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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. I Quantitation 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-diphosphoglycerate ...... 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. Infrared Differfffice Spectrum of Carboxyhemo globin S vs. Oxyhemoglobin...... 48

12. Infrared Spectrum of Cytochrome p“450camphor CO Complex vs. Water ...... 50

ix Figure Page 13. Infrared Spectra of Chloroperoxidase-CO Complex.as a. Function of pH andSolvent . . 52 14. Infrared Spectra of Horse Radish Peroxidase- CO Complex vs. Water as a Function of pH . 54 15. Infrared Spectra of Aqueous Cysteine, Cyan- methemoglobin, and Cytochrome p-450camphor in SH regi0n ...... 69 16. Infrared Spectra of Horse, Pig, and Cow Met- hemoglobin vs. 5% Aqueous n-butyl Am monium Chloride ...... 74 17. Infrared Spectra of Human Hemoglobin Deriva- . tives vs. 7.5% Aqueous n-butyl Ammonium C h l o r i d e ...... 76 18. Infrared Spectra of Human Hemoglobin Deriva tives vs. 7.5% Aqueous n-buytyl Ammonium C h l o r i d e ...... 77 19. Chromatographic Separation of HbLR and HbA on Carboxymethyl Cellulose ...... 99 20. Photographic Results of Agar Gel Electro phoresis of HbLR Hemolysate, Chromato- graphically Isolated HbLR, Chromatographi- cally Isolated HbA, and HbA hemolysate . . i q o 21. Effect of DPG on the Log p,-n and Hill's n of Isolated HbLR and H b A ...... 101 22. Effect of Chloride on the Log pg0 of Iso lated HbLR and HbA ...... 10 2 23. Effect of Potassium Phosphate on the log p5() of Isolated HbLR and HbA...... 103 24. Oxygen Equilibrium of Unseparated HbLR Hemo lysate Taken in 0.111 Potassium Phosphate Buffer (pH 7.15)...... 104

x GLOSSARY

a^ Absorbance extinction coefficient BISTRIS Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane DPG 2,3-Diphosphoglycerate Hb Hemoglobin IHP Inositol hexaphosphate Mb Myoglobin P-450 Cytochrome P-450oamphor

Apparent.integrated intensity extinc tion coefficient Ha If-Bandwidth (cm”*)

\> Frequency (cm"’*) GENERAL INTRODUCTION

Infrared spectroscopic instrumentation and technique have been improved greatly over the past several years to the point that Infrared spectroscopy can provide high qual ity information regarding biological systems which cannot presently be obtained in any other manner. This improve ment has largely been due to the development of a practi cal infrared interferometer and has permitted the observa tion of isolated groups within a variety of proteins under conditions which would have been impossible as recently as 1970. The present work deals exclusively with heme proteins and is subdivided into two major sections. The first deals with heme protein carbon monoxide complexes. The types of studies include the structure of the iron-carbon monoxide bond, as observed in the infrared, the anharmonicity of the carboxyhemoglobin CO stretching vibration, and the heme en-

* * • • vironment of a variety of denatured hemoglobin species and heme-containing enzymes. The second section deals with hemoglobin conformation in solution and interaction of anions with hemoglobin. It includes a study of hemoglobin conformation under the in fluence of organic phosphates using direct spectroscopic observation of sulfydryl groups as a probe. A second study deals with the oxygen equilibrium of a unique hemoglobin variant which has provided significant information re garding the allosteric control of hemoglobin oxygen affinity. I. INFRARED SPECTROSCOPY A. Principles of Infrared Spectroscopy Spectrophotometry in all regions of the electromagnet ic spectrum is based on observation of transitions between quantum energy levels caused by absorption of electromag netic radiation. In the mid-infrared region (600-5000 cm *), these transitions are vibrational and any transition causing a change in the dipole moment of a molecule may be observed as an absorption of infrared radiation. In gener al, there are two types of transition in this region: stretching and bending vibrations. The energy required to stretch a bond between two atoms is greater than that re quired to bend it and, because the frequency scale (in cm"'*’) is directly proportional to energy, stretching vibra tions occur at higher frequency than bending vibrations. The frequency (v) of a stretching vibration, if.the two atoms are assumed to vibrate as a perfect harmonic oscilla tor, is related to thie masses (M^ and Mfi) of the two atoms and the strength of the bond between them by the relation:

= 1 / F v 2 ttc y y in which c is the speed of light, y is the reduced mass (M-Mb/IM. + M^)) and k is the force constant, which is de- A B A d pendent upon the.electronic force field between the two atoms. Force constants for single, double, and triple bonds are approximately 5, 10, and 15 dynes/cm, respectively. If the assumption of a perfect harmonic oscillator were correct, interpretation of infrared data in terms of bond structure would be straightforward; however, molecular vibrations generally deviate substantially from this simple approximation. For example, the fundamental stretching frequency of HC1 gas occurs at a frequency over 100 cm"^ less than that predicted by the above relation (1). Con sequently, the anharmonicity of a vibration must be con sidered to obtain quantitative information regarding bond structure from infrared data. Frequencies may also be modified by the presence of other atoms in the system or by coupling to other vibrations. Useful information may also be obtained from the half bandwidth which is the bandwidth measured at half maximal absorbance. Ramsay (2) states that the major fac tor producing line broadening in the infrared is perturba tion by neighboring molecules. Consequently, the bandwidth is directly related to the randomness of the environment, a factor which, is important in the study of biological materi als.

B. Infrared Spectroscopy of Biological Systems The applications of infrared spectroscopy to the study of biological materials has been extensively reviewed by Parker (3). This technique has been of limited usefulness in examination of native biological specimens because of the complexity of macromolecules and because infrared absorp tions are generally rather weakf relative to those observed in the visible and ultraviolet regions of the spectrum. From theoretical considerations, a non-linear molecule may be shown to have 3N - 6 vibrational states, where N is the number of atoms. Macromolecules, such as proteins, usual ly have many thousands of atoms, giving rise to a great many potential absorptions. Not all of these will be in frared active and many will be very similar, e.g., the CH stretching vibration, and will simply cause a very borad band, making it difficult to isolate any particular absorp tion . If a particular absorption can be isolated, it may be difficult to obtain a sample sufficiently concentrated to observe it. For example, to observe the sulfydryl groups of hemoglobin, as will be described later, a saturated hemoglobin solution was prepared. This solution was only 0.027M in SH groups. . Further complications arise because many biological materials can be considered native only in aqueous solu tion. Water absorbs strongly through most of the infra red, making short path lengths (often 0.1 mm or less) necessary to get sufficient energy through the sample to obtain satisfactory detector response. Also, the fact that many absorptions of interest lie on the side of a major solvent or sample absorption requires precise matching of sample and reference concentrations and path lengths. . Consequently, all these factors combined, i.e, low absorption intensity, low sample concentration, short path length and low solvent transmittance, produce a require ment for very sophisticated and sensitive instrumentation and technique,

