Chapter -1

Introduction of Superoxide dismutase (SOD)

Superoxide dismutase (EC: 1.15.1.1) is an endogenous metalloenzyme mainly Cu-Zn SOD, Fe SOD and Mn SOD present in all aerobic system. It acts as an antioxidant and protects the cell from toxic, harmful ROS generated in biological systems. SOD plays major role of dismutation of ROS into non harmflil species O2 and H2O2. This is an essential redox enzyme having broad spectrum of applications such cosmetics, medicine, sports nutrition and anti-inflammatory drug etc. However the natural enzyme have certain limitations such as yield, time span, thermal stability and storage condition. This paradigm has been overcome up to certain extent using synthetic chemistry called as artificial or enzyme mimic. The SOD mimic are the stable, low molecular weight compounds possessing SOD like activity. SOD mimic could either be organic molecules, metal complexes, and nanoparticles, fiinctionalised NPs, or immobilized SOD on various supports. 1.1 Introduction All living beings on planet earth for their survival of life essentially require water, oxygen and light. In an aerobic ecosystem, in the presence of these factors a number of bio transformations takes place in the biological systems. A hundreds of multi-purpose specific enzymes are catalysing these bio transformations to run the smooth and efficient 'Chemistry of life'. Enzymes are a globular made up of linear chains of residues (ranges from 62 to over 2500 residues) folds to produce a three dimensional primary, secondary, tertiary and quaternary structure of proteins'. The specificity of the enzyme depends on the sequence of these 20 amino acids, coenzymes and which also determines the structure and catalytic activity. So the enzymes are highly specific in action and play a vital role in life. They are classified on the basis of general class of reactions that they catalyse.

Classification of enzyme Table no.l: Classification of enzymes

Class Reaction T\T)e Subclass Dehydrogenases, Peroxidases, 1 Oxidoerductase 1 reductases, oxidases, Mono and dioxygenases Glycosyl transferases, 1 Transferases 1 amino tran sfer ase s, AH (. A B t Phosphotransferases Esterases, Glycosidases, •BSCDJB^^^^H ^ n MyO A'^* R I'>»4 Hiosphatases, Peptidases, Amidases C-C , C-O, C-N and C-S bond 1 1 1 Lyases 1 0K-» lyases Epimerases, Cis trans isomerases ..- .. . . f" y^ 1 J I'.t-t-j^ L (•^ I 1

! *' X A C-..IJ,C >fM> C-C, C-O, C-N and C-S bond ^ ligases A B

The above pie chart^ indicates that, a class of enzymes Hydrolases, Transferases and oxidoreductase have a major contribution of compared to other classes of enzymes. Above all, Oxidoerductase is a major class of enzyme also known as antioxidants, acts as defence system in living beings. Antioxidants are distributed in two types namely Fig. 1.1: Pie chart of enzyme Primary defence antioxidants and Secondary defence antioxidants. Primary defence antioxidants directly react or catalyse the ROS. It includes Superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), glutathione reductase (GTx) and some minerals like Se, Mn, Cu, and Zn. Secondary defence antioxidants only scavenges the generated ROS. It includes as lipophilic enzymes, phospholipases, proteolytic enzymes, proteases, peptidases, DNA repair enzymes, endonuclease, exonuclease, and ligase enzyme^' '*.

In an aerobic system. Superoxide dismutase^'^ (EC: 1.15.1.1) is an endogenous enzyme, acts as primary antioxidant. It protects the cell and other parts from the harmful attack of ROS generated in biological transformation. It is dismutate ROSinto biologically essential dioxygen and hydrogen peroxide. In short, antioxidant SOD scavenges the harmful reactive oxygen species (ROS) and coverts into neutral molecules. Since 1976, there are around 30,701 pubmed-indexed research papers published on SOD enzyme out of which 16609 were published in last decade and 2260 in the last year 2016. There are around 209 US patents granted on SOD^. In present scenario the SOD related inventions and novelties are at its peak due to its property and uses.

