., Matsuoka, T., 2003. Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: Two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography. Proteomics 3, 616-626. Kumar, R., Simran Chhatwal, S., Sahiba Arora, S., Sharma, S., Jaswinder Singh, J., Narinder Singh, N., Vikram Bhandari, V., Khurana, A., 2013. Antihyperglycemic, antihyperlipidemic, anti-inflammatory and adenosine

302 | P a g e

Bibliography

deaminase & lowering effects of garlic in patients with type 2 diabetes mellitus with obesity. Diabetes, Metab. Syndr. Obes. Targets Ther. 6, 49- 56. Kunitz, M., 1947. Crystalline soybean trypsin inhibitor : II. General properties. J. Gen. Physiol. 30, 291-310. Kweon, S., Park, K.A., Choi, H., 2003. Chemopreventive effect of garlic powder diet in diethylnitrosamine-induced rat hepatocarcinogenesis. Life Sci. 73, 2515–2526. Kwiatkowska, M., Huras, B., Bukowska, B., 2014. The effect of metabolites and impurities of glyphosate on human erythrocytes (in vitro). Pestic. Biochem. Physiol. 109, 34-43. Laber, B., Krieglstein, K., Henschen, A., Kos, J., Turk, V., Huber, R., Bode, W., 1989. The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein. FEBS Lett. 248, 162-168. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lakowicz, J.R., 2006. Principles of fluorescence spectroscopy. Springer. Lalaouni, A., Henderson, C., Kupper, C., Grant, M.H., 2007. The interaction of chromium (VI) with macrophages: Depletion of glutathione and inhibition of glutathione reductase. Toxicology 236, 76-81. Lamb, C., Dixon, R.A., 1997. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 251-275. Langerholc, T., Zavasnik-Bergant, V., Turk, B., Turk, V., Abrahamson, M., Kos, J., 2005. Inhibitory properties of cystatin F and its localization in U937 promonocyte cells. FEBS J. 272, 1535-1545. Lanzotti, V., Maulana2006. The analysisAzad Library, of onion andAligarh garlic. Muslim J. Chromatogr. University A 1112, 3- 22. Lanzotti, V., Barile, E., Antignani, V., Bonanomi, G., Scala, F., 2012. Antifungal saponins from bulbs of garlic, Allium sativum L. var. Voghiera. Phytochemistry 78, 126-134. Lau, B.H., Woolley, J.L., Marsh, C.L., Barker, G.R., Koobs, D.H., Torrey, R.R., 1986. Superiority of intralesional immunotherapy with Corynebacterium parvum and Allium sativum in control of murine transitional cell carcinoma. J. Urol. 136, 701–705.

303 | P a g e

Bibliography

Launay, S., Hermine, O., Fontenay, M., Kroemer, G., Solary, E., Garrido, C., 2005. Vital functions for lethal caspases. Oncogene 24, 5137-5148. Laurent, T.C., Killander, J., 1964. A theory of gel filtration and its exeperimental verification. J. Chromatogr. A 14, 317-330. Lecaille, F., Kaleta, J., Bromme, D., 2002. Human and parasitic papain-like cy t p t : Th phy y p th y c t developments in inhibitor design. Chem. Rev. 102, 4459-4488. Ledezma, E., Marcano, K., Jorquera, A., De Sousa L, Padilla, M., Pulgar, M., Apitz-Castro, R., 2000. Efficacy of ajoene in the treatment of tinea pedis: a double-blind and comparative study with terbinafine. J. Am. Acad. Dermatol. 43, 829-832. Lee, M.J., Yu, G.R., Park, S.H., Cho, B.H., Ahn, J.S., Park, H.J., Song, E.Y., Kim, D.G., 2008. Identification of cystatin B as a potential serum marker in hepatocellular carcinoma. Clin. Cancer Res. 14, 1080-1089. Lemar, K.M., Turner, M.P., Lloyd, D., 2002. Garlic (Allium sativum) as an anti-Candida agent: a comparison of the efficacy of fresh garlic and freeze-dried extracts. J. Appl. Microbiol. 93, 398-405. Lenarcic, B., Ritonja, A., Dolenc, I., Stoka, V., Berbie, S., Pungerear, J., Strukelj, B., Turk, V., 1993. Pig leukocyte cysteine proteinase inhibitor (PLCPI), a new member of the stefin family. FEBS Lett. 336, 289-292. Lenney, J.F., Tolan, J.R., Sugai, W.J., Lee, A.G., 1979. Thermostable endogenous inhibitors of cathepsins B and H. Eur. J. Biochem. 101, 153- 161. Levinthal, C., 1968. Are there pathways for protein folding? J. Chim. Phys. Physico-Chimie Biol. 65, 44-45.

Lewis,Maulana W.J., van AzadLenteren, Library, J.C., Phatak, Aligarh S.C., Muslim Tumlinson, University J.H., 1997. A total system approach to sustainable pest management. Proc. Natl. Acad. Sci. 94, 12243-12248. Li, M., Ona, V.O., Guegan, C., Chen, M., Jackson-Lewis, V., Andrews, L.J., Olszewski, A.J., Stieg, P.E., Lee, J.P., Przedborski, S., Friedlander, R.M., 2000. Functional role of caspase-1 and caspase-3 in an ALS transgenic mouse model. Science 288, 335-339. Li, X., Zhou, J., 1996. Unfolding and aggregation-associated changes in the secondary structure of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride as monitored by FTIR.

304 | P a g e

Bibliography

Biospectroscopy 121–129. Lima, A.M., dos Reis, S.P., de Souza, C.R.B., 2015. Phytocystatins and their potential to control plant diseases caused by fungi. Protein Pept. Lett. 22, 104-111. Lin, J.G., Chen, G.W., Su, C.C., Hung, C.F., Yang, C.C., Lee, J.H., Chung, J.G., 2002. Effects of garlic components diallyl sulfide and diallyl disulfide on arylamine n-acetyltransferase activity and 2-aminofluorene- DNA adducts in human promyelocytic leukemia cells. Am. J. Chin. Med. 30, 315-325. Lineweaver, H., Burk, D., 2002. The determination of enzyme dissociation constants 56, 658-666. Lineweaver, H., Burk, D., 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658-666. Lipke, H., Fraenkel, G.S., Liener, I.E., 1954. Growth inhibitors. Effect of soybean inhibitors on growth of Tribolium confusum. J. Agric. Food Chem. 2, 410-414. Liu, C., Liu, Z., Wang, J., 2017. Uncovering the molecular and physiological processes of anticancer leads binding human serum albumin: A physical insight into drug efficacy. PLoS One 12, e0176208. Liu, Y., Lin, J., Chen, M., Song, L., 2013. Investigation on the interaction of the toxicant, gentian violet, with bovine hemoglobin. Food Chem. Toxicol. 58, 264-272. Lofberg, H., Grubb, A., Davidsson, L., Kjellander, B., Strömblad, L.G., Tibblin, S., Olsson, S.O., 1983. Occurrence of gamma-trace in the calcitonin-producing C-cells of simian thyroid gland and human medullary thyroid carcinoma. Acta Endocrinol. (Copenh). 104, 69-76. Maulana Azad Library, Aligarh Muslim University Lofberg, H., Grubb, A.O., Brun, A., 1981. Human brain cortical neurons contain y-trace. rapid isolation, immunohistochemical and physicochemical characterization of human y-trace. Biomed. Res. 2, 298- 306. Lofberg, H., Nilsson, K.E., Stromblad, L.G., Lasson, A., Olsson, S.O., 1982. Demonstration of gamma-trace in normal endocrine cells of the adrenal medulla and in phaeochromocytoma. An immunohistochemical study in monkey, dog and man. Acta Endocrinol. (Copenh). 100, 595-598. Lopez-Otin, C., Bond, J.S., 2008. Proteases: Multifunctional enzymes in life