C. Fourier Transform Infrared Spectroscopy A thorough discussion of the principles of infrared interferometry is beyond the scope of this dissertation. However, it should be mentioned that a Michelson inter ferometer collects data in multiplex form, i.e., it scans all frequencies simultaneously, as a function of mirror travel. The light intensity as a function of frequency (spectrum) can be shown to be the Fourier transform of the light intensity as a function of mirror travel (inter- ferogram). The ability to collect information in this manner gives rise to a number of advantages of the inter ferometer over a conventional scanning spectrophotometer: higher signal to noise ratio, greater optical throughout, no equivalent of stray light, and accurate frequency de termination, based on a laser frequency. The low heat ab sorption by the sample is also of critical importance in dealing with biological specimens. Furthermore, the requirement for a digital computer for performing Fourier transforms makes available the capa bility for extended signal averaging and the ability to manipulate and store individual single-beara spectra. There for, a series of sample spectra and a common reference can all be obtained using the same cell, thus minimizing the effects of variations of path length and window surfaces. There are, of course, disadvantages to interferometry. These include high cost and relatively frequent breakdowns of the more sophisticated instrumentation, compared to a scanning spectrophotometer. Also, if energy outside the computational frequency range of the interferometer is not carefully filtered out, it will be ''folded1' back into the spectrum and give rise to spurious absorptions.

D. Quantitation of Infrared Data 1. Isolated Absorptions The most straightiforward means of obtaining informa tion for an isolated, symmetrical absorption is to graphi cally measure the center frequency, absorbance, and half bandwidth. The area (apparent integrated intensity) above • the graphically estimated baseline may be measured by plan imetry. This procedure involves some uncertainty in measur ing the parameters other than frequency because the true baseline of the absorption may fall somewhat below the estimated baseline, since the wings extend to infinity (4). Consequently, a necessary restriction in using data obtained by this method is that it can only be compared with data measured by the same method. This procedure was used for quantitation of infrared absorptions in the studies of heme protein-bound carbon monoxide.

2. Overlapping Absorptions For analysis of band envelopes containing overlapping absorptions, a curve-fitting procedure is necessary. Ramsey (2) has shown that a Lorentzian function, i.e.:

A, = log i- . v - j-Io v - (v-v0 r 5 + b^ 2

provides a very reasonable fit to infrared absorptions and also has theoretical justification from statistical mechan ics . The terms a and b are related to experimentally ob- 2 servable quantities by: (maximum absorbance) = a/b , J R a X ^vl/2 (half-bandwidth) = 2b and the center frequency is The area (integrated intensity) enclosed by this function

is /Avav = (TT/2) (Amax x Av 1/2) . For curve-fitting to resolve overlapping bands, addi-' tional terms are added to describe each individual absorp tion, e.g., for two absorptions, the equation has the form:

al a2 A “ ■ * ■ V (v-v^2 + b2 (v-v2>2 + b2

Iteration was performed manually until a reasonable fit to the absorption envelope was obtained. The best apparent fits to experimental data were obtained by performing the computations on a baseline 10-12% of the maximal graphic abosrbance below the graphically estimated baseline.

3. Cell Path Lengths A determination of the cell path length is necessary for the accurate calculation of infrared extinction coef ficients by the Beer-Lambert Law. The infrared spectrum of the empty cell ratioed against that of air will show a sine wave pattern due to interference if the windows are flat and parallel. The cell path length (£, in microns) can be obtained from the relation:

o _ 10^ n - 2

UNIVERSITY MICROFILMS. 11 which differ somewhat in composition but have a tertiary structure remarkably similar to each other and to myoglobin. The four chains are tetrahedrally packed to form an approxi mately spherical molecule with three mutually perpendicular axes of twofold symmetry (5). Mammalian hemoglobin has a number of features not found in myoglobin or separated a or 3 chains, which enable hemoglobin to deliver oxygen more effectively and are apparently due to the presence of quaternary structure. These include: 1) the Bohr effect, by which hemoglobin has a lower oxygen affinity at lower pH, 2) heme-heme interaction or cooperativity, by which oxygen affinity is lowered with progressive deoxygenation and which causes the sigmoid oxygenation curve, and 3) allosteric control of oxygen affinity by organic phosphates such as 2,3-diphosphoglycerate (DPG). The three-dimensional structures of oxy and deoxy hemoglobins have been elucidated from X-ray structure anal ysis by Perutz and co-workers (7, 7). Individual chains were found to be composed of eight connected right-hand ex-helical segments wrapped around the heme and designated A through H starting at the N-terminal end. Approximately 75% of amino acid residues are in helical regions. Polar- groups are spread uniformly over the outer surface.of the molecule, with few being found in the interior or at inter subunit contacts. Non-polar side chains are generally directed towards the interior of the molecule, filling 12 interstices between adjacent helical segments, forming hydrophobic regions for the subunit interfaces, or provid ing the non-polar environment around the heme required for reversible binding of oxygen. As for myoglobin (8, 9), interaction between the protein and the heme is complex and consists of five general types: 1. A coordinate covalent bond between the "proximal” F8 histidine and the heme iron. 2. Salt bridges between the propionic acid side chains of the heme and ionic residues on the surface. 3. Van Der Waals contacts between the heme and many non-polar side chains. 4. Pi.interactions between the heme and several aro matic side chains oriented nearly parallel to the plane of the heme. 5. Hydrogen bonding between the "distal” E7 histidine and a ligand present at the sixth coordination site of the iron.