1.2 occurrence of SOD

i. Occurrence of SOD in plants

CallW«» Cu-2nSOO :5 ^ r) C

XaiiiiirtV- p \9»m*ei»nt9ooy (\Ukn9iaO J

Fig. 1.2: Schematic diagram of SOD in plant SOD is present in almost all parts of plants with varied concentration. Generally it is located in the mitochondria, chloroplast, apoplast, perioxosomes and cell wall of the nucleus^. The SOD enzyme is isolated from different parts of plant like leaves, roots, shoots, seeds, fhiits of numerous sources. The reported SOD sources were tobacco {Nicotiana tabaccumf, mung beans (Vigna mungoY^, watermelon and citrus {Citrullus vulgaris)^^, petals of carnations {Dianthus caryophyllus)^^, peas (Pisum sativumf^, s^mach{Spinacia oleracedf'*'^^, Jackson et al. The rhizomes of the Zingiberaceae (ginger) family rhizomes from C aeruginosa in dietary- based medical applications'^, Fe-SOD was isolated from tomato leaves {Lycopersicon esculentumY^, A peroxisomal Mn SOD was isolated in pea {Pisum sativumy^ for the removal

3 of O2 formed as a result of xanthine oxidase action. The seeds and seedHngs of com (Zea mays)'^, oats (Avena sativay^, and peas {Pisum sativumY^ were used for SOD isolation. Also SOD was isolated Soyabean roots nodule'^, Tissues of Spinach, kiwi Fruit^^ and apples and were used for assay of SOD enzyme.

Occurrence of SOD in animals and human beings

In an abundant molecular oxygen environment during body metabolism spin restriction and reduction of oxygen may occur generating a hazardous ROS as by-product. Superoxide dismutase catalyse, these ROS into O2 and H2O2. SOD is present in eukaryotes and prokaryotes of aerobic ecological system. Fe and Mn SOD are characteristic of prokaryotes. Cu-Zn SOD are characteristics of cytosol eukaryotes. There three types of SOD's are present in humans. The SOD activity varies with tissues. The highest SOD activity was observed in liver, adrenal gland, kidney and spleen. Mn SOD in mitochondrial matrix was isolated from yeast, chicken liver, rat liver, human liver, baboon liver and pig heart ^'. In cytosol both Cu-Zn SOD and Mn SOD were observed.

1.3 Classification of SOD's SOD (EC: 1.15.1.1) was first isolated in 1938 by Mann and Kleilin. Earlier it was considered as copper storage but in 1963, I. Fridovich^^ isolated a protein from eukaryotes and reported as SOD enzyme. Broadly SOD enzyme is classified into three types encompasses Fe- SOD, Cu-Zn SOD and Mn SOD depending on metal ion as cofactors. Copper-Zinc Superoxide dismutase (Cu-Zn SOD)

Fig.l .3: Ribbon diagram of Cu-Zn SOD

In plant cells, Cu-Zn SOD is a soluble enzyme and is located in the stroma, nucleus and apoplast ^^••^*. In green plants, several isozymes of Cu Zn-SOD have been detected and isolated from both extracellular and subcellular locations, such as the chloroplasts, cytosol, mitochondria and peroxisomes^^'^^. The Cu-Zn SODs in chloroplasts or the cytosol have been isolated from various green plants observed the difference in their amino acids residues, absorption spectra in visible light, circular dichroism spectra, sensitivity to inactivation by hydrogen peroxide and immunological properties'^. In animals'^ Cu-zn SOD is denoted as (SODl). A Cu-Zn SOD were found in both eukaryotes and prokaryotes. They are p barrel proteins with either homodimeric or monomeric binuclear metal ions at the centre.

Iron Superoxide dismutase (Fe SOD):

Fig. 1.4: Ribbon diagram of Fe SOD

Fe SOD is almost present in chloroplast in many plants, may associate with the plastid nucleoid and participate in signalling or gene regulation. It is also located in cytoplasm of cowpea. The presence of Fe SOD activity in Gingkoaceae, Nymphaceae, and Cruciferae was concluded to be a random occurrence of the enzyme in the plant kingdom'^. Comparative studies with Fe SODs showed that three primary chemical functions are necessary for Fe SOD to catalyse a dismutation reaction^":

1. Availability of at least one coordination site of Fe centre for binding O2" between two adjacent states.

2. The ion redox couple must be lie between redox potentials of O2" oxidation (0.16 v) and O2" reduction (0.89 v) in primary coordination sphere surrounded by protein matrix.

