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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 40106 I 73-26,766 \ BARE, George Harlow, 1942- j PHYSICAL STUDIES OF HH4E PROTEINS. ! The Ohio State University, Ph.D., 1973 [ Biochemistry | f f |t I University Microfilms, A XEROX Company, Ann Arbor, Michigan | i PHYSICAL STUDIES OF HEME PROTEINS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By George Harlow Bare, B.A. ***** The Ohio State University 1973 Reading Committee: Approved by John S. Rieske Richard H. Matthews Mser Department of Physiological Chemist ACKNOWLEDGMENTS I wish to thank my adviser, Dr. James 0: Alben, for his patient and understanding guidance of my research efforts. Also, I particularly wish to thank Dr. Philip A. Bromberg, who has kindly provided much support, assist­ ance and perceptive advice for this research, and my wife, who has endured many privations because of it. VITA November 13, 1942 . Born - Baltimore, Maryland 1964 ................ B.A. , University of North Caro­ lina, Chapel Hill, North Caro­ lina 1969-1973 ............ Teaching Associate, Department of Physiological Chemistry, The Ohio State University PUBLICATIONS "HAEMOGLOBIN LITTLE ROCK (fl143 His -*■ Gin; H21) : A High Oxy­ gen Affinity Variant with Unique Properties," Nature, in press "Fourier Transform Infrared Spectroscopy of Heme-Protein Carbonyls," 57th Annual Meeting of the Federation of American Societies of Experimental Biology, April, 1973, Atlantic City, New Jersey, Abstracts: 1586 "Infrared Studies of Structural Interactions with Ligands Bound to Hemoglobins and Other Metal Proteins," 162nd National Meeting of the American Chemical Society, Sept., 1971, Washington, D.C., Abstracts: Biol. 165 FIELDS OF STUDY Major Field: Biochemistry Physical Biochemistry of Heme Proteins. Professor James 0. Alben iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS 11• • VITA i• ■n • LIST OF TABLES v m LIST OF FIGURES ix GLOSSARY xi GENERAL INTRODUCTION 1 INFRARED SPECTROSCOPY ................... 3 A. Principles of Infrared Spectroscopy . 3 B, Infrared Spectroscopy of Biological Systems ........................ 4 C. Fourier Transform Infrared Spectroscopy . 6 D. QuantitationI of Infrared Data 7 II. HEME PROTEINS . 10 A. Hemoglobin 10 B. Peroxidases 17 C. Cytochrome P-450camphor 18 SECTION I. HEME PROTEIN CARBONYLS 19 I. INTRODUCTION 19 II. MATERIALS AND METHODS . 23 A. ^aterials ...... 23 T1. Chemicals .... 23 2. Proteins .... 23 iv Page B. Methods .............................. 24 1. Preparation of Native and Denatured Carboxyhemoglobin A ......... 24 2. Preparation of Abnormal Carboxyhemo- g l o b i n s ............................ 26 3. Preparation of Cytochrome p“450cainpkor C a r b o n y l .......................... 27 4. Preparation of Chloroperoxidase Car­ bonyl ............................. 27 5. Preparation of Horse Radish Peroxi­ dase Carbonyl ............ 27 6 . Determination of Infrared and Visible S p e c t r a ............................ 23 III. R E S U L T S ...................................... 29 A. Isotopic Frequency Shifts of Bound Carbon M o n o x i d e ................... 29 B. Extinction Coefficients of Carboxyhemo- g l o b i n s ............................. 40 C. Denaturation of Carboxyhemoglobin ........ 40 D. Abnormal Hemoglobins .................... 46 E. cytochrome P-«0camphor .................. 46 F. Chloroperoxidase ............. 49 G. Horse Radish Peroxidase ........ 53 IV. DISCUSSION ............ 56 SECTION II. HEMOGLOBIN CONFORMATION AND INTER­ ACTION WITH ANIONS .................. 60 PART I. INFRARED STUDIES OF HEMOGLOBIN SULFHYDRYL GROUPS ................................. 60 I. INTRODUCTION ................................ 60 V Page II. MATERIALS AND M E T H O D S ....................... 