Contacts between a and 8 subunits are largely of a non-polar nature, although several hydrogen bonds and other types of polar interactions also exist at the interfaces. The most stable contact is the interface, which has 34 side chains from helices B, G, and H involved in some form of interaction. The cc^ interface is formed by 19 residues in contact from helices C, F, and G. Significant 13 changes in the structure of this contact occur in the trans ition between the deoxy and oxy conformation. Contacts be tween the two a subunits consist of several salt bridges which exist only in the deoxy conformation and are partially responsible for the Bohr effect (10). There is apparent ly no interaction between the B chains. The X-ray structures show that deoxyhemoglobin under goes a large change of quaternary structure, consisting mainly of. a movement of the $ chains away from each other. Perutz has proposed explanations for both the Bohr effect (10) and heme-heme interaction (11) based on these observa tions. According to this scheme, the four C-terminal resi dues are involved in six interchain salt bridges in deoxy hemoglobin, stabilizing this form and causing a low oxygen affinity state. The HC2 tyrosine side chains are anchored in pockets between the F and H helices and cannot be moved without breaking the interchain salt bridges. Further, o the iron atoms of deoxyhemoglobin are about 0.75A out of the planes of the porphyrin rings and, in the 3 chains, ac- cess to the iron by a potential ligand is partially re stricted by the Ell valine side chains. Reaction of the first a chain with a ligand pulls the iron back into the plane of the porphyrin, causing movement of the F helix toward the H helix, expelling the HC2 tyrosine side chain from its pocket. This, in turn, breaks two of the six salt bridges and, because the pKs of the groups involved are altered, Bohr effect protons are released. Oxygenation of the second a chain has an identical effect. .At this point, the transition to the oxy quaternary structure is favored and the remaining two salt bridges break. The activation energy required for a ligand to bind to the B chains is now greatly reduced and the molecule is now in a high af finity state. Consequently, the 3 chains pick up oxygen very readily, undergoing the same changes in tertiary struc ture as described for the a chains. Thus, heme-heme interaction and the Bohr effect are proposed to result from the influence of the tertiary structure of one chain upon that of another, mediated by the quaternary conformation. Details of this scheme are presently open to question. Lindstrom and Ho (12) have observed that oxygen binds preferentially to a chains un der certain conditions, from NMR studies of ring-current shifted protons. Tyuma, et al. (13), have obtained similar evidence from oxygen equilibrium studies. Olson and Gibson (14), however, maintain.that the 3 chains are oxygenated first, based on kinetic evidence. .Raftery and Huestis (15) suggest that, while it is plausible that this mechanism contributes to cooperativity, it is probably oversimplified and that changes in hydrophobic interactions are at least as important to cooperativity as electrostatic interactions Perutz has further proposed (16) that an inverse re lationship exists between the electronic spin state of the 15 iron and oxygen affinity which is influenced by changes in tertiary and quaternary structure. The iron in deoxyhemoglobin is in a high spin state, having four unpaired elec trons, while in oxyhemoglobin, it is low spin, having no unpaired electrons. A high spin iron atom has a larger radius than a low spin atom and is not capable of fitting inside the porphyrin ring. Perutz (16) suggests that, for a partially liganded hemoglobin molecule having the deoxy quaternary structure, the liganded chains place a strain on the quaternary structure which is transmitted to the proximal F8 histidines of the unliganded chains and de creases the length of the histidine-iron bond. This is postulated to reduce the spin-of these iron atoms and, thereby, raise the oxygen affinity of the unliganded chains. He further proposes that DPG and IHP, which lower hemoglob in oxygen affinity, do so by binding to the protein in such a manner that the histidine-iron bond is lengthened, causing a higher net spin at the iron and a consequent lower affinity. These proposals, however, seem to be based on data of questionable interpretation and introduce fraction al changes of electron spin into a quantum system. The influence of anions on hemoglobin oxygen affinity has long been known (17) and it was observed that poly valent anions were able to loiter oxygen affinity more ef fectively than monovalent anions. Benesch and Benesch (18) observed in 1967 that 2,3-diphosphoglycerate (DPG), a 16 compound present in high concentrations in the erythrocytes of many mammals, was an extremely potent modulator of oxy gen affinity. DPG was shown chemically (19, 20) and by X- ray structure analysis (21) to bind in the central cavity of deoxyhemoglobin, between the 3 chains. Residues in volved in binding DPG were shown (21) to be the 32 and 3143 histidines, the 382 lysines, and the 3 chain N-terminal amino groups. The DPG molecule is stereochemically comple mentary to the binding site on deoxyhemoglobin and, because of the conformation change upon oxygenation, it binds much less tightly to oxyhemoglobin. Therefore, DPG, in most effectively binding to deoxyhemoglobin, stabilizes it and, consequently, lowers oxygen affinity. DPG has also been shown to interact with partially oxygenated hemoglobin intermediates (13, 22), probably being released upon binding of the third oxygen molecule. Other anions, including such small anions as chloride, can compete effectively for the DPG binding site in suf ficiently high concentration (23). Antonini, et al. (24), have suggested that different anions act similarly on hemo globin from consideration of anion interactions in terms of Wyman's (25) treatment of linked functions. The interaction of inositol hexaphosphate (IHP) with human hemoglobin has also been extensively investigated. IHP, which until recently was thought to be an analogue of DPG in the blood of birds and reptiles (26), lowers oxygen 17 affinity even more effectively than DPG (19). However, it apparently does this in a different manner than DPG, having been shown to affect the oxygen equilibrium (27) and kin etics (22) of fully oxygenated hemoglobin, as well as of

deoxyhemoglobin. It also appears to affect a chains, rather than 8 chains (14), as DPG does.

B. Peroxidases Peroxidases are a class of enzymes catalyzing the gen eral reaction:

ROOH + H2X *► ROH + H20 + X

in which ROOH is any type of peroxide and H2X is a hydro gen donor which undergoes oxidation to X by the transfer of two electrons to the peroxide and includes phenols, as- corbate, iodide, a variety of organic dyes and hydrogen peroxide. Most peroxidases contain high spin ferriproto- porphyrin IX as a prosthetic group and are fairly low molec ular weight glycoproteins. Peroxidases form four different complexes with peroxides, only two of which are catalytically active (28). The nature of these complexes has not been unequivocally determined. Peroxidases can be reversibly reduced to form a catalytically inactive carbon monoxide complex with visible spectral properties similar to those of carboxyhemoglobin (29). Chloroperoxidase is a special type of peroxidase found 18 in the. mold, Caldariomyces fumago, which catalyzes the peroxidative chlorination of 0-keto acids, 0-diketones, or phenols (30), as well as the classical peroxidative reac tion (31). It only performs the chlorination at low pH (31). The carbon monoxide complex has recently been re ported (32) to have remarkably similar visible and UV spec tral properties to those of Cytochrome p”^5®camphor*

C. Cytochrome P-450camphor

Cytochrome P-450 is a ubiquitous ferric heme protein which functions as the terminal oxidase in a mixed func tion oxidase system involved in the metabolism of drugs, steroids, fatty acids, and a variety of foreign substances (33). It is unusual because the Soret absorption of the re duced CO complex occurs at the very long wavelength of 450 nm. Mammalian cytochromes P-450 are difficult to solubil ize and denature readily to a catalytically inactive P-420 form. Cytochrome p“^50campjior' ^so-*-a'*:ed from Pseudomonas putida and which catalyzes the hydroxylation of camphor, is soluble and under intensive investigation. ESR studies (314) indicate that P-450, as isolated, is in a low spin form, becoming high spin on binding of substrate. It also exhibits an unusually large rhombic distortion of the iron environment when substrate is bound, which has been attrib uted to non-planarity of the heme. SECTION I. HEME PROTEIN CARBONYLS

I. INTRODUCTION The binding of carbon monoxide to zero valent transi tion metals to form a variety of ir-bonded complexes is a well established phenomenon. Simple ferrous heme carbonyl complexes have many similarities to Fe^-CO complexes. A series of heme carbonyls have been investigated by Alben and Caughey (35) using infrared spectroscopy and an inverse relationship was established between the CO stretching fre quency and the basicity of the trans ligand. The first infrared observation of CO bound to a de natured heme protein was made by Wang, et al. (36), and was performed with dry carboxyhemoglobin (HbCO) pressed into a KBr pellet. The CO stretching frequency occurred near 1970 cm”*'. Alben and Caughey (35) observed the CO ab sorption of native HbCO in packed red cells and in aqueous solution at 1951 cm~^. They found single, narrow (8 cm band which was interpreted as being due to a single, rather nonpolar and highly structured molecular environment of the CO moiety. Frequency shifts of isotopically labelled CO bound to hemoglobin were interpreted as indicating an iron- oxygen bond rather than an iron-carbon bond as had previous ly been assumed. A similar pattern of isotopic frequency 19 20 shifts was observed with myoglobin (37) and hemocyanin (38), a non-heme oxygen-carrying protein containing copper in stead of iron.