305 | P a g e

Bibliography

and disease. J. Biol. Chem. 283, 30433–30437. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Lutgens, S.P.M., Cleutjens, K.B.J.M., Daemen, M.J.A.P., Heeneman, S., 2007. Cathepsin cysteine proteases in cardiovascular disease. FASEB J. 21, 3029-3041. Ma, M., Lau, P.-S., Jia, Y.-T., Tsang, W.-K., Lam, S.K.., Tam, N.F.., Wong, Y.-S., 2003. The isolation and characterization of Type 1 metallothionein (MT) cDNA from a heavy-metal-tolerant plant, Festuca rubra cv. Merlin. Plant Sci. 164, 51–60. Ma, Y., Chen, Y., Petersen, I., 2017. Expression and epigenetic regulation of cystatin B in lung cancer and colorectal cancer. Pathol. Res. Pract. 213, 1568-1574. Macedo, M.L.R., Mello, G.C., das Gracas M. Freire, M., et al., 2002. Effect of a trypsin inhibitor from Dimorphandra mollis seeds on the development of Callosobruchus maculatus. Plant Physiol. Biochem. 40, 891–898. Machleidt, W., Borchart, U., Fritz, H., Brzin, J., Ritonja, A., Turk, V., 1983. Protein inhibitors of cysteine proteinases. II. Primary structure of stefin, a cytosolic protein inhibitor of cysteine proteinases from human polymorphonuclear granulocytes. Hoppe. Seylers. Z. Physiol. Chem. 364, 1481-1486. Makhatadze, G.I., Privalov, P.L., 1992. Protein interactions with urea and guanidinium chloride. A calorimetric study. J. Mol. Biol. 226, 491-505. Makin, O.S., Atkins, E., Sikorski, P., Johansson, J., Serpell, L.C., 2005. Molecular basis for amyloid fibril formation and stability. Proc. Natl. Acad. Sci. 102, 315-320. Maulana Azad Library, Aligarh Muslim University Manconi, B., Liori, B., Cabras, T., Vincenzoni, F., Iavarone, F., Castagnola, M., Messana, I., Olianas, A., 2017. Salivary cystatins: Exploring new post- translational modifications and polymorphisms by top-down high- resolution mass spectrometry. J. Proteome Res. 16, 4196–4207. Margis, R., Reis, E.M., Villeret, V., 1998. Structural and phylogenetic relationships among plant and animal cystatins. Arch. Biochem. Biophys. 359, 24-30. Margni, M., Rossier, D., Crettaz, P., Jolliet, O., 2002. Life cycle impact assessment of pesticides on human health and ecosystems. Agric. Ecosyst.

306 | P a g e

Bibliography

Environ. 93, 379-392. Martínez, M., Abraham, Z., Carbonero, P., Díaz, I., 2005. Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley. Mol. Genet. Genomics 273, 423-432. Martinez, M., Abraham, Z., Gambardella, M., Echaide, M., Carbonero, P., Diaz, I., 2005. The strawberry gene Cyf1 encodes a phytocystatin with antifungal properties. J. Exp. Bot. 56, 1821-1829. Martinez, M., Diaz-Mendoza, M., Carrillo, L., Diaz, I., 2007. Carboxy terminal extended phytocystatins are bifunctional inhibitors of papain and legumain cysteine proteinases. FEBS Lett. 581, 2914-2918. Martinez, M., Diaz, I., 2008. The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship. BMC Evol. Biol. 8, 1-12. Martinez, M., Lopez-Solanilla, E., Rodriguez-Palenzuela, P., Carbonero, P., Diaz, I., 2003. Inhibition of plant-pathogenic fungi by the barley cystatin Hv-CPI (Gene Icy ) is not associated with its cysteine-proteinase inhibitory properties. Mol. Plant-Microbe Interact. 16, 876-883. Maschalidi, S., Hassler, S., Blanc, F., Sepulveda, F.E., Tohme, M., Chignard, M., van Endert, P., Si-Tahar, M., Descamps, D., Manoury, B., 2012. Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation. PLoS Pathog. 8, e1002841. Maubach, G., Lim, M.C.C., Zhuo, L., 2008. Nuclear cathepsin F regulates activation markers in rat hepatic stellate cells. Mol. Biol. Cell 19, 4238- 4248. Mdegela, R.H., Mosha, R.D., Sandvik, M., Skaare, J.U., 2010. Assessment of acetylcholinesterase activity in Clarias gariepinus as a biomarker of organophosphateMaulana and Azad carbamate Library, exposure. Aligarh Ecotoxicology Muslim University19, 855-863. Mello, G.C., Oliva, M.L. V., Sumikawa, J.T., Machado, O.L.T., Marangoni, S., Novello, J.C., Macedo, M.L.R., 2001. Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds. J. Protein Chem. 20, 625-632. Merrifield, M.E., Ngu, T., Stillman, M.J., 2004. Arsenic binding to Fucus vesiculosus metallothionein. Biochem. Biophys. Res. Commun. 324, 127– 132. Metwally, A., Safronova, V.I., Belimov, A.A., Dietz, K.J., 2004. Genotypic

307 | P a g e

Bibliography

variation of the response to cadmium toxicity in Pisum sativum L. J. Exp. Bot. 56, 167-178. Mirhadi, S.A., Singh, S., Gupta, P.P., 1991. Effect of garlic supplementation to cholesterol-rich diet on development of atherosclerosis in rabbits. Indian J. Exp. Biol. 29, 162–168. Mitchinson, C., Pain, R.H., 1985. Effects of sulphate and urea on the stability v b f f β-lactamase from Staphylococcus aureus: I p c t f th f p thw y f β-lactamase. J. Mol. Biol. 184, 331-342. Moglich, A., Krieger, F., Kiefhaber, T., 2005. Molecular basis for the effect of urea and guanidinium chloride on the dynamics of unfolded polypeptide chains. J. Mol. Biol. 345, 153-162. Mokarizadeh, A., Faryabi, M.R., Rezvanfar, M.A., Abdollahi, M., 2015. A comprehensive review of pesticides and the immune dysregulation: mechanisms, evidence and consequences. Toxicol. Mech. Methods. 25, 258-278. M0ller, A., Hansen, B.L., Hansen, G.N., Hagen, C., 1985. Autoantibodies in sera from patients with multiple sclerosis directed against antigenic determinants in pituitary growth hormone-producing cells and in structures containing vasopressin/oxytocin. J. Neuroimmunol. 8, 177-184. Morita, M., Hara, Y., Tamai, Y., Arakawa, H., Nishimura, S., 2000. Genomic construct and mapping of the gene for CMAP (Leukocystatin/Cystatin F, CST7) and identification of a proximal novel gene, BSCv (C20orf3). Genomics 67, 87-91. Morshedi, D., Ebrahim-Habibi, A., Moosavi-Movahedi, A.A., Nemat-Gorgani, M., 2010. Chemical modification of lysine residues in lysozyme may dramaticallyMaulana Azad influence Library, its amyloid Aligarh fibrillation. Muslim Biochim. University Biophys. Acta - Proteins Proteomics 1804, 714-722. Mosimann, S.C., Cherney, M.M., Sia, S., Plotch, S., James, M.N.., 1997. Refined X-ray crystallographic structure of the poliovirus 3C gene product 1 1Edited By D. Rees. J. Mol. Biol. 273, 1032-1047. Mostafalou, S., Abdollahi, M., 2017. Pesticides: an update of human exposure and toxicity. Arch. Toxicol. 91, 549-599. Mostafalou, S., Abdollahi, M., 2013. Pesticides and human chronic diseases: Evidences, mechanisms, and perspectives. Toxicol. Appl. Pharmacol. 268, 157-177. 308 | P a g e

Bibliography

Mountain, R.D., Thirumalai, D., 2003. Molecular dynamics simulations of end- to-end contact formation in hydrocarbon chains in water and aqueous urea solution. J. Am. Chem. Soc. 125, 1950-1957. Muller-Esterl, W., Fritz, H., Machleidt, W., Ritonja, A., Brzin, J., Kotnik, M., Turk, V., Kellermann, J., Lottspeich, F., 1985. Human plasma kininogens t c w th α-cysteine proteinase inhibitors. Evidence from immunological, enzymological and sequence data. FEBS Lett. 182, 310- 314. Murachi, T., 1970. Bromelain enzymes. Methods Enzymol. 9, 273-284 Murachi, T., 1976. Bromelain enzymes. Methods Enzymol. 45, 475-485. Murphy, A., Taiz, L., 1995. Comparison of metallothionein gene expression and nonprotein thiols in ten Arabidopsis ecotypes. Correlation with copper tolerance. Plant Physiol. 109, 945-54. Naeem, A., Amani, S., 2013. Deciphering structural intermediates and genotoxic fibrillar aggregates of albumins: a molecular mechanism underlying for degenerative diseases. PLoS One 8, e54061. Naeem, A., Bhat, S.A., Iram, A., Khan, R.H., 2016. Aggregation of intrinsically disordered fibrinogen as the influence of backbone conformation. Arch. Biochem. Biophys. 603, 38-47. Naeem, A., Iram, A., Bhat, S.A., 2015. Anesthetic 2,2,2-trifluoroethanol induces amyloidogenesis and cytotoxicity in human serum albumin. Int. J. Biol. Macromol. 79, 726-735. Naeem, A., Khan, R.H., 2004. Characterization of molten globule state of cytochrome c at alkaline, native and acidic pH induced by butanol and SDS. Int. J. Biochem. Cell Biol. 36, 2281-2292.

