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Expression, Purification, and Characterization of Mammalian and Earthworm Hemoglobins

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Expression, Purification, and Characterization of Mammalian and Earthworm Hemoglobins Expression, Purification, and Characterization of Mammalian and Earthworm Hemoglobins Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jacob James Elmer, B.S. Chemical and Biomolecular Engineering Graduate Program The Ohio State University 2011 Dissertation Committee: Andre F. Palmer, Advisor David Wood Jessica Winter 1 Copyright by Jacob James Elmer 2011 2 Abstract The frequent shortages, risks of disease transmission, and storage issues associated with donated blood illustrate a significant demand for a red blood cell (RBC) substitute. Such a substitute should be able to effectively transport oxygen throughout the body with minimal side effects. Several hemoglobin-based oxygen carriers (HBOCs) have been developed and clinically tested, but they have all caused severe side effects. The problems associated with these HBOCs may all be attributed to removing hemoglobin from the RBC. Therefore, this work focuses on the use of the extracellular hemoglobin of the earthworm Lumbricus terrestris (LtEc) as a new class of HBOC. Since earthworms lack RBCs, their hemoglobin is freely dissolved in the bloodstream and has already adapted to solve many of the challenges facing modern synthetic HBOCs. It has a lower rate of oxidation, avoids harmful side reactions with nitric oxide (NO), and it is extremely stable. We have developed a novel purification technique to highly purify large amounts of LtEc at costs that are comparable to donated blood. The LtEc product also transports oxygen similarly to human blood. Transfusion of LtEc into hamsters does not elicit the harmful side effects observed with other HBOCs and preliminary studies have not revealed any immune or allergic reactions in vivo. Therefore, this work shows that LtEc might be an effective and safe oxygen carrier that warrants further study and suggests the need for a paradigm shift in the HBOC field from cellular to extracellular hemoglobins. ii Dedication I dedicate this work to my Mother, for her dedication to me. iii Acknowledgements This work is the product of collaborations between myself and many other individuals. I would like to thank Dr. Andre Palmer for giving me one of the best graduate experiences possible. I would also like to thank my lab mates for their continuous support and friendship: David Harris Guoyong Sun Ning Zhang Guo Chen Sharon Gunderson Alex Roth Jorge Fontes Shahid Rameez Yipin Zhou Uddyalok Banerjee I would also like to thank the faculty of the chemical engineering department for being excessively generous with their equipment and their advice. Specifically, I would like to thank Dr. David Wood for mentoring me towards the end of my graduate tenure. Much of this work would not have been possible without him. iv Last but not least, I would like to specifically thank all of my undergraduate assistants. Besides conducting experiments, my students also inspired me to work harder and perform some experiments which I wouldn’t have done otherwise. I wish them all the best (in chronological order): Henry White Mark Politz Katie Zorc Ilse Fernandez Zeinab Mohammed Parth Patel v Vita 1999-2003 ............................................................ Seckman Senior High School 2003-2007 ............................................................ B.S. Chemical Engineering B.S. Biological Sciences Missouri University of Science & Technology Summer, 2005 ...................................................... Trainee, NASA Space Flight and Life Sciences Training Program Summer, 2006 ...................................................... Intern, Donald Danforth Plant Sciences Center 2004-2007 ............................................................ Undergraduate Researcher, Missouri University of Science and Technology 2007-present ........................................................ Graduate Research Associate, Department of Chemical Engineering, The Ohio State University PUBLICATIONS Elmer J, Harris DR, Sun G, Palmer AF. Purification of hemoglobin by tangential flow filtration with diafiltration. Biotechnol Prog. 2009 25(5):1402-10. Elmer J, Buehler PW, Jia Y, Wood F, Harris DR, Alayash AI, Palmer AF. Functional comparison of hemoglobin purified by different methods and their biophysical implications. Biotechnol Bioeng 2010 106(1):76-85. Elmer J, Cabrales P, Wang Q, Zhang N, Palmer AF. Synthesis and biophysical properties of polymerized human serum albumin. Biotechnol Prog. 2011 27(1):290-6. vi Elmer J, Harris D, Palmer AF. Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography. J Chromatogr B Analyt Technol Biomed Life Sci. 2011 879(2):131-8. FIELDS OF STUDY Major Field: Chemical and Biological Engineering Specialization: Protein expression and purification vii TABLE OF CONTENTS Abstract ............................................................................................................................................ ii Dedication ....................................................................................................................................... iii Acknowledgements......................................................................................................................... iv Vita……. ........................................................................................................................................... vi List of Tables ................................................................................................................................... xi List of Figures .................................................................................................................................xiii CHAPTER 1: INTRODUCTION TO TRANSFUSION, HEMOGLOBIN, AND BLOOD SUBSTITUTES ..... 1 1.1- The Past, Present, and Future of Transfusion ............................................................. 1 1.2 - Hemoglobin................................................................................................................. 2 1.3 – Hb Dissociation, Oxidation, and Interactions with Nitric Oxide ................................ 7 1.4 - Previous Generations of HBOCs................................................................................ 13 1.5 - References ................................................................................................................ 14 CHAPTER 2: PURIFICATION OF HEMOGLOBIN BY TANGENTIAL FLOW FILTRATION .................. 22 2.1 – Introduction ............................................................................................................. 22 2.2 - Materials and Methods ............................................................................................ 25 2.3 – Advantages and Disadvantages of Diafiltration ....................................................... 32 2.4 – Comparison of TFF-Purified Hbs and Commercially Prepared Hbs .......................... 36 2.5 - Conclusion ................................................................................................................. 45 2.6 – References ............................................................................................................... 45 CHAPTER 3: PURIFICATION OF HEMOGLOBIN BY IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY ..................................................................................................................... 48 3.1 – Introduction ............................................................................................................. 48 3.2 – Materials and Methods ............................................................................................ 51 viii 3.4 - Conclusion ................................................................................................................. 60 3.5 - References ................................................................................................................ 60 CHAPTER 4: INTRODUCTION TO ERYTHROCRUORIN .................................................................. 62 4.1 - Extracellular Hbs: A New Paradigm .......................................................................... 62 4.2 - Structure and Stability of LtEc .................................................................................. 63 4.3 - O2 Transport by LtEc ................................................................................................. 66 4.4 - Autoxidation of LtEc .................................................................................................. 67 4.5 - Interactions between LtEc and other Ligands .......................................................... 69 4.6 - Preliminary Animal Studies with LtEc ....................................................................... 70 4.7 - Conclusion ................................................................................................................. 70 4.8 - References ................................................................................................................ 71 CHAPTER 5: PURIFICATION AND IN VITRO CHARACTERIZATION OF ERYTHROCRUORIN .......... 76 5.1 – Introduction ............................................................................................................. 76 5.2 – Materials and Methods ............................................................................................ 78 5.3 – Results and Discussion ............................................................................................
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