Caughey, et al. (39), also examined several variant hemoglobins and found that amino acid substitutions remote from the heme had no apparent effect on the CO stretching absorption while for hemoglobins in which the distal histidine was replaced by tyrosine or arginine was shifted to higher frequency. The CO derivatives of separated, PMB-labelled a and 8 chains of hemoglobin have also been examined (37) and was found to be the same as that for normal HbCO. These derivatives were unstable and denatured when allowed to become heated in the infrared beam over a period of time. During this time, a band at about i967 cm * was seen to increase in intensity. Carboxymyoglobin has also been extensively investi gated (37, 39) and shows the CO absorption at a substanti ally lower frequency (about 1945 cm ^) than HbCO. The ex act frequency varies somewhat with the species from which the MbCO was prepared. Carboxysulfmyoglobin (40) has vco at 1953 cm This was interpreted as being due to withdrawal of electrons from the iron and consequently, from the CO, presumably by a sulfur atom associated with the heme. The carbon monoxide complex of cytochrome oxidase has 21 been reported (41) to have an absorption at 1963 cm”'*' with an extremely narrow half-bandwidth of 3 cm”'*'. This was interpreted to mean that the environment required for the biochemical reduction of oxygen is even less polar than that required for reversible dissociation, as in Hb or Mb. Molecular orbital calculations by Magnusson and Rippon, as quoted by Caughey (42) and Antonini and Brunori (43), suggest that CO bound perpendicularly to the heme with an iron-carbon bond should have a stretching frequency in the region of 19 70 cm This is consistent with recent obser vations that \>co is about 1970 cm ^ for the N-methylimida- zole complex of hemecarbonyl (44) and an imidazole-iron tetraphenylporphyrin carbonyl complex bonded to a polysty rene matrix (45). The calculations suggest that bending of the carbon monoxide molecule away from the z axis (per pendicular to the heme plane) should lower vCQ substanti ally. Bending of the iron-CO bond would presumably be caused by steric hindrance in the heme pocket. A bent iron- CO bond has been observed in an X-ray structure analysis of erythrocruorin (46), an insect larval hemoglobin with a. tertiary structure similar to myoglobin. However, the present studies indicate that variations in the CO stretch- • * ing frequency caused by differences in the solvation shell of the heme-CO are also very important. Solvation effects may arise from Van der Waals contacts, hydrogen bonding, and other types of interactions. For example. X-ray structure analysis (47) of azidometmyoglobin suggested a hydrogen bond between of the azide ion and the distal histidine. Because MbN^ and MbCO are isomor- phous, a similar hydrogen bond may also exist in MbCO and HbCO. This hydrogen bond is expected to influence the CO stretching frequency. The molecular orbital calculations (42) further sug gested that bending of the iron-CO bond may also be re sponsible for the anomalous isotopic frequency shifts, for which Alben and Caughey (35) had proposed an iron-oxygen bond. In the present studies, the pattern of isotopic frequency shifts of bound CO will be shown to be a general observation with metal carbonyls, and cannot be used to determine the orientation of the bound-CO. Evidence will also be presented that this pattern of isotopic shifts is not due to anharmonicity. These studies, covering native, abnormal, and denatured hemoglobins and several types of heme-containing enzymes, provide some unique new insights into structural interac tions around the heme and give substantive information • about denaturation processes. 23 II. MATERIALS AND METHODS A. Materials 1. Chemicals Hydrochloric acid, glacial acetic acid, sodium carbon ate, and tripotassium phosphate were purchased from Allied Chemical Company. Potassium hydroxide, sodium dithionite, and carbon monoxide ( 12 * C 16 0) were purchased from J. T. Baker Chemical Company. Deuterium oxide (99.8% was purchased from Biorad Laboratories. Carbon monoxide, 90% enriched in 13 16 C 0, was purchased from Monsanto Chemical Corporation and 90.8% 12 C 18 0 was purchased from Miles Research Labora- ' tories. Argon gas was obtained from Burdette Oxygen Com pany and sodium benzoate was purchased from Hooker Chemi- cal Company. Sodium dodecylsulfate (SDS) was obtained from Sigma. Recrystallized sodium dithionate was provided by Dr. R. Chiang (Dept, of Chemistry, University of Illinois). Unless otherwise specified, all chemicals were reagent or analytical grade and were used without further purifica- * tion. Glass redistilled water was used for all experiments.

2. Proteins Hemoglobin A solutions were prepared from normal human blood, freshly collected in heparin. Abnormal hemoglobin hemolysates were prepared from fresh blood in acid-citrate- dextrose from appropriate subjects. Cytochrome p“^5®camphor (P-450) was prepared from Pseudomonas putida in the 24 laboratory of Dr. I. C. Gunsalus and was furnished to us as a lyophilized powder by Dr. Karl Dus (Department of Chemis try, University of Illinois). Chloroperoxidase (CPO) was prepared from the mold Caldariomyces fumago in the labora tory of Dr. L. P. Hager and was provided as a ImM (heme) solution in 0.05M phosphate buffer (pH 3) by Dr. R. Chiang (Department of Chemistry, University of Illinois). Horse . radish peroxidase was purchased from Boehringer by Dr. J. Peisach (Department of Pharmacology, Albert Einstein Col lege of Medicine, Yeshiva University) and was furnished as a 5mM (heme) solution in 0.1M phosphate buffer (pH 7).