3. The rapid conversion between the Fe (II) and Fe (III) oxidation states is must at Fe centre than the spontaneous dismutation reaction^'. Manganese Superoxide dismutase (Mn SOD):

Fig. 1.5: Ribbon diagram of Mn SOD

Mn SOD's are located in mitochondria, chloroplasts and in the peroxisomes with homo di or tetrameric subunit at its active centre^^. Mn and Fe SODs have a similarity in the structure, but differ in their activity^^. The mechanism of Mn SOD was well explained by Bowler et al. 1994. by Mn SODs is through the attraction of negatively charged O2" molecules to a site formed from positively charged amino acids present at the active site of the enzyme, similar to Cu-Zn SODs^"*. The metal present in the active site reduces one O2" molecule on an electron donation, which in turn forms H2O2 with a proton.

Nickel Superoxide dismutase (Ni SOD):

Fig. 1.6: Ribbon diagram of Ni SOD

An entirely new class of SOD is established as Ni SOD found in Streptomyces and cyanobacteria with a different metal centre and distinct protein fold. Ni SOD is small protein having 117 amino acids with no any homology sequence compared to other SOD's^^. A tetramer of Ni is functioned as Ni SOD where its monomeric form is helpless. It was observed from X-ray and EPR studies that Ni is in oxidised Ni^^ form in Streptomyces seoulensis^^. Its biological function helps in regulation of reactive oxygen species.

1.3 Applications of SOD In aerobic life, nature has SOD enzyme as most potent antioxidant enzyme to fight against ROS. It plays crucial role of protection of a cell and extra cellular parts fi-omharmfu l reactive oxygen species (ROS). Decrease or less concentration of SOD causes many hazards to living beings. It is administered parentally because of its bio-availability. It acts as a catalyst during dismutation of ROS indicates high affinity and rate of reaction. It has enormous applications in both plants and human beings.

1. First and foremost application of SOD is to use as superoxide O2" sensor'^.

2. It is used for preservation of fhiits and vegetables. It avoids early ripening on exposure to sunlight ^l

3. SOD is extensively used in cosmetic industry, for commercial formulations of moisturizers, sunscreens, nail polish, anti-hair fall sprays skin-lightening creams, and eye creams. Frequently used in lipsticks and facial creams. In sunlight exposure, these cream have tendency to generate ROS which further causes haemolysis and lipid peroxidation in human erythrocytes^^

4. SOD formulations are used in products of cigarette and tobacco to avoid the damage caused by ROS in oropharynx and respiratory tract *° also used as a refresher for alcoholic to prevent alcohol induced hangover'*'.

5. Beyond this it has been widely used as wound healing, Anti-aging and sports nutrition by decreasing lactic acid formation.

6. It is used for elongating the time span of transplantation organs ''^ and sperms '^^.

7. In plants, increased SOD concentration helps to protect the plants against environmental stress (drought, chilling, salinity and over exposure to sunlight etc.) and chemical stress (ozone, free radicals, metal ions and oxygen generating herbicides etc.)'*'*.

8. Implementation of high dose of SOD enzyme will improve the biomass production with its flora^5,46,47

9. In microorganism assisted fermentation industries where air oxygen was used as a flux caused the generation of ROS level was increased. So to avoid attack on such food stuff has been avoided using SOD application i.e. It acts as a preservative. Hence we have focused our studies on Superoxide dismutase (SOD) isolation from some new source and synthesis of models of SOD as a SOD mimics.