Nagler, D.K.,Maulana Tam, W., Storer,Azad A.C.,Library, Krupa, Aligarh J.C., Mort, Muslim J.S., M Universityenard, R., 1999. Interdependency of sequence and positional specificities for cysteine proteases of the papain family. Biochemistry 38, 4868-4874. Nakajima, Y., Suzuki, S., 2013. Environmental stresses induce misfolded protein aggregation in plant cells in a microtubule-dependent manner. Int. J. Mol. Sci. 14, 7771–7783. Nakanishi, H., 2003. Neuronal and microglial cathepsins in aging and age- related diseases. Ageing Res. Rev. 2, 367–381. Nandi, P.K., Robinson, D.R., 1984. Effects of urea and guanidine hydrochloride on peptide and nonpolar groups. Biochemistry 23, 6661–

309 | P a g e

Bibliography

6668. Napper, A.D., Bennett, S.P., Borowski, M., Holdridge, M.B., Leonard, M.J., Rogers, E.E., Duan, Y., Laursen, R.A., Reinhold, B., Shames, S.L., 1994. Purification and characterization of multiple forms of the pineapple-stem- derived cysteine proteinases ananain and comosain. Biochem. J. 301, 727– 735. Naveenraj, S., Raj, M.R., Anandan, S., 2012. Binding interaction between serum albumins and perylene-3,4,9,10-tetracarboxylate – A spectroscopic investigation. Dye. Pigment. 94, 330-337. Neuberger, A., Brocklehurst, K., 1987. Hydrolytic enzymes. First edition. Elsevier. 16. Neurath, H., 1999. Proteolytic enzymes, past and future. Proc. Natl. Acad. Sci. 96, 10962-10963. Neurath, H., 1984. Evolution of proteolytic enzymes. Science 224, 350-357. Neuteboom, L.W., Matsumoto, K.O., Christopher, D.A., 2009. An extended AE-rich N-terminal trunk in secreted pineapple cystatin enhances inhibition of fruit bromelain and is posttranslationally removed during ripening. Plant Physiol. 151, 515-527. Ni, J., Abrahamson, M., Zhang, M., Alvarez Fernandez, M., Grubb, A., Su, J., Yu, G.-L., Li, Y., Parmelee, D., Xing, L., Coleman, T.A., Gentz, S., Thotakura, R., Nguyen, N., Hesselberg, M., Gentz, R., 1997. Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins. J Biol Chem. 272, 10853-10858. Ni, J., Alvarez Fernandez, M., Danielsson, L., Chillakuru, R.A., Zhang, J., Grubb, A., Su, J., Gentz, R., Abrahamson, M., 1998. Cystatin F Is a glycosylated human low molecular weight cysteine proteinase inhibitor. J BiolMaulana Chem. 273, Azad 24797 Library,-24804. Aligarh Muslim University Nick Pace, C., Scholtz, J.M., Grimsley, G.R., 2014. Forces stabilizing proteins. FEBS Lett. 588, 2177-2184. Nissen, M.S., Kumar, G.N.M., Youn, B., Knowles, D.B., Lam, K.S., Ballinger, W.J., Knowles, N.R., Kang, C., 2009. Characterization of solanum tuberosum multicystatin and its structural comparison with other cystatins. The Plant Cell 21, 861-875. Nusrat, S., Siddiqi, M.K., Zaman, M., Zaidi, N., Ajmal, M.R., Alam, P., Qadeer, A., Abdelhameed, A.S., Khan, R.H., 2016. A comprehensive

310 | P a g e

Bibliography

spectroscopic and computational investigation to probe the interaction of antineoplastic drug nordihydroguaiaretic acid with serum albumins. PLoS One 11, e0158833. O'Brien, E.P., Dima, R.I., Brooks, B., Thirumalai, D., 2007. Interactions between hydrophobic and ionic solutes in aqueous guanidinium chloride and urea solutions: lessons for protein denaturation mechanism. J. Am. Chem. Soc. 129, 7346-7353. O'Gara, E.A., Hill, D.J., Maslin, D.J., 2000. Activities of garlic oil, garlic powder, and their diallyl constituents against Helicobacter pylori. Appl. Environ. Microbiol. 66, 2269-2273. Obata-Onai, A., Hashimoto, S.I., Onai, N., Kurachi, M., Nagai, S., Shizuno, K.I., Nagahata, T., Matsushima, K., 2002. Comprehensive gene expression analysis of human NK cells and CD8+ T lymphocytes. Int. Immunol. 14, 1085-1098. Obayomi, A., Adeola, S.A., Bankole, H.A., Raimi, O.G., 2015. Characterization of partially purified cysteine protease inhibitor from Tetracarpidium conophorum (African walnut). African J. Biochem. Res. 9, 26–34. Oda, K., 2012. New families of carboxyl peptidases: serine-carboxyl peptidases and glutamic peptidases. J. Biochem. 151, 13-25. Ojima, A., Shiota, H., Higashi, K., Kamada, H., Shimma, Y., Wada, M., Satoh, S., 1997. An extracellular insoluble inhibitor of cysteine proteinases in cell cultures and seeds of carrot. Plant Mol. Biol. 34, 99–109. Oliva, M.L., Souza-Pinto, J.C., Batista, I.F., Araujo, M.S., Silveira, V.F., Auerswald, E.A., Mentele, R., Eckerskorn, C., Sampaio, M.U., Sampaio, C.A., 2000. Leucaena leucocephala serine proteinase inhibitor: primary structureMaulana and action Azad on bloodLibrary, coagulation, Aligarh kininMuslim rele aseUniversity and rat paw edema. Biochim. Biophys. Acta 1477, 64-74. Oliveira, A.S., Xavier-Filho, J., Sales, M.P., 2003. Cysteine proteinases and cystatins. Brazilian Arch. Biol. Technol. 46, 91-104. Olsson, M., Zhivotovsky, B., 2011. Caspases and cancer. Cell Death Differ. 18, 1441-1449. Omar, S.H., 2013. Garlic and cardiovascular diseases. In "Natural products". (Ed: Ramawat K., Merillon J.M.). Springer; Berlin:Heidelberg, 3661– 3696.

311 | P a g e

Bibliography

Ota, S., Moore, S., Stein, W.H., 1964. Preparation and chemical properties of purified stem and fruit . Biochemistry 3, 180-185. Otto, H.H., Schirmeister, T., 1997. Cysteine proteases and their inhibitors. Chem. Rev. 97, 133-172. Ouchterlony, O., 1962. Diffusion-in-gel methods for immunological analysis. II. Prog. Allergy 6, 30-154. Pace, C.N., 1986. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131, 266-80. Padiya, R., Banerjee, S.K., 2013. Garlic as an anti-diabetic agent: recent progress and patent reviews. Recent Pat. Food. Nutr. Agric. 5, 105-27. Page, M. J., Di Cera, E., 2008. Serine peptidases: Classification, structure and function. Cell. Mol. Life Sci. 65, 1220-1236. Page, M. J, Cera, E.D., 2008. Evolution of peptidase diversity. J. Biol. Chem. 283, 30010–30014. Palsdottir, A., Snorradottir, A.O., Thorsteinsson, L., 2006. Hereditary cystatin C amyloid angiopathy: genetic, clinical, and pathological aspects. Brain Pathol. 16, 55-59. Pandey, N., Sharma, C.P., 2002. Effect of heavy metals Co2+, Ni2+and Cd2+ on growth andmetabolism of cabbage. Plant Sci. 163, 753-758. Pannetier, C., Giband, M., Couzi, P., Le Tan, V., Mazier, M., Tourneur, J., Hau, B., 1997. Introduction of new traits into cotton through genetic engineering: insect resistance as example. Euphytica 96, 163-166. Papadopoulou, A., Green, R. J., Frazier, R.A., 2004. Interaction of flavonoids w th b v b : f c c q ch t y J. Agric. Food Chem. 53, 158-163. Maulana Azad Library, Aligarh Muslim University Parween, T., Jan, S., Mahmooduzzafar, S., Fatma, T., Siddiqui, Z.H., 2016. Selective effect of pesticides on plant—a review. Crit. Rev. Food Sci. Nutr. 56, 160-179. Pavlova, A., 2003. Studies of inhibition of cysteine endo- and exopeptidases by cystatins A and C. In "Mechanism of action of mammalian cystatins". Swedish University of Agricultural Sciences. Pawar, S.A., Deshpande, V. V, 2000. Characterization of acid-induced unfolding intermediates of glucose/xylose . Eur. J. Biochem. 267, 6331-6338.