B . Methods . 1. Preparation of Native and Denatured Carboxyhemoglobin A ~ a. Native Carboxyhemoglobin Native human hemoglobin A was prepared by addition of 1 volume of water and 0.5 volumes of toluene to fresh, saline-washed red cells. The clear hemolysate obtained after centrifugation was lOmM in heme, determined as cyanmethemoglobin, and was used without further purification. Isotopically labelled carbon monoxide complexes were formed following evacuation of the hemolysate in a stoppered tube and flushing with Argon. The appropriate isotopically en riched CO gas was then injected with a syringe. These so lutions were allowed to equilibrate with CO at least 8 hr before use; 25 B. Denatured Carboxyhemoglobin Acid denatured HbCO isotope complexes were prepared by titration of the appropriate HbCO solution to pH 2.5 with 2N HC1.. Partially denatured complexes were prepared by addition to the HbCO solution of glacial acetic acid to a final concentration of 0.11-1 at pH 4.5 or by addition of 2N HC1 sufficient to precipitate a substantial amount of pro tein. The precipitate was sampled for the infrared study. Base denatured HbCO complexes were prepared by.titra tion of the appropriate HbCO solution to pH 11.9 with 2N KOH. Heat denatured HbCO isotope complexes were prepared by immersion of the appropriate solution in a boiling water bath for 20 min or for 5 hr, under a CO atmosphere. This formed a loose red precipitate. Sodium dodecylsulfate denatured HbCO was prepared by addition of SDS to a solution of HbCO to a final concen tration of 0.5M. SDS/acetic acid denatured HbCO was pre pared by addition of SDS to a concentration of 0.1M and glacial acetic acid to a concentration of 0.5M. A reddish- black precipitate was formed. Benzoate denatured HbCO was prepared by addition of sodium benzoate to a final concen tration of 2M. Except where otherwise indicated, all types of de natured HbCO were clear, very viscous solutions and, 26 because of the viscosity, no particular precautions were necessary to exclude air. c. Carboxyhemoglobin in D2 O Native human hemoglobin was prepared by hemolysis of saline-washed red cells in 2 volumes of D2 O and 0.5 volumes of toluene. The hemolysate was.concentrated to half its original volume by pressure ultrafiltration. An equal volume of D 2 O was added and the solution was again concen trated. This step was repeated. The final hemoglobin con centration was 18mM (heme) in about 90% D2 O. Isotopic CO derivatives were prepared as described for native HbCO in water.

2. Preparation of Abnormal Carboxy- hemoglobins Hemoglobin S was prepared by freezing and thawing saline-washed red cells in 1 volume of water. The CO com plex was formed by blowing CO gas over the surface of the solution. Hemoglobin Little Rock was prepared chromatographically as described in the legend to Figure 19. The solution was concentrated by vacuum ultrafiltration and the CO complex was formed by blowing CO gas over the surface of the solu tion. 27 3. Preparation of Cytochrome p~^^Qcamphor Carbonyl

The P-450 CO complex was prepared by dissolving the lyophilized protein in 6mM aqueous camphor. This was re duced with Na2S204 under a CO atmosphere. The heme concen tration was estimated to be about 3mM.

4. Preparation of Chloroperoxidase Carbonyl CPO-CO at pH 3 was prepared directly from the stock solution by reduction with approximately 1.5 equivalents of Na2S20g under a CO atmosphere. CPO-CO at pH 4.2 was prepared by dialysis of the stock solution against glycerol for 9 0 min. The resulting con centrated solution was reduced as described above. The solution was about 4mM in heme and about 50% in glycerol. Isotopic CO derivatives of CPO-CO at pH 5.9 were pre pared as described for the pH 4.2 derivative except that the stock solution was dialyzed against 0.1M phosphate buf fer (pH 6.0) before the glycerol concentration step. The heme concentration was about 2.5mM in 35% glycerol.

5. Preparation of Horse Radish Peroxidase HRP-CO isotope complexes were prepared by reduction under a CO atmosphere with about 1 equivalent of Na2S20^ dissolved anaerobically in 0.1M phosphate buffer (pH 7.0). For further studies, the pH of HRP was adjusted to 8.7 with 1M Na2C03/K3P04 or to pH 4.9 with 1M sodium acetate 28 (pH 4.5). The pH of the latter sample was readjusted to 8.2 with 1M K 3P04 .

6. Determination of Infrared and Visible Spectra Infrared spectra of most samples were taken on a Digi- lab FTS-14 interferometer which was usually equipped with a liquid nitrogen cooled Indium-Antimonide detector. In some instances, a triglycine sulfate detector was used. The spectrum of HbS-CO was taken on a Perkin-Elmer 102 scanning spectrometer. These spectra were taken in in frared cells equipped with CaF2 windows and having a path length of 0.1 mm. Cells for the spectra of HbCO in D20 had a path length of 0.2 mm while those used for the abnormal hemoglobin spectra had a path length of 0.05 mm. Cell path lengths were measured from interference fringes as described in the General Introduction. Most spectra were taken at ambient temperature (28° C) except HRP and CPO which were cooled to about 5° C. Infrared spectra were taken at 2 cm"^ resolution except for measurement of the HbCO overtone, which required duplicate data collections at 2 and 8 cm*'*' resolution to eliminate the possibility of instrumental artifacts. Quantitation of infrared data was performed graphically as described in the General Introduction. All infrared frequencies given here are corrected to vacuum. Visible absorption spectra were taken in the infrared cells on a Perkin-Elmer 4000A spectrophotometer. 29 III. RESULTS A. Isotopic Frequency Shifts of Bound Carbon Monoxide The spectra of the three isotopic CO derivatives of normal hemoglobin (Figure 1) duplicate and verify the ob servations of Alben and Caughey (35) and were taken to es tablish the frequency shifts of hemoglobin-bound CO with a higher degree of accuracy than had formerly been possible. Isotopic frequency shifts may conveniently be expressed - as the difference (A) between the v*/v ratio of the bound CO and that of the free gas. The v* and v terms are the CO stretching frequencies of the isotopically labelled CO and 12 IS C ' 0, respectively. The v*/v ratios for the free iso topic gases are the same as those dictated by the mass-only frequency shift, which may be calculated from the relations

M*M*/(M* + M*) 1 / 2 — = M M 7YM +"31 r V A B A in which M is the mass of atom A or B and * indicates the heavier isotope. HbCO in H20 or D20 shows the same pattern of frequency shifts (Table 1) as was previously observed (1), i.e., v*/v for the 13C160 derivative is essentially the same as that for the free gas, while that for the 12 C 18 0 isotope is significantly larger than that of the free gas. The shift of vCQ for ^2c180, relative to 12C160 amounts to a frequency difference of about 3.5 cm ^ less than that predicted for the mass-only shift of an harmonic oscillator,

v (0 - 1) - “e - 2“exe

v (0 * 2) " 2“e ’ 6“exe in which and v^q- ^ 2 ) are tlie fundamental stretch ing frequency and that of its first overtone, respectively.

The harmonic fundamental frequency is w 6 , and x* 6 is the anharmonicity constant. Infrared difference spectra taken at 2 and 8 cm’^ res olution of the isotopic CO derivatives of HbCO in D20 (Fig ure 7) show the presence of a single absorption which can be identified as the CO overtone. Absorptions at lower frequency in the 8 cm~* resolution spectra do not appear to have the same intensity in the 2 cm~*^ resolution spec tra and may be artifacts. The areas of the overtone bands are about 0,5% of that of the fundamental, which is very similar to the observations of Penner and Weber (50), who found that the intensity of the overtone of the free gas was 0,33% of that of the fundamental. The fundamental frequencies corrected for anharmonicity (Table 2) have an isotopic shift pattern, u>*/oi , very similar to that observed for the uncorrected fundamental (v*/v). Therefore, anhar- 12 18 monicity cannot be invoked to explain why the C 0 fre quency shift is less than that predicted by mass effects. eolbnv. xhmgoi. o: b 0 Center: 0, C Hb Top: oxyhemoglobin. vs. hemoglobin b210, otm IIb12C 160. Bottom: Hb12C180.,