1.5 Generation and significance of ROS in plants and animals

0, + e- O, (1) .02 + .02- + 2H^ 21£iSODW H2O2+ O2 (K2 = 2.4 X 10'm-^s 1) (2)

H2O2 + •Oi -^ OH-+ "OH +0, (3)

CAT H2O2 + H2O2 -• 2H2O + O2 (Ki = 1.7xl0''in-'s-0 (4)

Fig. 1.7: Generation of ROS

In plants, ROS are the by-products generated in different compartments of the cells including mitochondria, chloroplasts, microsomes, glyoxyomes, peroxyomes, apoplast and cytosol during electron transport activities, in metabolic pathways and various biochemical reactions occurred. These are the molecules derived from O2 includes free radicals, ion and reactive species such as 'O2, O2'", H2O2 and 'OH''^. ROS plays two different major roles in plants; in low concentrations they act as signalling molecules that mediate several plant responses in plant cells, including responses imder stresses, whereas in high concentrations they cause harmful damage to cellular components'*^. In plants enhanced production of ROS was caused due to ozone exposure, metal toxicity, exposure to radiation, wounding, chilling, drought, salinity, heat, abiotic and biotic stress, pathogens and senescence^^ which results in membrane rigidity, lipid peroxidation, protein denaturation and DNA mutation^' as well as leaf senescence, wilting of cut flowers and post harvested fruit spoilage^^' ^^' ^'*' ^^. So these excess production of ROS are prevented by several defence mechanisms, both enzymic and nonenzymic. These mechanisms are emerged to protect the cells against these destructive oxidative injury. The nonenzymic antioxidants are GSH, cysteine, hydroquinone's, mannitol, vitamins C and E, alpha-tocopherol, and phenolic compounds, flavonoids, some alkaloids, p- carotene, taimins and lignin precursors'^. In human beings, ROS are the most dangerous free radicals firmed as by-products formed in biological reactions and initiate many chain reactions. ROS can be generated in body endogenously and exogenously. The endogenous sources are mitochondria, cytochrome P450 metabolism, peroxisomes, neutrophils, eosinophil and macrophages and inflammatory cell activation^^, whereas exogenous sources are metal catalysed redox reactions, xenobiotics, chlorinated compounds, UV light. X-rays, y-rays and atmospheric pollutants^^. These ROS plays dual role similar to plants. They are either beneficial or destructive depends on their environment and concentration^^'^^. Beneficial ROS plays fimctionalrol e in cellular responses such as defence against infections and in signalling system. In reverse, excess concentration of ROS leads to Oxidative stress and causes damage to cells, lipids, membranes, proteins and also to nucleic acids. Many highly detrimental effects are observed. It has been associate with aging and age related diseases such as cardiovascular diseases, eye disorders, joint disorders, neurodegenerative disorders (Parkinson, Alzheimer, lateral Sclerosis) cancer, lungs disorders, damages kidney, liver and pancreas, diabetes, damages male and female reproductive system with infertility observed and other chronic conditions^'.

Fig. 1.8: General mechanism of SOD SuperoxidI e 02'- Superoxide dismutase SOD

Oxygen /s Hydrogen Peroxide O2 IH2O 2 Catalase CAT 7^ Oxygen Water 02 H20 1.6 Role of Superoxide dismutase enzyme (SOD)

Action of SOD

In an aerobic ecosystem, the organisms survive in an oxygen rich environment. In aqueous media, during normal cell metabolism highly reactive ROS are generated as product of single electron reduction of molecular oxygen. Mitochondria is the major source for ROS generation. These superoxide radical carmot accumulated in aqueous media, it spontaneously undergo disproportion in dismutation. Fridovich observed that, at pH 7.4 the rate constant for the spontaneous dismutation (2 x 10^ m'/sec') is 10'* times advantageous the rate constant for 02- generation is about 2 x 10^ M'Vsec"'. The concentration of dismutase enzyme is much higher than the steady state concentration of generation of O2 • radical. This leads to keep the dismutation rate higher than collision of O2 • radicals among them.

Action of SOD in plants

Superoxide dismutase (EC 1.15.1.1) is an ambiguous class of metalloenzyme present in all aerobic organisms as an effective first line of cell defence against superoxides produced while following single electron reductions of molecular oxygen^. In an oxygen rich atmosphere SOD plays a pivotal role to disproportionate those potentially harmful and deleterious superoxides or Reactive Oxygen Species (ROS) to non-harmful oxygen and hydrogen peroxide by following the mechanism of sequential reduction and oxidation at metal centre.

