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The Investigation of Lactoferrin Nitration: Quantification, Unction,F and Inhibition

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The Investigation of Lactoferrin Nitration: Quantification, Unction,F and Inhibition University of Denver Digital Commons @ DU Electronic Theses and Dissertations Graduate Studies 1-1-2018 The Investigation of Lactoferrin Nitration: Quantification, unction,F and Inhibition Amani Yahya Alhalwani University of Denver Follow this and additional works at: https://digitalcommons.du.edu/etd Part of the Biochemistry Commons, and the Environmental Chemistry Commons Recommended Citation Alhalwani, Amani Yahya, "The Investigation of Lactoferrin Nitration: Quantification, unction,F and Inhibition" (2018). Electronic Theses and Dissertations. 1452. https://digitalcommons.du.edu/etd/1452 This Dissertation is brought to you for free and open access by the Graduate Studies at Digital Commons @ DU. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital Commons @ DU. For more information, please contact [email protected],[email protected]. THE INVESTIGATION OF LACTOFERRIN NITRATION: QUANTIFICATION, FUNCTION, AND INHIBITION __________ A Dissertation Presented to the Faculty of Natural Sciences and Mathematics University of Denver __________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy __________ by Amani Yahya Alhalwani June 2018 Advisor: John Alexander Huffman, PhD ©Copyright by Amani Yahya Alhalwani 2018 All Rights Reserved Author: Amani Yahya Alhalwani Title: THE INVESTIGATION OF LACTOFERRIN NITRATION: QUANTIFICATION, FUNCTION, AND INHIBITION Advisor: John Alexander Huffman, PhD Degree Date: June 2018 Abstract Lactoferrin (LF) is an iron-binding glycoprotein of molecular mass ca. 80 kDa that is predominantly found in mammalian body fluids. Lactoferrin is a multifunctional protein that has a wide range of properties such as antibacterial, antiviral, anti- inflammatory, and anti-allergic functions. Tyrosine residues in the protein play a part in many lactoferrin functions. Protein tyrosine nitration modification represents an oxidative and nitrosative stress process which can be caused by the exposure of proteins to oxidants from air pollution or disease. Understanding the way nitrated lactoferrin interacts with the biochemical environment of the body is thus important to the broader goal of understanding and preventing lactoferrin-related diseases, including certain ocular diseases. The main goal of this set of linked studies was to investigate the process by which LF may be nitrated, to observe changes in its protein structure and function, and to investigate a possible route to block the nitration process. In the first project discussed here, nitrated lactoferrin was synthesized using two different nitration agents: sodium peroxynitrite and tetranitromethane. After nitration, spectroscopic analysis showed formation of nitrotyrosine with an increase in absorbance intensity at 350 nm, as well as attenuation in fluorescence intensity at an excitation wavelength of 280 nm. A sandwich ELISA protocol was developed to quantify the amount of nitrotyrosine in nitrated lactoferrin samples. The results show that the increase ii in the amount of nitrotyrosine depends on the molar ratio of nitrating agents added to the reaction. This study represents the first time that nitrotyrosine in lactoferrin had been selectively detected. In the second project, a broth microplate antibacterial susceptibility assay was applied to determine changes in the antibacterial function of lactoferrin brought about after nitration. The results reveal a reduction in the naturally present antibacterial activity of LF, which scales linearly with the increase in nitration molar ratios. In the third project, ergothioneine was used to inhibit the nitration reaction using several protein models, as well as to examine it as a potential therapeutic target for nitrosative stress-related diseases. The results illustrated that ergothioneine diminished the nitration production and protected the protein antibacterial activity. Collectively, these findings suggest that the nitration of tyrosine residues within lactoferrin can be important with respect to several physiological processes. Thus, further understanding the interaction between nitrated LF and the biochemical environment may aid in future research toward clinical diagnoses and in the development of therapies for diseases involving nitration of lactoferrin. iii Acknowledgements I would like to express appreciation to the mentorship of Dr. J. Alex Huffman. I sincerely appreciate his patience throughout my graduate studies and his precious advice. I am extremely grateful to committee members; Dr. Michelle K. Knowles, Dr. Brian Majestic, and Dr. Rebecca Powell who assisted and guided me through the process to accomplish this graduate study. Also, a big thanks to Dr. Michelle K. Knowles, Dr. John E. Repine, Dr. Brian Cowen, Dr. John Latham, Dr. Scott Horowitz, Dr. Scott Barbee, and Dr. Angila Hebel for their contributions, thoughtful sharing of knowledge and information. I am grateful to Dr. Ying Chen for her invaluable scientific advice and friendship. I would like to thank the funding support from a grant by the University of Denver Knoebel Institute for Healthy Aging (KIHA) and the scholarship support from King Saud bin Abdulaziz University for Health Sciences (KSAU-HS). A special thanks to my friends who provided skillful help and friendship. I am grateful to all past and current members of the Huffman group for their support at all times. I dedicate this work to the memory of my mother in heaven, Mrs. Weedad T. Nusier. I would like to give sincerest thanks to my father, Mr. Yahya A. Alhalwani for his emotional support no matter what path I choose. I would like to extend special thanks to my sisters and brothers for the constant support they've provided to me. Heartfelt thanks to my husband, Dr. Mohammed Khojah, for his patience and continual support of my academic endeavors over the past years. I would like to warmly thank my children who bring me such happiness and keep me on my toes. My children iv have grown up watching me study and manage both family and work. Each has contributed immeasurably to our family happiness in their own special way. I hope I have been a good mother and that I have not lost too much during my studies. v Table of contents Chapter one: Introduction ................................................................................................... 1 1.1 Overview of lactoferrin ..................................................................................... 1 1.1.1 Background of lactoferrin .................................................................. 1 1.1.2 Structure of lactoferrin ....................................................................... 1 1.1.3 Iron binding sites on lactoferrin structure .......................................... 2 1.2 Lactoferrin function .......................................................................................... 3 1.2.1 Lactoferrin function dependent on iron ............................................. 3 1.2.2 Lactoferrin function independent of iron ........................................... 3 1.2.3 The surface properties of lactoferrin .................................................. 3 1.3 Lactoferrin functions. ........................................................................................ 4 1.3.1 Anti-inflammatory properties ............................................................ 4 1.3.2 Antioxidant properties ....................................................................... 4 1.3.3 Antibacterial mechanisms of lactoferrin ............................................ 5 1.4 Protein nitration ................................................................................................ 5 1.4.1 Tyrosine nitration reaction ................................................................. 5 1.4.2 Nitration protein and biochemical properties .................................... 5 1.4.3 Protein nitration health implications .................................................. 6 1.5 LF nitration modification .................................................................................. 6 1.5.1 The effect of chemical modifications to LF function ........................ 6 1.6 Analytical techniques to characterize protein nitration ................................... 7 1.7 Nitration inhibitor ............................................................................................ 8 1.8 Lactoferrin and health ....................................................................................... 9 1.8.1 The effect of peroxynitrite in eye ....................................................... 9 1.8.2 The effect of protein nitration in ocular surfaces ............................... 9 1.8.3 Lactoferrin and disease .................................................................... 10 1.8.4 Lactoferrin role in tears .................................................................... 10 1.8.5 LF role in cornea .............................................................................. 11 1.8.6 The relation between ocular surface health and nitration ................ 11 1.9 Motivation ....................................................................................................... 12 1.10 Hypotheses .................................................................................................... 13 1.11 Objectives ..................................................................................................... 14 1.12 Research Aims .............................................................................................
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