Enhancing the Nitrite Reductase Activity of Modified Hemoglobin: Bis-Tetramers and their PEGylated Derivatives by Francine Evelyn Lui A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Chemistry University of Toronto © Copyright by Francine E. Lui 2011 Abstract Enhancing the Nitrite Reductase Activity of Modified Hemoglobin: Bis-Tetramers and their PEGylated Derivatives Francine Evelyn Lui, Doctor of Philosophy, 2011 Department of Chemistry, University of Toronto The need for an alternative to red cells in transfusions has led to the creation of hemoglobin-based oxygen carriers (HBOCs). However, evaluations of all products tested in clinical trials have noted cardiovascular complications, raising questions about their safety that led to the abandonment of all those products. It has been considered that the adverse side effects come from the scavenging of the vasodilator – nitric oxide (NO) by the deoxyheme sites of the hemoglobin derivatives. Another observation is that HBOCs with lower oxygen affinity than red cells release oxygen prematurely in arterioles, triggering an unwanted homeostatic response. Since the need for such a product remains critical, it is important to understand the reactivity patterns that contribute to the observed complications. Various alterations of the protein have been attempted in order to reduce HBOC-induced vasoconstriction. Recent reports suggest that a safe and effective product should be pure, homogenous and have a high molecular weight along with appropriate oxygenation properties. While these properties are clearly important, vasodilatory features of hemoglobin through its nitrite reductase activity may also act as an in situ source of NO. It follows that HBOCs with an enhanced ability to produce NO from endogenous nitrite may serve to counteract vasoactivity associated with NO- scavenging by hemoglobin. ii Here we characterize the effects of different protein modifications on the nitrite reductase activity of hemoglobin. We produced a variety of HBOCs that include cross- linked tetramers, polyethylene glycol (PEG) conjugates and bis-tetramers of hemoglobin. We report that the rate of NO production strongly depends on the conformational state of the protein, with R-state stabilized proteins (PEG-Hbs), exhibiting the fastest rates. In particular, we found that PEGylated bis-tetramers of hemoglobin (BT-PEG) exhibit increased nitrite reductase activity while retaining cooperativity and stability. Animal studies of BT-PEG demonstrated that this material is benign: it did not cause significant increases in systemic blood pressure in mice, the major side effect associated with existing HBOCs. BT-PEG exhibits an enhanced nitrite reductase activity together with sample purity and homogeneity, molecular size and shape, and appropriate oxygenation properties, characteristics of a clinically useful HBOC. iii Acknowledgements Professor Ronald Kluger supervised and directed this research. The achievements in this research and my personal growth as a scientist are a result of his care, constant guidance and indispensible instruction. I truly thank him, both as a supervisor and as a mentor, for the memorable and irreplaceable time that has been my Ph.D. in the Kluger Lab. In addition, my committee members, the Kluger lab members, and many fellow classmates have also been central to my training as a graduate student. As well, the opportunity to collaborate with Dr. Warren Zapol at Massachusetts General Hospital, Harvard Medical School has been imperative to my research and understanding of physiological animal models. Jonathan S. Foot, Ying Yang and DongXin Hu from the Kluger lab as well as Binglan Yu and David Baron from the Zapol lab worked directly with me on this research and imparted much of their knowledge upon me. Maksims N. Volkovs wrote the program for the deconvolution of spectral data, a fundamental part of this research. Adelle Vandersteen and Sohyoung Her have been my chemistry comrades and will leave me with a lifetime’s worth of priceless memories. Scott Mundle, Raj Dhiman, Harini Kaluarachchi, Andrew Sydor and Kim Chan Chung all played crucial roles in this research, contributing their opinions and ideas, moulding and shaping the direction of my work. Finally and most importantly, I am especially grateful to my parents, sister and Maksims N. Volkovs. They were unquestionably the most essential contributors to this work, in their unwavering support and enthusiastic encouragement. They are the inspiration and motivation in my academic pursuits, and the source of any of my accomplishments in life. iv For M.N.V v Table of Contents Abstract _____________________________________________________________ ii Acknowledgements ____________________________________________________ iv Table of Contents ______________________________________________________ vi List of Tables _________________________________________________________ ix List of Schemes _______________________________________________________ x List of Schemes _______________________________________________________ x List of Figures ________________________________________________________ xi Abbreviations ________________________________________________________ xv Abbreviations ________________________________________________________ xv Statement of Authorship and Publication Status ____________________________ xvii Chapter 1 : HBOCs – Safety and Efficacy ___________________________________ 1 1.1 Blood, Transfusions, Hemoglobin, and Oxygen Delivery ..................................... 1 1.2 Hemoglobin-Based Oxygen Carriers (HBOCs)..................................................... 3 1.3 HBOC-Induced Vasoconstriction: Hypotheses ..................................................... 4 1.4 Chemical Approaches that Deal with HBOC-induced Vasoconstriction .............. 6 1.5 Future Prospects: Understanding HBOC-induced Vasoconstriction ..................... 9 1.6 ChemBioChem Review: Reviving Artificial Blood: Meeting the Challenge of Dealing with NO Scavenging by Hemoglobin .......................................................... 11 1.7 Purpose of Thesis ................................................................................................. 21 Chapter 2 : PEG-Hemoglobins and Nitrite Reductase Activity __________________ 23 2.1 Introduction .......................................................................................................... 23 2.2 Biochemistry (ACS) Article: Polyethylene Glycol Conjugation Enhances the Nitrite Reductase Activity of Native and Cross-Linked Hemoglobin ....................... 24 2.3 Detailed ................................................................................................................ 33 vi 2.2.1 Chemical Modification of Hemoglobin: Cross-linking and PEGylation ...... 33 2.2.2 Analytical Methods: HPLC, SDS-PAGE, CD spectroscopy, Oxygen Binding ................................................................................................................................ 35 2.2.3 Kinetic Measurements .................................................................................. 36 2.2.4 Deconvolution of Spectra ............................................................................. 37 2.4 Supplementary Information ................................................................................. 38 2.4.2 CD Spectroscopy .......................................................................................... 38 2.4.2 Kinetic Measurements: Nitrite Reductase Activity ...................................... 39 2.5 Summary .............................................................................................................. 42 Chapter 3 : Enhancing Nitrite Reductase Activity – PEGylated Bis-Tetramers _____ 44 3.1 Introduction .......................................................................................................... 44 2.2 Biochemistry (ACS) Article: Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives .................................. 46 3.3 Detailed Experimental Methods ......................................................................... 55 3.3.1 General Methods ........................................................................................... 55 3.3.2 Chemical Modification of Hemoglobin: Cross-linking and PEGylation ...... 55 3.3.3 Analytical Methods: HPLC, SDS-PAGE, CD spectroscopy, Oxygen Binding ................................................................................................................................ 57 3.3.4 Kinetic Measurements .................................................................................. 59 3.4 Supplementary Information ................................................................................. 59 3.4.1 Kinetic Measurements .................................................................................. 59 3.5 Summary .............................................................................................................. 61 Chapter 4 : Physiological Responses of Bis-Tetramers and their PEGylated Derivatives ___________________________________________________________________ 65 4.1 Introduction .......................................................................................................... 65 4.2 Experimental ........................................................................................................ 66 4.2.1 General Methods and