In stroma of chloroplast, electron was removed from H2O and transferred to weak electron acceptor CO2 in sunlight during photosynthesis. The carbohydrates and oxygen were the product formed. This called Calvin Photosynthetic Carbon Reduction (PCR) Cycle. In this reduction process there are fare chances of occurrence of ROS production and consecutively its dismutation by SOD.

Fig. 1.9: Photosynthesis with mechanism

10 The e" transfer mechanism in chloroplast involved photosystem II (PS-II), cytochrome be-/ complex and photosystem I (PS-I). A chloroplast consist of thylakoid membrane having pigments which captured the light as a source of energy from sunlight. The absorbed light excites the electron of pigments from photosystem II to photosystem I using cytochrome b6 as e" carrier. In a short time, (few nano seconds) an excited state e' emits energy in terms of heat / fluorescence and comes to ground state. In this process ADP is converted to ATP with loss of e'. So two electron transfer process occurred. If Ferridoxin content in chloroplast is low resuhs into production of ROS. They act as both oxidant and reductant. As a reductant they react further with O2 and generate more ROS such as superoxide O2", peroxide, OH", singlet O2. Whereas, as oxidant they carried out oxidation of polyphenols, thiols, and tocopherols, denatures catalase, peroxidase. Hence these dangerous ROS can be easily dismutate into usefiil O2 and H2O2 using SOD enzyme. Finally, similar to mitochondria, electron is transferred reduction of NADP to NADPH takes place^l

The enzymatic antioxidant defences include Superoxide dismutase (SOD), ascorbate peroxidase and GSH reductase, Catalase, the CATs and peroxidases removes H2O2 very efficiently and SODs scavenges the superoxide anion O2 • to H2O2 and water to avoid cell damage^^ . The CATs and SODs are the most efficient antioxidant enzymes. The enzymatic antioxidant defences include enzymes capable of removing, neutralizing, or scavenging free radicals and oxyintermediates. In progression, the enhanced SOD level was observed in correlation with increase in oxidative stress in plants for the cell protection. Consequently, the combined action of SOD and CAT terminate the formation of the most toxic and highly reactive oxidant^'*. Another function of superoxide dismutase is to protect dehydratases (dihydroxy acid dehydratase, aconitase, 6-phosphogluconate dehydratase and fumarases A and B) against inactivation by the free radical superoxide^^. The current status of SOD in plant, need of critical overview and employing more interdisciplinary approach, applications in coming days. In current review we focused on the role of an endogenous antioxidant SOD enzyme in plant in terms of its occurrence, basic mechanism, increase and decrease in SOD level and its significance. We also put forth with some new insights and challenges of SOD.

Action of SOD in animals and human beings SOD enzyme is an endogenous enzyme present in nearly all cells of body. The percent of SOD level is higher in the liver^^ of human being. The acts as antioxidant i.e. dismutation of ROS into non harmful H2O2 and O2. Decrease in SOD level will be the cause for various other diseases. So it's very essential to maintain the SOD level in body.

11 1.7 Biological Assay for SOD enzyme

A specific enzyme assay is the basic requirement of productive study of any enzyme. It should be comprehensive and reveaUng. In aqueous medium, production rate of O2" is high (short Hfe span) and very low scavenging rate, which may damage the cells during this nonspecific oxidation. So relatively short life span of O2" radical cannot be reacted with substrate like usual enzyme assays. The SOD activity is determined by two ways: a) Direct method which is based upon decay kinetics of pulse generated by O2' and b) Indirect method which is based on inhibition of oxidation sensitive substrates like xanthine, NBT, Cytochrome c. In literature more than 20 different direct and indirect methods were described for SOD activity^^' ^^. Pulse radiolysis is the only direct method for the SOD assay. It is based on paramagnetic and U.V. absorption properties of O2". The order and the rate constant of a decay of concentration O2" has been determined using stop flow and continuous flow method in presence of compound being tested. This is the most accurate but inconvenient method over the other methods^^. In 1969, McCord and Fridovich^" introduced first spectrophotometric SOD assay with Xanthine / Xanthine oxidase system and native cytochrome c as indicating scavenger. Further the assay has been modified and developed for the extracellular SOD and SOD in mitochondria using ferrichrome c as indicating scavenger at pH 10 by Kuthan" [et al 1986]. Ascorbic acid was also used as superoxide radical scavenger for SOD activity^^. A distinctive property of SOD enzyme is to convert reduced oxygen 02' (superoxide anion radical) to molecular oxygen and H2O2 extremely rapidly. This autoxidation property of superoxide radical helps as a usefiil probe for the study of involvement of radical using oxygen. Thus involving superoxide prone autoxidations is the convenient and rapid aid for SOD enzyme assay. At suitable pH the rate of autoxidation varied with the concentration of SOD enzyme. Marklund and co-workers^' described SOD assay based on ability of enzyme to inhibit autoxidation of pyrogallol in presence of EDTA at pH 7. Similar SOD assay was performed using epinephrine, DPPH^'' etc as a substrate. Buchmap and Fridovich^^ developed a photochemical method using indicator nitro blue tetrazolium (NBT). KO2 in dmso was used as source for superoxide generation in presence or absence of crown ether. The reduction of yellow coloured NBT to blue coloured formazan in phosphate buffer was monitored at 560 nm.