312 | P a g e

Bibliography

Peixoto, F., Alves-Fernandes, D., Santos, D., Fontaínhas-Fernandes, A., 2006. Toxicological effects of oxyfluorfen on oxidative stress enzymes in tilapia Oreochromis niloticus. Pestic. Biochem. Physiol. 85, 91-96. Perfus-Barbeoch, L., Leonhardt, N., Vavasseur, A., Forestier, C., 2002. Heavy metal toxicity: cadmium permeates through calcium channels and disturbs the plant water status. Plant J. 32, 539-48. Pernas, M., Lopez-Solanilla, E., Sanchez-Monge, R., Salcedo, G., Rodriguez- Palenzuela, P., 1999. Antifungal activity of a plant cystatin. MPMI 12, 624-627. Pernas, M., Sanchez-Monge, R., Gomez, L., Salcedo, G., 1998. A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests. Plant Mol. Biol. 38, 1235-1242. Perrin, B.J., Huttenlocher, A., 2002. Calpain. Int. J. Biochem. Cell Biol. 34, 722-725. Peterson, E.A., Sober, H.A., 1962. Column chromatography of proteins: Substituted celluloses. Methods Enzymol. 5, 3-27. Petkov, V., 1979. Plants and hypotensive, antiatheromatous and coronarodilatating action. Am. J. Chin. Med. 7, 197-236. Petrovska, B.B., Cekovska, S., 2010. Extracts from the history and medical properties of garlic. Pharmacogn. Rev. 4, 106-10. Pfeil, W., Privalov, P.L., 1976. Thermodynamic investigations of proteins. II. Calorimetric study of lysozyme denaturation by guanidine hydrochloride. Biophys. Chem. 4, 33-40. Pirovani, C.P., da Silva Santiago, A., dos Santos, L.S., Micheli, F., Margis, R., da Silva Gesteira, A., Alvim, F.C., Pereira, G.A.G., de Mattos Cascardo, J.C., 2010.Maulana Theobroma Azad cacao Library, cystatins Aligarh impair MuslimMoniliophthora University perniciosa mycelial growth and are involved in postponing cell death symptoms. Planta 232, 1485–1497. Potempa, J., Dubin, A., Korzus, G., Travis, J., 1988. Degradation of elastin by a cysteine proteinase from Staphylococcus aureus. J. Biol. Chem. 263, 2664-2667. Povarova, O.I., Kuznetsova, I.M., Turoverov, K.K., et al., 2010. Differences in the pathways of proteins unfolding induced by urea and guanidine hydrochloride: molten globule state and aggregates. PLoS One 5, e15035. Prajapati, S., Bhakuni, V., Babu, K.R., Jain, S.K., 1998. Alkaline unfolding and

313 | P a g e

Bibliography

salt-induced folding of bovine liver catalase at high pH. Eur. J. Biochem. 255, 178-84. Prasad, C.K.P.S., Hiremath, P.C., 1985. Varietal screening and chemical control in fenugreek against foot-rot and damping off caused by Rhizoctonia solani. Pesticides 19, 34-36. Privalov, P.L., 1979. Stability of proteins: small globular proteins. Adv. Protein Chem. 33, 167-241. Privalov, P.L., Khechinashvili, N.N., 1974. A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J. Mol. Biol. 86, 665-684. Priyadarshini, M., Bano, B., 2010. Cystatin like thiol proteinase inhibitor from pancreas of Capra hircus: purification and detailed biochemical characterization. Amino Acids 38, 1001-1010. Priyadarshini, M., Khan, R.H., Bano, B., 2010. Physicochemical properties of thiol proteinase inhibitor isolated from goat pancreas. Biopolymers 93, 708-717. Ptitsyn, O.B., Finkelstein, A. V., Adman, E.T., et al., 1980. Similarities of protein topologies: evolutionary divergence, functional convergence or principles of folding? Q. Rev. Biophys. 13, 339-386. Puente, X.S., Sanchez, L.M., Overall, C.M., Lopez-Otin, C., 2003. Human and mouse proteases: a comparative genomic approach. Nat. Rev. Genet. 4, 544-558. Qiu, J., Ai, L., Ramachandran, C., Yao, B., Gopalakrishnan, S., Fields, C.R., Delmas, A.L., Dyer, L.M., Melnick, S.J., Yachnis, A.T., Schwartz, P.H., Fine, H.A., Brown, K.D., Robertson, K.D., 2008. Invasion suppressor cystatin E/M (CST6): High-level cell type-specific expression in normal brainMaulana and epigenetic Azad Library, silencing inAligarh gliomas. Muslim Lab. Investig. University 88, 910-925. Rabbani, G., Khan, M.J., Ahmad, A., Maskat, M.Y., Khan, R.H., 2014. Effect f c pp x p t c th c f t ct v ty f β- galactosidase. Colloids Surfaces B Biointerfaces 123, 96-105. Rahman, K., Lowe, G.M., 2006. Garlic and cardiovascular disease: a critical review. J. Nutr. 136, 736S-740S. Rahman, Y., Afrin, S., Husain, M.A., Sarwar, T., Ali, A., Shamsuzzaman, Tabish, M., 2017. Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug, with calf thymus DNA: An in vitro and

314 | P a g e

Bibliography

molecular modelling study. Arch. Biochem. Biophys. 625-626, 1–12. Rahman, Y., Afrin, S., Tabish, M., 2018. Interaction of pirenzepine with b v b ff ct f β-cyclodextrin on binding: A biophysical and molecular docking approach. Arch. Biochem. Biophys. 652, 27-37. Rakash, S., a, Rana, F., Rafiq, S., Masood, A., Amin, S., 2012. Role of proteases in cancer: A review. Biotechnol. Mol. Biol. Rev. 7, 90-101. Ramasubbu, N., Reddy, M.S., Bergey, E.J., Haraszthy, G.G., Soni, S.D., Levine, M.J., 1991. Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva. Biochem. J. 280, 341-352. Rao, M.B., Tanksale, A.M., Ghatge, M.S., Deshpande, V. V, 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62, 597-635. Rashid, A., Khan, H.H., 1985. The mechanism of hypotensive effect of garlic extract. J. Pak. Med. Assoc. 35, 357-362. Rashid, F., Sharma, S., Bano, B., 2005. Comparison of guanidine hydrochloride (GdnHCl) and urea denaturation on inactivation and unfolding of human placental cystatin (HPC). Protein J. 24, 283-292. Rassam, M., Laing, W.A., 2004. Purification and characterization of phytocystatins from kiwifruit cortex and seeds. Phytochemistry 65, 19–30. Rawlings, N.D., Barrett, A.J., 1994. Families of serine peptidases. Methods Enzymol. 244, 19-61. Rawlings, N.D., Barrett, A.J., 1990. Evolution of proteins of the cystatin superfamily. J. Mol. Evol. 30, 60-71. Rawlings, N.D.,Maulana Barrett, Azad A.J., Bateman,Library, A., Aligarh 2011. Asparagine Muslim University Peptide Lyases. J. Biol. Chem. 286, 38321-38328. Rawlings, N.D., Barrett, A.J., Bateman, A., 2010. MEROPS: the peptidase database. Nucleic Acids Res. 38, D227-D233. Rawlings, N.D., Barrett, A.J., Finn, R., 2016. Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 44, D343-D350. Rawlings, N.D., Barrett, A.J., Finn, R., 2015. Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic

315 | P a g e

Bibliography

Acids Res. 44, 343-350. Rawlings, N.D., Morton, F.R., Kok, C.Y., Kong, J., Barrett, A.J., 2007. MEROPS: the peptidase database. Nucleic Acids Res. 36, D320-D325. Rawlings, N.D., Tolle, D.P., Barrett, A.J., 2004. Evolutionary families of peptidase inhibitors. Biochem. J. 378, 705-16. Rees, L.P., Minney, S.F., Plummer, N.T., Slater, J.H., Skyrme, D.A., 1993. A quantitative assessment of the antimicrobial activity of garlic (Allium sativum). World J. Microbiol. Biotechnol. 9, 303-307. Rehman, M.T., Faheem, M., Khan, A.U., 2015. An insight into the biophysical ch ct t f ff t t t f c f t x hy y β-lactamase 15 (CTX-M-15). J. Biomol. Struct. Dyn. 33, 625-638. Reis, E.M., Margis, R., 2001. Sugarcane phytocystatins: Identification, classification and expression pattern analysis. Genet. Mol. Biol. 24, 291- 296. Remans, T., Opdenakker, K., Guisez, Y., Carleer, R., Schat, H., Vangronsveld, J., Cuypers, A., 2012. Exposure of Arabidopsis thaliana to excess Zn reveals a Zn-specific oxidative stress signature. Environ. Exp. Bot. 84, 61- 71. Rezus, Y.L.A., Bakker, H.J., 2006. Effect of urea on the structural dynamics of water. Proc. Natl. Acad. Sci. 103, 18417–18420. Ried, K., Frank, O.R., Stocks, N.P., 2013. Aged garlic extract reduces blood pressure in hypertensives: a dose - response trial. Eur. J. Clin. Nutr. 67, 64- 70. Riley, M.B., Keese, R.J., Camper, N.D., Whitwell, T., Wilson, P.C., 1994. Pendimethalin and oxyfluorfen residues in pond water and sediment from containerMaulana plant Azad nurseries. Library, Weed Aligarh Technol. Muslim 8, 299-303. University Rinne, A., Jarvinen, M., Alavaikko, M., Martikainen, J., Hopsu-Havu, V.K., 1985. Occurrence of cysteine proteinase inhibitors in cells of the monocytic-histiocytic system. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 49, 153-159. Rinne, A., Jarvinen, M., Räsänen, O., 1978. A protein reminiscent of the epidermal SH-protease inhibitor occurs in squamous epithelia of man and rat. Acta Histochem. 63, 183-192. Rinne, A., Jarvinen, M., Rasanen, O., Hopsu-Havu, V.K., 1984. Acid and neutral cysteine proteinase inhibitor in normal uterine portio and in