Fig. 1. Infrared difference spectra of native carboxy- native of spectra difference Infrared 1. Fig. ABSORBANCE .4 0 0.8 1.2 I860 Y C N E U Q E R F 1940 (cm-*) 13

19801900 16 31 globin denatured at pH 2.5 with HC1 vs. oxyhemoglobin. oxyhemoglobin. vs. HC1 with 2.5 pH at denatured globin o: b3 6, etr H1C8, otm Hb12C160. Bottom: Hb12C180, Center: 160, Hb13C Top: ABSORBANCE 0.04 Fig. 2. Infrared difference spectra of carboxyhemo- carboxyhemo- of spectra difference Infrared 2. Fig. 0.08 0.12 0.16 8 1900 0 I86 RQEC (cm-') FREQUENCY 901980 1940 CD DENATURED ACID HEMOGLOBIN

2020 32 33

BASE DENATURED HEMOGLOBIN 1.6 13. 16

1.2

ot i l 2 m< or O co m <

12- 16. 0.4

I860 1900 1940 1980 2020 FREQUENCY (cm-*)

-Fig. 3. Infrared difference spectra of carboxyhemoglobin denatured at pH 11.9 with KOH vs. oxyhemoglobin. Top: Hb13C160, Center: Hb12C180, Bottom: Hb12C160. globin denatured at 100° C for 20 min vs. oxyhemoglobin. oxyhemoglobin. vs. min 20 for C 100° at denatured globin o: b310 Cne: b210 Bto: IIb12C160. Bottom: Hb12C180, Center: Hb13C160, Top:

ABSORBANCE 0.5 2.0 Fig. 4. Infrared difference spectra of carboxyhemo- carboxyhemo- of spectra difference Infrared 4. Fig. 1.0 860 6 I8 1900 RQEC (cm-') FREQUENCY ET DENATURED HEAT HEMOGLOBIN 19801940

2020 34 35

0.08

0.06

Ot i l z 00 OC (/)O CO 0.04 <

0.02

(900 1950 . . 2000 FREQUENCY {cm-')

Fig. 5. Infrared spectra of chloroperoxidase-CO com plex at pH 5.9 vs. 35 % aqueous glycerol. Dots indicate •uncompensated water vapor absorptions. Top: CP0— Center: CPO-12C180, Bottom: CPO-12C160. 36

0.16

0.12 lii o z

0.04

1900 1950 FREQUENCY (cm-')

Pig. 6. Infrared spectra of horse radish peroxidase- C0 complex at pH 7.0 vs. water. Dots indicate uncompen- 13 16 sated water vapor absorptions. Top: HRP- C 0, Center: HIIP-12C180, Bottom: HRP~12C160. Table 1. Isotopic Shifts of the CO Stretching Frequency of Heme Protein Carbonyls

Vl3c16o 13_16 A Vl2C150 4 v12C160 v12C180 v C O 10 A 104A Vl2c16o Vl2c16o

CO (gas)a 2143.8 2092.1 2096.1 0.9759 0.9777 Hb 1950.9+0.2 1906.8 1906.5 .9774 15 .9772 -5 Hb (in D^O) 1950.6±0.2 1906.7 1906.3 .9775 16 .9773 -4 Acid Denatured hb 1967.6+1.0 1922.9 1921.5 .9773 14 .9766 -11 Base Denatured hb 1965.6±0.5 1921.3 1921.1 .9775 16 .9774 -3 Heat Denatured hb 1966.3±0.5 1921.5 1921.5 .9772 13 .9772 -5 CPO (pH 5.9) 1960.8±0.5 1915.5 1915.3 .9769 10 .9768 -9 HRP I 1904.8+0.5 1863.3 1861.1 .9782 23 .9771 -6 HRP II 1933.4±0.5 1890.5 1889.8 .9778 19 .9775 -2 Ni (CO) 4 ( v ^ 13 2125.0 . 2078.3 2075.9 .9780 21 .9769 -9 Ni(CO)4 (v5)b 2044.5 1997.4 1998.4 .9770 11 .9775 -2

a. Mills, I. M. and Thompson, H. W., Trans. Faraday Soc., 49, 224 (1953) and Pyler, E. K., Blaine, L. R., and Tidwell, E. D., J. Res. Natl. Bur. Std., 55, 183 (1955). b. Jones, L. H., McDowell, R. S., and Goldblatt, M., J. Chem. Phys., 48, 2663 (1968). 38

0 .0 4

UJ o 0 .0 3 < CD or o CD 0.02 <

0.01

3 7 0 0 3800 3900 FREQUENCY (cm-1)

Fig. 7. Infrared difference spectra of isotopically 12 16 labelled HbCO comples in D20 vs. lib C 0 in CO overtone region. Upward arrow indicates position of CO overtone of isotopically labelled HbCO complexes. Downward arrow marks 12 16 position of negative absorption due to Hb C 0. Top: n if i io _i Hb C 0, Upper center: Hb- C 0, both at 2 cm resolu- i ^ 1 fi i 0 1 a tion. Lower center: Hb C 0, Bottom: Hb C 0, both at 8 cm ^ resolution. 39

Table 2. HbCO Overtone Characteristics and Fundamental Frequencies Corrected for Anharmonicity

12c16o 12c18o ^8C^80

HbCO in D20

V (0 2) 3877 ± 1 3789 ± 1 3790 ± 1

Av 1/2 21.0 20.5 21.0

V (0 - 1) 1950.6 1906.7 1906.3 8.8 8.7 8.7 Av 1/2 6.0 6.2 5.9 1974.3 1930.6 1921.1 we ------0.9773 0.9774

104 A ------14 -3

Ni(CO)4a • 103xe (Vl) 5.05 4.95 4.96

10 3X (v5) 8.30 8.15 8.15

CO (gas)

103Xe 6.13b 5.97° 5.96d

a. Jones, L. H., McDowell, R. S., and Goldblatt, M. J., J. Chem. Phys., 413, 2663 (1968) b. Rank, D. H., Guenter, A. H., Saksena, G. D., Shearer, J. N., and Wiggins, Tv A., J. Opt. Soc. Am., 47, 686 (1957) c. Mills, X. M. and Thomp son, H. W., Trans. Faraday Soc., 49, 224 (1953) d. Pyler, E. K., Benedict, W. S., and Silverman S., J. Chem. Phys., 20, 175 (1952). 40. B. Extinction Coefficients of Carboxy- hemoglobins Intensities of absorptions due to isotopically substi tuted molecules are expected to be different from those of the unsubstituted molecule because of bond moment changes (51). Vibrational coupling of one atom of CO to the iron

Table 3. Extinction Coefficients3 of HbCO Isotope Com plexes in D 2 O

12c 16o 12c 18o 13c 1 6 o amM (aksorkance) 2 .88±0.08 2.88+0.08 2.8710.08 emM (a r e a ) 32.2±0.9 31.3±0.9 31.210.9

a. Calculated from graphic measurements of absorb- and and area might be expected to produce a perturbation of the extinc- . tion coefficient of CO isotopically labelled at that atom. Consequently, extinction coefficients (Table 3) were ac curately calculated for both abosrbance and area of the fundamental absorptions of the HbCO derivatives in D2 O. . No significant differences between the isotopes were observed,