12 1.8 Characterization of SOD enzyme

The enzyme was characterized using various techniques. Now a days highly sophisticated instruments helps to analyse the compounds in short time with accuracy. A UV-Vis. Spectrophotometer is the best tool for the early predictions of proteins.

Determination of proteins: Routinely Lowry et al?^ (1951) method and Bradford method^^ was used for protein determination using bovine serum albumin as a standard.

Determination of Carbohydrates: Anthrone's reagent^^ and Phenol-sulphuric acid method ^^ were practised to estimate the carbohydrate content of the natural source.

Determination of molecular weight (Mr): To determine the molecular weight {Mr), a slab gel electrophoresis supported on Sodium dodecyl sulphate (SDS) permeated Polyacrylamide gels (12-5%) SDS-PAGE method describe by Laemmli (1970)^'. Commassie blue containing Phosphorylase b, BSA, ovalbumin, trypsinogen and lysozyme are use as Mr markers.

Determination of metal content: Atomic Absorption Spectrophotometer is use to determine presence of the metal and can be calculated as described by Sawada et al (1972)^^.

Determination of protein structure: Single crystal technique is used to determine the exact structure of enzyme.

1.9 SOD mimics

Biomimics is an emerging interdisciplinary field in chemistry and biology in which lessons learned from biology form the backbone applied for novel chemical materials. It involves intrinsic investigation of both structures and physical fianctions of biological molecules mainly of interest for human well-being with the goal of designing and synthesizing new and improved molecules. Natural enzymes are proteins with high specific activity, efficiently catalyses many biotransformations successflilly used in many fields to achieve targets. Unfortunately, these enzymes are of high molecular weight, highly unstable, easily denatured by environment condition. In short, high cost of purification and harsh storage conditions of these enzymes narrowed its spectrum of applications. This difficulty is overcome up to certain extent by applying the biomimicking of enzymes. A chemist will design and synthesize a molecule which should be nontoxic, stable, a low molecular weight, high cell permeability with retention of specific activity in vivo. For in vivo studies the molecules should possess the properties like