316 | P a g e

Bibliography

squamo-epithelial metaplasia, dysplasias and infiltrative carcinoma of the uterine portio. Exp. Pathol. 26, 67-70. Robbins, B.H., 1930. A Proteolytic enzyme in ficin, the anthelmintic principle of Leche de Higueron. J. Biol. Chem. 87, 251-257. Roseman, M., Jencks, W.P., 1975. Interactions of urea and other polar compounds in water. J. AM. Chem. Soc. 97, 631-640. Rousseau, F., Serrano, L., Schymkowitz, J.W.H., 2006. How evolutionary pressure against protein aggregation shaped chaperone specificity. J. Mol. Biol. 355, 1037-1047. Roy, A.S., Dinda, A.K., Dasgupta, S., 2012. Study of the interaction between fisetin and human serum albumin: a biophysical approach. Protein Pept. Lett. 19, 604-615. Ryan, C.A., 1989. Proteinase inhibitor gene families: Strategies for transformation to improve plant defenses against herbivores. BioEssays 10, 20-24. Ryan, C.A., Walker-Simmons, M., 1981. Plant Proteinases. Proteins and Nucleic Acids 321-350. Sagardoy, R., Morales, F., Lopez-Millán, A.F., Abadia, A., Abadía, J., 2009. Effects of zinc toxicity on sugar beet ( Beta vulgaris L.) plants grown in hydroponics. Plant Biol. 11, 339-350. Sagardoy, R., Morales, F., Rellán-Alvarez, R., Abadía, A., Abadia, J., Lopez- Millan, A.F., 2011. Carboxylate metabolism in sugar beet plants grown with excess Zn. J. Plant Physiol. 168, 730-733. Sagardoy, R., Vazquez, S., Florez-Sarasa, I.D., Albacete, A., Ribas-Carbo, M., Flexas, J., Abadia, J., Morales, F., 2010. Stomatal and mesophyll conductancesMaulana to CO2 Azad are theLibrary, main limitations Aligarh Muslimto photosynthesis University in sugar beet (Beta vulgaris) plants grown with excess zinc. New Phytol. 187, 145- 158. Salminen-Mankonen, H.J., Morko, J., Vuorio, E., 2007. Role of cathepsin K in normal joints and in the development of arthritis. Curr. Drug Targets 8, 315-323. Salvesen, G., Parkes, C., Abrahamson, M., Grubb, A., Barrett, A.J., 1986.

Human low-Mr kininogen contains three copies of a cystatin sequence that are divergent in structure and in inhibitory activity for cysteine proteinases. Biochem. J. 234, 429-434.

317 | P a g e

Bibliography

Samac, D.A., Smigocki, A.C., 2003. Expression of oryzacystatin I and II in Alfalfa increases resistance to the root-lesion nematode. Phytopathology 93, 799-804. Sanita Di Toppi, L., Gabbrielli, R., 1999. Response to cadmium in higher plants. Environ. Exp. Bot. 41, 105-130. Santamaria, M., Diaz-Mendoza, M., Diaz, I., Martinez, M., 2014. Plant protein peptidase inhibitors: an evolutionary overview based on comparative genomics. BMC Genomics 15, 1-14. Santos-Rosa, H., Kirmizis, A., Nelson, C., Bartke, T., Saksouk, N., Cote, J., Kouzarides, T., 2009. Histone H3 tail clipping regulates gene expression. Nat. Struct. Mol. Biol. 16, 17-22. Sato, N., Ishidoh, K., Uchiyama, Y., Kominami, E., 1990. Molecular cloning and sequencing of cDNA for rat cystatin beta. Nucleic Acids Res. 18, 6698. Sawaya, M.R., Sambashivan, S., Nelson, R., Ivanova, M.I., Sievers, S.A., Apostol, M.I., Thompson, M.J., Balbirnie, M., Wiltzius, J.J.W., McFarlane, H.T., Madsen, A.O., Riekel, C., Eisenberg, D., 2007. Atomic structures of amyloid cross-β p v v t c pp N t 447, 453-457. Schellman, J.A., 1955. The thermodynamics of urea solutions and the heat of formation of the peptide hydrogen bond. C. R. Trav. Lab. Carlsberg. Chim. 29, 223-229. Schuttelkopf, A.W., Hamilton, G., Watts, C., Van Aalten, D.M.F., 2006. Structural Basis of Reduction-dependent Activation of Human Cystatin F. J. Biol. Chem. 281, 16570 –16575. Semisotnov, G. V., Rodionova, N.A., Razgulyaev, O.I., Uversky, V.N., Gripas, Maulana F Azad h Library, I 1991 Aligarh t y fMuslim th t University b t t state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31, 119-128. Sen, L.C., Whitaker, J.R., 1973. Some properties of a ficin-papain inhibitor from avion egg white. Arch. Biochem. Biophys. 158, 623-632. Senthilkumar, R., Cheng, C.P., Yeh, K.-W., 2010. Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco. Plant Biotechnol. J. 8, 65-75. Shah, A., Priyadarshini, M., Khan, M.S., Aatif, M., Amin, F., Bano, B., 2013.

318 | P a g e

Bibliography

Biochemical, immunological and kinetic characterisation of thiol protease inhibitor (cystatin) from liver. Appl. Biochem. Biotechnol. 171, 667-75. Shamsi, A., Ahmed, A., Bano, B., 2016. Glyoxal induced structural transition of buffalo kidney cystatin to molten globule and aggregates: Anti- fibrillation potency of quinic acid. IUBMB Life 68, 156-166. Sharma, S., Rashid, F., Bano, B., 2006. Unfolding during urea denaturation of a low molecular weight phytocystatin (thiol protease inhibitor) purified from Phaseolus mungo (Urd). Protein Pept. Lett. 13, 323-339. Sharma, S., Rashid, F., Bano, B., 2005. Biochemical and biophysical changes induced by fungicide sodium diethyl dithiocarbamate (SDD), in phytocystatin purified from Phaseolus mungo (Urd): a commonly used Indian legume. J. Agric. Food Chem. 53, 6027-6034. Sharma, V.D., Sethi, M.S., Kumar, A., Rarotra, J.R., 1977. Antibacterial property of Allium sativum Linn.: in vivo & in vitro studies. Indian J. Exp. Biol. 15, 466-468. Sheela, C., Kumud, K., Augusti, K., 1995. Anti-diabetic effects of onion and garlic sulfoxide amino acids in rats. Planta Med. 61, 356-357. Shortle, D., 1996. The denatured state (the other half of the folding equation) and its role in protein stability. FASEB J. 10, 27-34. Shukla, Y., Kalra, N., 2007. Cancer chemoprevention with garlic and its constituents. Cancer Lett. 247, 167-181. Shutov, A.D., Do, N.L., Vaintraub, I.A., 1982. Purification and partial characterization of protease B from germinating vetch seeds. Biokhimiia 47, 814-821. Shyu, D.J.H., Chyan, C.-L., Tzen, J.T.C., Chou, W.-M., 2004. Molecular cloning, Maulana expression, Azad and functionalLibrary, Aligarh characterization Muslim of University a cystatin from pineapple stem. Biosci. Biotechnol. Biochem. 68, 1681-1689. Sial, A.Y., Ahmad, S.I., 1982. Study of the hypotensive action of garlic extract in experimental animals. J. Pak. Med. Assoc. 32, 237-239. Siddiqui, A.A., Khaki, P.S.S., Bano, B., 2017. Interaction of almond cystatin with pesticides: Structural and functional analysis. J. Mol. Recognit. 30, 1- 10. Siddiqui, A.A., Khaki, P.S.S., Sohail, A., Sarwar, T., Bano, B., 2016. Isolation and purification of phytocystatin from almond: Biochemical, biophysical, and immunological characterization. Cogent Biol. 2, 1-17.