C. Denaturetion of Carboxyhemglobin Carboxyhemoglobin was denatured using four general types of denaturant: acid, base, heat, and detergents. 12 16 Comparing only the acid, base, and heat denatured Hb C 0 spectra (Figures 2, 3 and 4) with the detergent denatured 41 HbCO spectra (Figure 8), it can be seen'that all types of denaturation tested produce a hemoglobin species with simi lar frequency and half-bandwidth (Table 4). These similar ities indicate a general similarity of the environment

Table 4. Carbon Monoxide Stretching Frequencies (v ^q ) and Half-Bandwidths (Av]y2) Native and Various Types of Denatured HbCOa

VCO *Vl/2

Native 1950.9 8.3 Acid (pH 2.5) 1967.1 17.4 Base (pH 11.9) 1965.6 17.4 Heat (20 min boiling) 1966.3 19.6 SDS (0.5M) 1969.0 16.8 SDS (0.1M)/HOAc (0.5M) 1973.0 16.9 Benzoate (2M, pH 7.2) 1964.1 18.5

a. Values for denatured HbCO taken by graphic reso lution of the absorption envelopes into two symmetrical absorptions. around the CO molecule and, therefore, around the heme. It is also apparent, from the slight asymmetry to lower fre quency and from examination of the partially acid denatured HbCO spectra (Figure 9), that only two major species exist: a component with an absorption band at the native HbCO fre quency of 1951 and one representing the major denatured -1 form with v_n between 1964 and 1973 cm • Intermediate frequency species are not seen in the equilibrium mixture. How ever, the component at 1951 cm ^ could not be entirely 42

0.4 0.1 M SOS 0.5M HOAc

0.3

UJ o 0.5M SDS m cc ^ ^ o 0.2 co m

0.1

_ 2 M Na BENZOATE

0 — 1900 1940 1980 2020 FREQUENCY (cm*')

Fig. 8. Infrared spectra of detergent denatured car- boxyhemoglobins. Top: HbCO in 0.111 SDS and 0.5M acetic acid vs. oxyhemoglobin. Center: HbCO in 0.5M SDS vs. oxy hemoglobin. Bottom: HbCO in 2M .sodium benzoate (pH .7.2) vs. 2M sodium benzoate. 43

PARTIALLY ACID DENATURED 0.4 HEMOGLOBIN

0.3

ui o z < CO o 0.2 mto <

0.1

I860 1900 1940 I960 2020 FREQUENCY ( c r 1)

Fig. 9. Infrared difference spectra of partially acid denatured carboxyhemoglobin vs. oxyhemoglobin. Top: Hb13C160 denatured with IIC1. Center: Hb12C160 denatured 10 If* with HC1. Bottom: Hb C ' 0 denatured with acetic acid. 44 eliminated; i.e., extending the boiling period from 20 min to 5 hr for the heat denatured HbCO produced no further changes in the absorption envelope. This component may be, therefore a second, minor denatured hemoglobin species, or possibly indicate a slight reversibility of denaturation under these conditions. Consideration of the frequencies and half-bandwidths of these denatured hemoglobin species leads to the conclu sion that the denaturants other than SDS and benzoate pro duce a collapse of the protein structure around the heme, enclosing it in a randomized environment of rather non polar amino acid side chains, i.e., a protein micelle. The striking similarity of the frequencies of the acid, base, and heat denatured hemoglobins suggest that neither acid nor base was able to affect the heme itself under these conditions. The half-bandwidth of the denatured hemo globins is substantially larger than that of native HbCO and indicates that considerable randomization has taken place, but it does not approach the value that would be expected if the heme were exposed to water. For example, aqueous azide ion, a completely solvated species, has a -1 half-bandwidth of about 25 cm (52). Tetraphenylporphin -1 carbonyls in ^H^/CHCl^ have half-bandwidths of 30 cm (53) while heme carbonyls in pyridine/CHBr^ have half- -1 bandwidths of about 35 cm (54). The frequencies and half-bandwidths of the SDS, SDS/ 45 HOAc, and benzoate denatured hemoglobins suggest that these agents are included in the protein micelle around the heme. The two types of SDS denatured HbCO have the highest fre quencies and narrowest bandwidths of all the denatured hemoglobins. Inclusion of SDS in the protein micelle would create a somewhat less polar environment around the heme and, consequently, cause a smaller half-bandwidth. Ben zoate, on the other hand, produces.the lowest frequency and broadest half-bandwidth of all types of denatured HbCO. Benzoate is much smaller than SDS and is expected to associ ate directly with the heme through ir-interactions, intro ducing a polar carboxylate anion into the protein micelle. The resultant destabilization of the micelle would tend to expose the heme-CO complex more to the aqueous solvent, creating a broader half-bandwidth.’ These data also indicate that exchange of the trans heme ligand (histidine) does not occur. Allis and Stein- hardt (55) have postulated detachment and dimerization of the heme to explain the kinetics of acid denaturation of dilute hemoglobin solutions. However, ligand exchange is expected to produce substantial changes in \>co and is not expected to cause the same frequency for each type of de naturation. Additional evidence bearing on this point is the stoichiometry. If ligand exchange were occurring, free heme-CO would be liberated and aggregation with loss of CO would be a competing process, especially at acid pH. 46 Calculation of the amount of CO remaining after denaturation (Table 5) indicates that heme aggregation is not a major effect. Losses which occur are reasonably due to autoxidation.

Table 5. Percentage of Bound CO Remaining after De- naturation of HbCO

12C160 12C180 13c16o

Acid 76 57 51 Base 79 78 89 SDS 104 —

Benzoate 97 --- —

a. Calculated using area extinction coefficients in Table 3.

D. Abnormal Hemoglobins Infrared spectra of the CO derivatives of hemoglobins Little Rock, 6143 His •+• Gin, and S, 36 Glu Val, were also examined (Figures 10 and 11) and appeared to be nearly identical to that taken for normal human HbCO. This is consistent with previous observations (39) that substitu tions at residues removed from the heme pocket do not af fect the CO stretching frequency.

E. cytochrome P-450camphor

The infrared spectrum of the P-450-CO complex (Figure lbnLtl Rc s oxyhemoglobin. vs. Rock Little globin ABSORBANCE 0.004 0.008 0.012 0.016 i. 10. Pig. 1900 d e r a r f n i 1920 FREQUENCY FREQUENCY difference_ 1940 spectrum of carboxyherao-of spectrum (cm-') 960 6 I9 1980 47 lbn s oxyhemoglobin. vs. S globin

ABSORBANCE i Fig. 11. Infrared difference spectrum of carboxyherac carboxyherac of spectrum difference Infrared 11. Fig. 0.08 0.04 0.16 0.12 1980 I960 FREQUENCY 1940 (cm-') 1920 48 49 12) shows a single large absorption with a much smaller, very broad absorption at higher frequency, attributed to denaturation products. The frequency of the main absorption (Table 6) is lower than that found for HbCO or MbCO. This is consistent with the presence of a stronger base than histidine as the trans ligand, although other explanations, i.e., differences in the heme pocket of the protein or bend ing of the Fe-CO bond cannot be rejected. The broader half bandwidth, relative to native HbCO indicates a more random ized heme environment and suggests a wider heme pocket than is present in HbCO.