13 electrostatic, steric, solvation, lipophilicity, bio distribution and stability. The pharmacokinetic studies will help for their therapeutic uses^^. The modelling studies that mimic the active sites of metalloenzymes are important for understanding the reaction mechanism of the and for developing small molecular weight mimetic catalysts^^. However, models are readily cleared by the kidney and are unable to enter cells because of their high molecular weight thus it is difficult, to employ SODs in vivo. But metal ions are responsible for the gaining of energy and maintenance of homeostasis within the cell^''. Transition metal ions in a variety of ligand environments have been implicated in the formation as well as the scavenging of primary and secondary ROS. The presence of copper, manganese, and iron at the active sites of SODs led many investigators to search for low molecular weight complexes of these metals having SOD activity^^. Even though many low molecular weight SOD mimics were reported earlier most of them were not based on the active site structures of Fe, Mn, Cu/Zn and Ni-SOD. Only a few SOD mimics were synthesized by using ligands that have similar donor environments present in the Fe and Mn-SOD active sites^^ In literature search, several reports are available for SOD mimics. There are many methods to make SOD mimic. Researchers reported organic molecules^^, metal complexes^^, nanoparticles^^, biomolecules supported nanoparticles^*' as SOD functional mimic. Mn(III)(salen) complexes, Mn(III)(porphyrinato) complexes, Mn(II) (1,4,7,10,13- pentaazayclopentadecane) and Mn(II) bis(cyclohexylpyridine)- substituted derivatives are fiiture active dugs in pharmacology against superoxide radicals^'. Mn porphyrins complexes are the most efficient model among the other mimics. Mn(III) N-alkylpyridyl porphyrins with were reported for SOD mimics especially when they do not possess any donor sets of ligands and specific coordination geometry at the metal centre. But these complexes showed higher SOD activity if the redox cycle occurs between the oxidation states Mn(III)/Mn(II) or Mn(IV)/Mn(III), when an intermediate achieved redox potential obtained between El/2 close to 0.36 V vs. NHE (0.12 vs. SCE). A very few of these Mn (II) complexes have SOD activity in vivo studies because of the property of lipophilicity and bio distribution in cell^^'^^. Beyond that dinuclear Mn compoimds are even better alternative for pharmaceuticals over reported mononuclear Mn compounds.

14 H I r vCi I H H. I r •!^' I ,H N——Mn—N

^N-—Mn—N M40401 M40404

M40403

Mn"—N

H '^—' H H ^ ^ H Mn(Pyane)Cl2 Mn(Pydine)Cl2

CI Ni

CI CI MnLjCIj MnLjClz

Fig. 1.10: Clinical trial competent SOD mimics of Mn complexes SOD models of Cu (II) imidazole bridged complexes of [(Bipy)2Cu-Im- Cu(Bipy)2](C104)3. CH30H,[(Phen)2Cu-Im-Cu(Phen)2] (BF4)3. 2CH3OH, [(Bipy)2Cu-Im-Zn(Bipy)2](BF4)3 and [(Phen)2Cu-Im-Zn(Phen)2](BF4)3 has been synthesised. These complexes were stable for wide range of pH 8.5-10.5 like the pH range stability for the proteins correlating with biological activation. For the abiotic models was better described in the literature^'*' ^^. A series of Cu(II) complexes^^' ^^' ^^ were prepared with the tridentate ligand bis(2- picolyl)amine as ligand and various anions (CI", Br", CH3C00', 804^', NO3' and CIO4') as a promising fiinctional model of SOD. Out of these, a water soluble chloro complex has a good anticancer activity, ability to cleave the genomic DNA under aerobic condition at room temperature.

15 + ©2 + HX

+ H2O2

Fig. 1.11: Mechanism of Cu (II) complexes The reaction sequence for the catalytic disproportionation of O2 • by copper (II) complexes. Recently, nanoparticle^^' ^^ showing intrinsic enzyme like activity is growing area of interest. Compared with natural enzymes they are stable, nontoxic, low cost and importantly resistant to high concentrations. Even though SOD is a potent antioxidant, it has certain problems yet to improve. Generally SOD is administered orally but the now modified to other ways. The enzyme stability was extended on adsorption on various bio inspired molecules such as nanoparticles of Cds were used as a supported for cytochrome P450 enzyme"". SOD was encapsulated with semipermeable vesicles designed as a nanoreactor'^^. A modified compounds SOD good results in oral and respiratory administration. The membrane of our triblock copolymer Nano vesicles plays a dual role, both to shield the sensitive protein and to selectively allowed superoxide and dioxygen penetrate to its irmer space. It helps to improve the oral administration of SOD which can be easily destroyed by gastric acidity earlier.

Fig. 1.12: Schematic representation of Liposome encapsulated SOD Similar ways, in future biomolecules like cyclodextrin, chitosan, d-glucose like natural will make better SOD model for more easy and proficient administration of SOD not only for SOD enzyme but also for metal complexes.

16 ";Zn OH,

a.

Fig.1.13: Encapsulation of complexes with biomolecules

A nanoparticles such as CdS"'^, Ti "^'*, SOD immobilzed on supermagnetic'"^ Fe nanoparticles were also reported as effective SOD mimic. Au, Ag nps were used a peroxidase model.