319 | P a g e

Bibliography

Siddiqui, A.A., Sohail, A., Bhat, S.A., Rehman, M.T., Bano, B., 2015. Non- enzymatic glycation of almond cystatin leads to conformational changes and altered activity. Protein Pept. Lett. 22, 449-459. Siddiqui, M.F., Ahmed, A., Bano, B., 2017. Insight into the biochemical , kinetic and spectroscopic characterization of garlic ( Allium sativum ) phyt cy t t : I p c t f c v c I t J B Macromol. 95, 734-742. Siddiqui, M.F., Bano, B., 2018a. Exposure of carbendazim induces structural and functional alteration in garlic phytocystatin: An in vitro multi- spectroscopic approach. Pestic. Biochem. Physiol. 145, 66-75. Siddiqui, M.F., Bano, B., 2018b. Insight into the functional and structural transition of garlic phytocystatin induced by urea and guanidine hydrochloride: A comparative biophysical study. Int. J. Biol. Macromol. 106, 20-29. Silva, D., Cortez, C.M., Cunha-Bastos, J., Louro, S.R.., 2004. Methyl parathion interaction with human and bovine serum albumin. Toxicol. Lett. 147, 53- 61. Sims, A.H., Dunn-Coleman, N.S., Robson, G.D., Oliver, S.G., 2004. Glutamic protease distribution is limited to filamentous fungi. FEMS Microbiol. Lett. 239, 95-101. Siqueira-Junior, C.L., Fernandes, K.V.S., Machado, O.L.T., da Cunha, M., Gomes, V.M., Moura, D., Jacinto, T., 2002. 87 kDa tomato cystatin exhibits properties of a defense protein and forms protein crystals in prosystemin overexpressing transgenic plants. Plant Physiol. Biochem. 40, 247-254. Skiles, J.W., Gonnella, N.C., Jeng, A.Y., 2004. The design, structure, and clinicalMaulana update Azad of Library, small molecular Aligarh weightMuslim matrix University metalloproteinase inhibitors. Curr. Med. Chem. 11, 2911-2977. Sloane, B.F., Honn, K. V, 1984. Cysteine proteinases and metastasis. Cancer Metastasis Rev. 3, 249-263. Soares-Costa, A., Beltramini, L.M.M., Thiemann, O.H.H., Henrique-Silva, F., 2002. A sugarcane cystatin: recombinant expression, purification, and antifungal activity. Biochem. Biophys. Res. Commun. 296, 1194-1199. Soh, H., Venkatesan, N., Veena, M.S., Ravichandran, S., Zinabadi, A., Basak, S.K., Parvatiyar, K., Srivastava, M., Liang, L.-J., Gjertson, D.W., Torres, J.Z., Moatamed, N.A., Srivatsan, E.S., 2016. Cystatin E/M suppresses 320 | P a g e

Bibliography

tumor cell growth through cytoplasmic retention of NF-κB Biol. 36, 1776-1792. Sohail, A., Faraz, M., Arif, H., Bhat, S.A., Siddiqui, A.A., Bano, B., 2017. Deciphering the interaction of bovine heart cystatin with ZnO nanoparticles: Spectroscopic and thermodynamic approach. Int. J. Biol. Macromol. 95, 1056-1063. Solomon, M., Belenghi, B., Delledonne, M., Menachem, E., Levine, A., 1999. The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11, 431-444. Sommers, P.B., Kronman, M.J., 1980. Comparative fluorescence properties of bovine, goat, human and guinea pig alpha lactalbumin. Characterization of the environments of individual tryptophan residues in partially folded conformers. Biophys. Chem. 11, 217-232. Soper, A.K., Castner, E.W., Luzar, A., 2003. Impact of urea on water structure: a clue to its properties as a denaturant. Biophys. Chem. 105, 649-666. Sorimachi, H., Hata, S., Ono, Y., 2011. Impact of genetic insights into calpain biology. J. Biochem. 150, 23-37. Sotiropoulou, G, Anisowicz, A., Sager, R., 1997. Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down- regulated in breast cancer. J. Biol. Chem. 272, 903-910. Sotiropoulou, Georgia, Anisowicz, A., Sager, R., 1997. Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer. J. Biol. Chem. 272, 903-910. Soto, C., 2003. Unfolding the role of protein misfolding in neurodegenerative diseases. Nat. Rev. Neurosci. 4, 49–60.

Souza, E.M.T.,Maulana Mizuta, Azad K., Library, Sampaio, Aligarh M.U., Muslim Sampaio, University C.A.M., 1995. Purification and partial characterization of a Schizolobium parahyba chymotrypsin inhibitor. Phytochemistry 39, 521-525. Souza, T.P., Dias, R.O., Silva-Filho, M.C., 2017. Defense-related proteins involved in sugarcane responses to biotic stress. Sparnins, V.L., Mott, A.W., Barany, G., Wattenberg, L.W., 1986. Effects of allyl methyl trisulfide on glutathione S‐ activity and BP‐induced neoplasia in the mouse. Nutr. Cancer 8, 211-215. Stabler, S.N., Tejani, A.M., Huynh, F., Fowkes, C., 2012. Garlic for the prevention of cardiovascular morbidity and mortality in hypertensive 321 | P a g e

Bibliography

patients. Cochrane Database Syst. Rev. 15, CD007653. Stenman, G., Astrom, A.K., Roijer, E., Sotiropoulou, G., Zhang, M., Sager, R., 1997. Assignment of a novel cysteine proteinase inhibitor (CST6) to 11q13 by fluorescence in situ hybridization. Cytogenet. Genome Res. 76, 45-46. Stern, V.M., Smith, R.F., van den Bosch, R., Hagen, K.S., 1959. The integration of chemical and biological control of the spotted alfalfa aphid: The integrated control concept. Hilgardia 29, 81-101. Stoka, V., Turk, B., Turk, V., 2005. Lysosomal cysteine proteases: Structural features and their role in apoptosis. IUBMB Life 57, 347-353. Stubbs, M.T., Laber, B., Bode, W., Huber, R., Jerala, R., Lenarcic, B., Turk, V., 1990. The refined 2.4 Å X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J. 9, 1939-1947. Sugawara, H., Shibuya, K., Yoshioka, T., Hashiba, T., Satoh, S., 2002. Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers. J. Exp. Bot. 53, 407-413. Suguna, K., Padlan, E.A., Smith, C.W., Carlson, W.D., Davies, D.R., 1987. Binding of a reduced peptide inhibitor to the aspartic proteinase from Rhizopus chinensis: implications for a mechanism of action. Proc. Natl. Acad. Sci. 84, 7009-7013. łk w k 2002 I t ct f w th b v h albumin. J. Mol. Struct. 614, 227-232. łk w k ow ck J B k B łk w k W 2003 I t ct f anticancer drugs with human and bovine serum albumin. J. Mol. Struct. 651,Maulana 133-140. Azad Library, Aligarh Muslim University Sumiyoshi, H., Wargovich, M.J., 1990. Chemoprevention of 1,2- dimethylhydrazine-induced colon cancer in mice by naturally occurring organosulfur compounds. Cancer Res. 50, 5084-5087. Suryawanshi, V.D., Walekar, L.S., Gore, A.H., Anbhule, P. V., Kolekar, G.B., 2016. Spectroscopic analysis on the binding interaction of biologically active pyrimidine derivative with bovine serum albumin. J. Pharm. Anal. 6, 56-63. Szymona, M., 1952. Effect of phytoncides of Allium sativum on growth and

322 | P a g e

Bibliography

respiration of certain pathogenic fungi. Acta Microbiol. Pol. 1, 5–23. Tadi, P.P., Lau, B.H., Teel, R.W., Herrmann, C.E., 1991. Binding of aflatoxin B1 to DNA inhibited by ajoene and diallyl sulfide. Anticancer Res. 11, 2037-2041. Tadi, P.P., Teel, R.W., Lau, B.H.S., 1991. Organosulfur compounds of garlic modulate mutagenesis, metabolism, and DNA binding of aflatoxin B1. Nutr. Cancer 15, 87-95. Tajima, N., Kawai, F., Park, S.-Y., Tame, J.R.H., 2010. A novel intein-like autoproteolytic mechanism in autotransporter proteins. J. Mol. Biol. 402, 645-656. Tanaka, T., Inazawa, J., Nakamura, Y., 1996. Molecular cloning of a human cDNA encoding putative cysteine protease (PRSC1) and its chromosome assignment to 14q32.1. Cytogenet. Genome Res. 74, 120-123. Tanford, C., 1968. Protein denaturation. Adv. Protein Chem. 23, 121-282. Tansey, M.R., Appleton, J.A., 1975. Inhibition of fungal growth by garlic extract. Mycologia 67, 409-13. Tartaglia, G.G., Pawar, A.P., Campioni, S., Dobson, C.M., Chiti, F., Vendruscolo, M., 2008. Prediction of aggregation-prone regions in structured proteins. J. Mol. Biol. 380, 425-436. Thiare, D.D., Khonte, A., Diop, A., Mendy, A., et al., 2015. Spectrofluorimetric analysis of the fungicide carbendazim and its metabolite 2-aminobenzimidazole in natural water. Am. J. Anal. Chem. 06, 767-775. Thomson, M., Ali, M., 2003. Garlic (Allium sativum): a review of its potential use as an anti-cancer agent. Curr. Cancer Drug Targets 3, 67-81. Thornberry, Maulana N.A., Bul Azadl, H.G., Library, Calaycay, Aligarh J.R., Muslimet al., University1992. A novel heterodimeric cysteine protease is required for interleukin- 1β processing in monocytes. Nature 356, 768-774. Tian, F.-F., Jiang, F.-L., Han, X.-L., Xiang, C., Ge, Y.-S., Li, J.-H., Zhang, Y., Li, R., Ding, X.-L., Liu, Y., 2010. Synthesis of a novel hydrazone derivative and biophysical studies of its interactions with bovine serum albumin by spectroscopic, electrochemical, and molecular docking methods. J. Phys. Chem. B 114, 14842-14853. Tian, J., Liu, J., Hu, Z., Chen, X., 2005. Interaction of wogonin with bovine serum albumin. Bioorg. Med. Chem. 13, 4124-4129.