64 A. M a t e r i a l s ......................... 54 1. Chemicals ............................ 2. Proteins............................ 64 B. M e t h o d s ............................... 65 1. Preparation of Potassium 2,3-diphospho- glycerate ......... ........ 65 2. Preparation of Human Hemoglobin Deriva­ tives ............................. 65 3. Preparation of Animal Hemoglobins . 67 4. Preparation of Cytochrome p“^^^camphor* ^ .5, Determination of Infrared and Visible Spectra .................... 67 6 . Quantitation of Infrared Data ........ 68 III. RESULTS ............. 71 A. Aqueous Cysteine...................... 71 B. Hemoglobin .............................. 71 C. Cytochrome P-450camphor .................. 78 IV. DISCUSSION ................... ....... 80 PART II. HEMOGLOBIN LITTLE ROCK: ANOMALOUS EFFECTS OF AN AMINO ACID SUBSTITUTION AT THE 2,3-DIPIIOSPHOGLYCERATE BINDING SITE . 86 I. INTRODUCTION ............................... g6 II. MATERIALS AND METHODS ................. 88 III. R E S U L T S ..................................... 91 A. Effect of D P G ............................ 91 B. Effect of Chloride and Phosphate ........ 94 C. Oxygen Equilibrium of Unseparated HbLR and H b A ................................... 97 Page XV. DISCUSSION ................................. 105 APPENDIX A ..................................... Ill BIBLIOGRAPHY................................. 115 LIST OF TABLES Table Page * 1. Isotopic Shifts of the CO Stretching Fre­ quency of Heme Protein Carbonyls ...... 37 2. HbCO Overtone Characteristics and Fundamen­ tal Frequencies Corrected for An- harmonic ity ............................... ,39 3. Extinction Coefficients of HbCO Isotope Complexes . ........................... 40 4. Carbon Monoxide Stretching Frequencies (vco) and Half Bandwidths of Na“tive and Various Types of Denatured HbCO .... 41 5. Percentage of Bound CO Remaining After Dena- turation of H b C O ......................... 46 6. CO Frequencies and Half Bandwidths of Carbon Monoxide Complexes of Heme-Containing E n z y m e s ................................. 49 7. Frequency (vSH) and Half Bandwidths (Av-jy2) Parameters Giving Best Fit to Infrared Sulfhydryl Spectra and Sulfydryl Group Ex­ tinction Coefficients ........ 72 8. Ratio of Area of al0 4/3 93 SH Absorption to Area of 8112 Absorption for Hemoglobin Derivatives ............................. 9. DPG Titration Curve Parameters and Calculated Dissociation Constants for HbLR and HbA . 93 10. Chloride and Phosphate Titration Curve Para­ meters and Calculated Dissociation Con­ stants for IlbLR and H b A ......... 95 11. Assumed Log p 5q Values for HbLR Hemolysate . 9 8 viii LIST OF FIGURES Figure Page 1. Infrared Difference Spectra of Native Car­ boxyhemoglobin vs. oxyhemoglobin ...... 31 2. Infrared Difference Spectra of Carboxy­ hemoglobin Denatured at pH 2.5 with HC1 vs. o x y h e m o g l o b i n ....................... 32 3. Infrared Difference Spectra of Carboxyhemo­ globin Denatured at pH 11.9 with KOH vs. Oxyhemoglobin ........................... 33 4. Infrared Difference Spectra of Carboxyhemo­ globin Denatured at 100° C for 20 Min vs. Oxyhemoglobin .................. ..... 34 5. Infrared Spectra of Chloroperoxidase-CO Com­ plex at pH 5.9 vs. 35% Aqueous Glycerol . 35 6. Infrared Spectra of Horse Radish Peroxidase- CO Complex at pH 7.0 vs. Water ....... 36 7. Infrared Difference Spectra of Isotopically Labelled HbCO complexes in D^O vs. Hbi2cl6o in CO Overtone R e g i o n ......... 38 8. Infrared Spectra of Detergent Denatured Car- boxyhemoglobins ......................... 42 9. Infrared Difference Spectra of Partially Acid Denatured Carboxyhemoglobin vs. Oxy­ hemoglobin ................................. .43 10. Infrared Difference Spectrum of Carboxyhemo­ globin Little Rock vs. Oxyhemoglobin .... 47 11.