Q Ag*

\» Ag ^ A ]«StAg Ag^ gjAg AChE ChOx H n lg^8 "2":

: ACh • : choline ^ : Au NP ——. : PEG • : ATP -f : AUR ^ : AUR product

Fig. 1.14: Au an Ag nps peroxisdase activity'^^

In future, other several nps, oxide nanoparticles will serve as good SOD mimic. It will be active at high temperature and helps in targeted drug delivery.

1.10 Summary

Enzymes are essential in biological systems. It plays a key role in biological transformations. They are classified into 7 types, out of these oxidoreductase contribute majorly. In aerobic ecology Superoxide dismutase (SOD) is a primary defence antioxidant enzyme from oxidoreductase class. It converts the harmful reactive oxygen species (ROS) to neutral molecules O2 and H2O2. SOD enzyme is a metalloenzyme divided into three type Cu-Zn SOD, Fe SOD and Mn SOD. Endogenous SOD enzyme is present in a cell of plants and animals with varied concentration. Cu-Zn SOD and Mn SOD's were usually found in plants []. In plants, SOD is present in chloroplast, apoplast, cytosol and mitochondria. In all, SOD concentration is high in mitochondria due to high oxygen source. Also, in seeds the SOD concentration level

17 is quite high to protect the embryo from dangerous, deleterious ROS species and natural calamities. In animals and human, Cu-Zn SOD (SODl) is generally present in major amoimt in a body. Mn SOD was located in extracellular part as EC-SOD. Occurrence of Fe SOD (SOD 2) is also observed with varied oxidation states. Similarly SOD enzyme is present various parts in animals.

Action of SOD was to convert generated superoxide into non harmfiil oxygen and hydrogen peroxide, in short dismutate the harmful ROS into usefiil oxygen and hydrogen peroxide.

Direct and indirect assays were developed for accurate SOD activity. Pulse radiolysis is a direct assay gives accurate SOD activity but it's not preferred. In indirect method auto-oxidations were carried out using superoxide source. Inhibition of these autoxidation is the measure of SOD activity. In this NBT assay using KO2 in dmso as superoxide source is most promising and easy assay compared with the others. The SOD enzyme can be characterized using techniques such as SDS Gel electrophoresis using Commassie marker for molecular weight determination, Atomic Absorption Spectrometry (AAS) for metal determination, Lowrys method for protein determination, U.V.-visible spectrophotometer. Circular dichroism (CD), MALDI, Electron spin resonance (ESR) etc. These enzymes are active at specific pH and temperature. They are highly unstable. So it's difficult to maintain their activity. This difficulty was partly overcome by biomimicking. It's an interdisciplinary branch, forming a bridge between chemistry and biology. In this small molecules are design and synthesize. Further the properties of these molecules are correlate with the properties of natural enzymes. The good biomimic compoimd should be stable, nontoxic, low molecular weight and importantly should possess the biological activity in both in vitro and in vivo studies. Various ways have been applied to achieve the target. They are organic molecules, metal complexes, nanoparticles, bio supported nanoparticles.

18 1.11 Objective of research project:

Outline of Research work 1 Isolation of SOD SOD mimics Conjugates of SOD from plant

/ \ Cliitosan Oi*ganic molecules Nano materials Metal complexes Graphene oxide

Fig. 1.15: Outline of research work

After carrying through literature search, we have designed our line of research. The project is divided into three parts viz. isolation of SOD enzyme, synthesis of SOD mimics and its application. In first part, we are going to isolate and purify the SOD enzyme from novel plant source. According to properties of plants, a source is going to be selected. The isolated SOD will optimized for temperature, pH and concentration. As efforts were made to increase the stability of SOD enzyme by carrying out adsorption studies on various supports. Concurrently we have designed and synthesized model compounds of SODs. Model compounds are organic molecules, metal complexes and nanoparticles. Model compounds are going to be well characterized using sophisticated techniques. Both isolated SOD and model compounds were followed NBT assay for SOD activity. The assay will be supported with DPPH assay and anticancer activity. In last part, we will carry out application study of all compounds. Finally, we will try to correlate all the study and find out which is better SOD mimic.

19 /12 References

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