323 | P a g e

Bibliography

Tirado-Rives, J., Orozco, M., Jorgensen, W.L., 1997. Molecular dynamics simulations of the unfolding of barnase in water and 8 M aqueous urea. Biochemistry 36, 7313-7329 Torrent, J., Marchal, S., Ribó, M., Vilanova, M., Georges, C., Dupont, Y., Lange, R., 2008. Distinct unfolding and refolding pathways of ribonuclease a revealed by heating and cooling temperature jumps. Biophys. J. 94, 4056-4065. Tortella, G.R.R., Mella-Herrera, R.A.A., Sousa, D.Z.Z., Rubilar, O., Briceño, G., Parra, L., Diez, M.C.C., 2013. Carbendazim dissipation in the biomixture of on-farm biopurification systems and its effect on microbial communities. Chemosphere 93, 1084-1093. Toyoda, H., Nicklin, M.J.H., Murray, M.G., Anderson, C.W., Dunn, J.J., Studier, F.W., Wimmer, E., 1986. A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein. Cell 45, 761- 770. Tryselius, Y., Hultmark, D., 1997. Cysteine proteinase 1 (CP1), a cathepsin L- like enzyme expressed in the Drosophila melanogaster haemocyte cell line mbn-2. Insect Mol. Biol. 6, 173-181. Tsai, Y., Cole, L.L., Davis, L.E., Lockwood, S.J., Simmons, V., Wild, G.C., 1985. Antiviral properties of garlic: in vitro effects on influenza B, herpes simplex and coxsackie viruses. Planta Med. 51, 460-461. Turk, B., 2006. Targeting proteases: successes, failures and future prospects. Nat. Rev. Drug Discov. 5, 785-799. Turk, B., Bieth, J.G., Bjork, I., Dolenc, I., Turk, D., Cimerman, N., Kos, J., Colic, A., Stoka, V., Turk, V., 1995a. Regulation of the activity of lysosomal cysteine proteinases by pH-induced inactivation and/or endogenousMaulana proteinAzad Library,inhibitors, Aligarhcystatins. MuslimBiol. Chem. University Hoppe. Seyler. 376, 225-230. Turk, B., Colifb, A., Stoka, V., Turk, V., 1994. Lzm Kinetics of inhibition of bovine cathepsin S by bovine stefin B, FEBS Lett. 339, 155-159. Turk, B., Krizaj, I., Kralj, B., Dolenc, I., Popovic, T., Bieth, J.G., Turk, V., 1993. Bovine stefin C, a new member of the stefin family. J. Biol. Chem. 268, 7323-7329. Turk, B., Krizaj, I., Turk, V., 1992. Isolation and characterization of bovine stefin B. Biol. Chem. Hoppe. Seyler. 373, 441-446.

324 | P a g e

Bibliography

Turk, B., Stoka, V., 2007. Protease signalling in cell death: caspases versus cysteine cathepsins. FEBS Lett. 581, 2761-2767. Turk, B., Stoka, V., Björk, I., Boudier, C., Johansson, G., Dolenc, I., Colic, A., Bieth, J.G., Turk, V., 1995b. High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen. Protein Sci. 4, 1874-1880. Turk, B., Turk, D., Salvesen, G.S., 2002. Regulating cysteine protease activity: essential role of protease inhibitors as guardians and regulators. Curr. Pharm. Des. 8, 1623-37. Turk, B., Turk, D., Turk, V., 2000. Lysosomal cysteine proteases: more than scavengers. Biochim. Biophys. Acta 1477, 98-111. Turk, B., Turk, V., Turk, D., 1997. Structural and functional aspects of papain- like cysteine proteinases and their protein inhibitors. Biol. Chem. 378, 141-50. Turk, V., Bode, W., 1991. The cystatins: Protein inhibitors of cysteine proteinases. FEBS Lett. 285, 213-219. Turk, V., Brzin, J., Longer, M., Ritonja, A., Eropkin, M., Borchart, U., Machleidt, W., 1983. Protein inhibitors of cysteine proteinases. III. Amino-acid sequence of cystatin from chicken egg white. Hoppe. Seylers. Z. Physiol. Chem. 364, 1487-1496. Turk, V., Stoka, V., Vasiljeva, O., Renko, M., Sun, T., Turk, B., Turk, D., 2012. Cysteine cathepsins: From structure, function and regulation to new frontiers. Biochim. Biophys. Acta - Proteins Proteomics 1824, 68-88. Turk, V., Turk, B., Guncar, G., Turk, D., Kos, J., 2002. Lysosomal cathepsins: structure, role in antigen processing and presentation, and cancer. Adv. Enzyme Regul. 42, 285-303. Maulana Azad Library, Aligarh Muslim University Turk, V., Turk, B., Turk, D., 2001. Lysosomal cysteine proteases: facts and opportunities. EMBO J. 20, 4629-4633. Urwin, P.E., Lilley, C.J., McPherson, M.J., Atkinson, H.J., 1997. Resistance to both cyst and root-knot nematodes conferred by transgenic Arabidopsis expressing a modified plant cystatin. Plant J. 12, 455-461. Ussuf, K.K., Laxmi, N.H., Mitra, R., 2001. Proteinase inhibitors: Plant-derived genes of insecticidal protein for developing insect-resistant transgenic plants. Curr. Sci. 80, 847-853. Uversky, V.N., 2002. Natively unfolded proteins: A point where biology waits

325 | P a g e

Bibliography

for physics. Protein Sci. 11, 739-756. Vain, P., Worland, B., Clarke, M.C., Richard, G., Beavis, M., Liu, H., Kohli, A., Leech, M., Snape, J., Christou, P., Atkinson, H., 1998. Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-IΔ 86) f nematode resistance in transgenic rice plants 96, 266–271. Valdes-Rodriguez, S., Cedro-Tanda, A., Aguilar-Hernandez, V., Cortes- Onofre, E., Blanco-Labra, A., Guerrero-Rangel, A., 2010. Recombinant amaranth cystatin (AhCPI) inhibits the growth of phytopathogenic fungi. Plant Physiol. Biochem. 48, 469-475. Valeur, B., Berberan-Santos, M., 2012. Molecular fluorescence: principles and applications. Second Edition. Wiley-VCH. Vallee, B.L., Auld, D.S., 1990. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29, 5647-5659. Valueva, T.A., Revina, T.A., Gvozdeva, E.L., Gerasimova, N.G., Ozeretskovskaya, O.L., 2003. Role of protease inhibitors in potato protection. Russ. J. Bioorganic Chem. 29, 454-458. Van Acker, G.J.D., Saluja, A.K., Bhagat, L., Singh, V.P., Song, A.M., Steer, M.L., 2002. Cathepsin B inhibition prevents trypsinogen activation and reduces pancreatitis severity. Am. J. Physiol. Liver Physiol. 283, G794- G800. Van Assche, F., Clijsters, H., 1986. Inhibition of photosynthesis in Phaseolus vulgaris by treatment with toxic concentration of zinc: Effect on ribulose- 1,5-bisphosphate carboxylase/ oxygenase. J. Plant Physiol. 125, 355-360. Van der Werf, H.M.G., 1996. Assessing the impact of pesticides on the environment. Agric. Ecosyst. Environ. 60, 81-96.

Varlan,Maulana A., Hillebrand, Azad Library, M., 2010. Aligarh Bovine Muslim and humanUniversity serum albumin interactions with 3-carboxyphenoxathiin studied by fluorescence and circular dichroism spectroscopy. Molecules 15, 3905-3919. Vasiljeva, O., Papazoglou, A., Krüger, A., Brodoefel, H., Korovin, M., Deussing, J., Augustin, N., Nielsen, B.S., Almholt, K., Bogyo, M., Peters, C., Reinheckel, T., 2006. Tumor cell–derived and macrophage-derived cathepsin b promotes progression and lung metastasis of mammary cancer. Cancer Res. 66, 5242-5250. Vatamaniuk, O.K., Bucher, E.A., Ward, J.T., Rea, P.A., 2002. Worms take the “phyt ” t f “phyt ch t ” T B t ch 20 61-64.

326 | P a g e

Bibliography

Villar-Pique, A., Sabate, R., Lopera, O., Gibert, J., Torne, J.M., Santos, M., Ventura, S., 2010. Amyloid-like protein inclusions in tobacco transgenic plants. PLoS One 5, e13625. Voller, A., Bartlett, A., Bidwell, D.E., 1978. Enzyme immunoassays with special reference to ELISA techniques. J. Clin. Pathol. 31, 507-520. Waldron, C., Wegrich, L.M., Ann Owens Merlo, P., Walsh, T.A., 1993. Characterization of a genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor. Plant Mol. Biol. 23, 801-812. Walsh, T.A., Strickland, J.A., 1993. Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains. Plant Physiol. 103, 1227-1234. Wang, C.-X., Yan, F.-F., Zhang, Y.-X., Ye, L., 2007. Spectroscopic investigation of the interaction between rifabutin and bovine serum albumin. J. Photochem. Photobiol. A Chem. 192, 23-28. Wang, H.C., Pao, J., Lin, S.Y., Sheen, L.Y., 2012. Molecular mechanisms of garlic-derived allyl sulfides in the inhibition of skin cancer progression. Ann. N. Y. Acad. Sci. 1271, 44-52. Wang, J., Chen, C., 2009. Biosorbents for heavy metals removal and their future. Biotechnol. Adv. 27, 195–226. Wang, J., Zhang, H., Zhang, T., Zhang, R., Liu, R., Chen, Y., 2015. Molecular mechanism on cadmium-induced activity changes of catalase and superoxide dismutase. Int. J. Biol. Macromol. 77, 59-67. Wang, J., Zhang, W., Qi, S., Guan, Y., Lu, J., 2014. Preparation and luminescence characteristics of Eu-doped calcium chloride silicate

Ca7Si2O8Cl6. J. Alloys Compd. 589, 120-124. Wang, J., Yang,Maulana X., Wang, Azad J., Library,Xu, C., Zhang, Aligarh W., Muslim Liu, R., University Zong, W., 2016. Probing the binding interaction between cadmium( II ) chloride and lysozyme. New J. Chem. 40, 3738-3746. Wang, K.M., Kumar, S., Cheng, Y.S., Venkatagiri, S., Yang, A.H., Yeh, K.W., 2008. Characterization of inhibitory mechanism and antifungal activity between group-1 and group-2 phytocystatins from taro (Colocasia esculenta). FEBS J. 275, 4980-4989. Wargovich, M.J., Woods, C., Eng, V.W., Stephens, L.C., Gray, K., 1988. Chemoprevention of N-nitrosomethylbenzylamine-induced esophageal cancer in rats by the naturally occurring thioether, diallyl sulfide. Cancer

327 | P a g e

Bibliography

Res. 48, 6872-6875. Watlaufer, D.B., Malik, S.K., Stoller, L., Coffin, R.L., 1964. Nonpolar group participation in the denaturation of proteins by urea and guanidinium salts. model compound studies. J. Am. Chem. Soc. 86, 508-514. Wattenberg, L.W., Sparnins, V.L., Barany, G., 1989. Inhibition of N- nitrosodiethylamine carcinogenesis in mice by naturally occurring organosulfur compounds and monoterpenes. Cancer Res. 49, 2689-2692. Weber, K., Osborn, M., 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412. Weber, N., Andersen, D., North, J., Murray, B., Lawson, L., Hughes, B., 1992. In vitro virucidal effects of Allium sativum (Garlic) extract and compounds. Planta Med. 58, 417-423. Wills, E.D., 1956. Enzyme inhibition by allicin, the active principle of garlic. Biochem. J. 63, 514-520.

Wisniewski, K., Zagdanska, B., 2001. Genotype‐dependent proteolytic response of spring wheat to water deficiency. J. Exp. Bot. 52, 1455-1463. Wolfson, J.L., Murdock, L.L., 1987. Suppression of larval Colorado potato beetle growth and development by digestive proteinase inhibitors. Entomol. Exp. Appl. 44, 235-240. Wu, J, Haard, N.F., 2000. Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 127, 209-220. Wu, T., Wu, Q., Guan, S., Su, H., Cai, Z., 2007. Binding of the environmental pollutant naphthol to bovine serum albumin. Biomacromolecules 7, 1899-1906. Yamamoto,Maulana M., Toda,Azad M.,Library, Tanaka, Aligarh K., Sugita, Muslim T., Sasaki, University S., Uneyama, C., Morikawa, K., 2007. Study on usage of pesticides in various countries. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku 92–100. Yang, J., Wang, T., Yang, J., Rao, K., Zhan, Y., Chen, R.B., Liu, Z., Li, M.C., Zhuan, L., Zang, G.H., Guo, S.M., Xu, H., Wang, S.G., Liu, J.H., Ye, Z.Q., 2013. S - allyl cysteine restores erectile function through inhibition of reactive oxygen species generation in diabetic rats. Andrology 1, 487- 494. Yasuda, Y., Kaleta, J., Bromme, D., 2005. The role of cathepsins in osteoporosis and arthritis: Rationale for the design of new therapeutics. 328 | P a g e

Bibliography

Adv. Drug Deliv. Rev. 57, 973-993. Yeh, Y.-Y., Liu, L., 2001. Cholesterol-lowering effect of garlic extracts and organosulfur compounds: Human and animal studies. J. Nutr. 131, 989S- 993S. Yoshida, S., Kasuga, S., Hayashi, N., Ushiroguchi, T., Matsuura, H., Nakagawa, S., 1987. Antifungal activity of ajoene derived from garlic. Appl. Environ. Microbiol. 53, 615-617. Yoshimura, N., Kikuchi, T., Sasakis, T., Kitahara, A., Hatanaka, M., Murachiv, T., 1983. Two distinct Ca2+ proteases (Calpain I and Calpain 11) purified concurrently by the same method from rat kidney. J. Biol. Chem. 258, 8883-8889. Yousuf, S., Ahmad, A., Khan, A., Manzoor, N., Khan, L.A., 2011. Effect of garlic-derived allyl sulphides on morphogenesis and hydrolytic enzyme secretion in Candida albicans. Med. Mycol. 49, 444-448. Yu, W., Santhanagopalan, V., Sewell, A.K., Jensen, L.T., Winge, D.R., 1994. Dominance of metallothionein in metal ion buffering in yeast capable of synthesis of (gamma EC)nG isopeptides. J. Biol. Chem. 269, 21010- 21015. Yue, Y., Dong, Q., Zhang, Y., Li, X., Yan, X., Sun, Y., Liu, J., 2016a. Synthesis of imidazole derivatives and the spectral characterization of the binding properties towards human serum albumin. Spectrochim. Acta. A. Mol. Biomol. Spectrosc. 153, 688-703. Yue, Y., Sun, Y., Yan, X., Liu, J., Zhao, S., Zhang, J., 2016b. Evaluation of the binding of perfluorinated compound to pepsin: Spectroscopic analysis and molecular docking. Chemosphere 161, 475-481. Yue, Y., Wang, Zhiyue, Wang, Zhixian, Zhang, Y., Liu, J., 2018. A comparativeMaulana study Azad of binding Library, properties Aligarh of Muslim different University coumarin-based compounds with human serum albumin. J. Mol. Struct. 1169, 75-80. Yue, Y., Zhang, Y., Zhou, L., Qin, J., Chen, X., 2008. In vitro study on the binding of herbicide glyphosate to human serum albumin by optical spectroscopy and molecular modeling. J. Photochem. Photobiol. B Biol. 90, 26-32. Yusa, V., Millet, M., Coscolla, C., Roca, M., 2015. Analytical methods for human biomonitoring of pesticides. A review. Anal. Chim. Acta 891, 15- 31.

329 | P a g e

Bibliography

Zeeuwen, P.L.J.M., van Vlijmen-Willems, I.M.J.J., Hendriks, W., Merkx, G.F.M., Schalkwijk, J., 2002. A null mutation in the cystatin M/E gene of ichq mice causes juvenile lethality and defects in epidermal cornification. Hum. Mol. Genet. 11, 2867-2875. Zenk, M.H., 1996. Heavy metal detoxification in higher plants - a review. Gene 179, 21-30. Zhang, G., Que, Q., Pan, J., Guo, J., 2008a. Study of the interaction between icariin and human serum albumin by fluorescence spectroscopy. J. Mol. Struct. 881, 132-138. Zhang, G., Wang, A., Jiang, T., Guo, J., 2008b. Interaction of the irisflorentin with bovine serum albumin: A fluorescence quenching study. J. Mol. Struct. 891, 93-97. Zhang, H.M., Chen, J., Zhou, Q.H., Shi, Y.-Q., Wang, Y.Q., 2011. Study on the interaction between cinnamic acid and lysozyme. J. Mol. Struct. 987, 7-12. Zhang, H., Jiang, Y., He, Z., Ma, M., 2005a. Cadmium accumulation and oxidative burst in garlic (Allium sativum). J. Plant Physiol. 162, 977-984. Zhang, H., Xu, W., Guo, J., He, Z., Ma, M., 2005b. Coordinated responses of phytochelatins and metallothioneins to heavy metals in garlic seedlings. Plant Sci. 169, 1059-1065. Zhang, X., Li, L., Xu, Z., Su, J., Li, B., Huang, J., 2014. Studies on the interaction of naringin palmitate with lysozyme by spectroscopic analysis. J. Funct. Foods 8, 331-339. Zhang, X., Liu, S., Takano, T., 2008. Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance. Plant Mol. Biol. 68, 131-143. Maulana Azad Library, Aligarh Muslim University Zhao, Y., Botella, M.A., Subramanian, L., Niu, X., Nielsen, S.S., Bressan, R.A., Hasegawa, P.M., 1996. Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitory activities than a constitutive homolog. Plant Physiol. 111, 1299-306. Zhou, J., Ueda, M., Umemiya, R., Battsetseg, B., Boldbaatar, D., Xuan, X., Fujisaki, K., 2006. A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity. Insect Biochem. Mol. Biol. 36, 527-535.

330 | P a g e