Table 6. CO Frequencies and Half-Bandwidths of Carbon Mon- oxide Complexes of Heme- Containing Enzymes

v II vCO 1 Vl/2 1 CO vV 2 II

P-450 (aqueous) 19 39 14.8 ------CPO (pH 3) (aqueous) 19 36 (11) 19 50 (11) CPO (pH 4.2) (50% glycerol) 1932 (11.5) 1943 (13.0) CPO (pH 5.p) (35% glycerol) 1961.3 12.3 ----- HRP (pH 7.0) (aqueous) 1904.8 14.8 1933.4 10.8

F. Chloroperoxidas e Infrared spectra of CPO-CO at pH 3 and 4,2 show much C ope s water. vs. complex -CO ABSORBANCE 0.06 0.02 0.04 Fig. 12. 3i*"'*50campjior indicates 52

0.16

0.12 oUJ z <00 DC O CO00 < 0.08

0.04

1900 1950 2000 FREQUENCY (cm-')

Fig. 13. Infrared spectra of chloroperoxidase-CO com plex as a function of pH and solvent. Dots indicate uncom pensated water vapor absorptions. Top: CP0-C0 at pH 5.9 in 35% glycerol vs. 35% aqueous glycerol. Center: CP0-C0 at pH 4.2 in 50% glycerol vs. 50% aqueous glycerol. Bottom: CP0-C0 at pH 3 in 0.5M phosphate buffer vs. water. 53 that the similarities observed between the two enzymes (32) may be rather superficial.

G-. Horse Radish Peroxidase The spectra of HRP-CO (Figure 14) also show the pres ence of two absorptions. There is a pH dependent transi tion between the two species, the lower frequency component being greatly diminished at pH 8.7. However, titration to pH 4.9 did not produce the same effect on the high frequen cy band; it merely restored the ratio of the areas of the two bands to its value at pH 7, with both bands becoming unsymmetrical. Back titration of this sample to pH 8.2 did not produce any further significant changes in the area ra tio. These observations are explained in terms of isozymes,, which are well documented for HRP (57) and have been shown to have different catalytic activities (58). From these data, one type of isozyme is pH independent and contributes only to the high frequency absorption. The other type has a pH dependent transition near the heme and is converted in to a species similar to the pH independent isozyme within creasing pH. However, an irreversible modification to at . least the pH dependent isozyme occurs at pH 4.9. The low frequency CO band of HRP is nearly 30 cm ^ low er than any other CO absorption which has been observed for metal protein carbonyls. The pH dependence of this band suggests a unique explanation for its low frequency. This absorption becomes larger with decreasing pH and is therefore 54

0.16

0J2 UJo z < CO so c CO •< 0.0 8

0.0 4

1900 1950 FREQUENCY (cm-')

Fig. 14. Infrared spectra of horse radish peroxidase- C0 complex vs. water as a function of pH. Top: HRP-C0 at pH 8.7. Center: HRP-C0 at pH 7.0. Bottom: HRP-C0 at pH 4.9. due to a protonated HRP species. Protonation of a group which-can then hydrogen bond to the CO molecule or of a group already hydrogen bonded to it, will lower vCQ by re ducing electron density in the CO bonding molecular orbi tals. The other alternatives, protonation of the trans ligand or of a group at the periphery of the porphyrin ring, will decrease electron density in the CO antibonding molecu lar orbitals, resulting in a higher v^0 (35). The 4 cm ^ increase in the half-bandwidth of HRP-CO upon protonation also indicates a substantial change in the heme environment for the protonated HRP species. It sug gests a major change of the protein conformation in the vicinity of the active site upon protonation, producing a wider heme pocket, which is more accessible to water. 56 IV. DISCUSSION Alben and Caughey (35) and Fager and Alben (38) pro posed that carbon monoxide is bound to metal proteins by means of a metal-oxygen bond because the isotopic frequen- 12 18 cy shift of bound C 0 was quite different from that of the free gas. This was presumed to arise from vibration al coupling between the carbon-oxygen and the metal-oxygen bonds and would reduce the effect of a heavier isotope on the CO stretching frequency. However, literature data on simple metal carbonyls (48), which has recently become avail able, indicates that this phenomenon also exists for carbon yls known to have a linear metal-carbon bond. The fact that this phenomenon is general does not make the hypothesis of a metal-oxygen bond untenable. The X-ray structure of erythrocruorin clearly shows a bent bond (46) and recent molec ular orbital calculations by Politzer (59) indicate that CO bound to heme iron may have only two stable conforma tions : a linear iron-carbon bond and a bent iron-oxygen bond. The bond structure and orientation of CO bound to heme proteins is not well defined at the present time and more sophisticated techniques will have to be developed to clarify this. The observation of a heme protein carbonyl overtone absorption is an exceedingly difficult experimental under taking, largely because of the very low intensity of the absorption. This is complicated by the presence of a large 57 number of intense water vapor absorptions in this spectral

region, low transmittance of the solvent (D2 ° ) t and by the possibility of spectral "folding,” which may occur with an interferometer operating near its frequency cutoff limits. However, observation of the overtone is the only realistic way of approaching the question of anharmonicity for bound carbon monoxide in native heme proteins and further research of this nature will certainly be of assistance in defining the bond structure of heme protein carbonyls. The studies on denatured hemoglobin are of particular interest because they represent a model system for all heme proteins and demonstrate that factors other than trans lig and effects can exert a major influence on the CO stretching frequency. The CO frequencies observed for the denatured hemoglobins are consistent with those observed for model imidazole-heme-CO complexes (44, 45)> but vary from 1964 to 1973 cra-^. This 9 c m ” '1' variation is interpreted as be- . ing due to differences in the solvation shell of the CO molecule. Native hemoglobin and myoglobin, however, have vCQ at 1951 and about 1945 cm \ respectively. For the • native proteins, variations of vCQ caused by differences in the solvation shell cannot be distinguished from those caused by the possible bending of the iron-CO bond. How ever, these large changes in vCQ can certainly be attributed to differences in the heme environment because the trans ligand is the same in all cases. 58 The acid denaturation study of HbCO is also useful in interpreting the low frequency absorption of HRP-CO. This absorption was attributed to protonation of a group associ ated with the CO moiety. However, the possibility also exists that the CO molecule itself might be protonated. For hemoglobin, neither acid denaturation with HC1 at pH 2,5 (Figure 2) or with acetic acid at pH 3.7 (Figure 9) produced any hint of an absorption which could be attributed to a protonated carbonyl. The fact that acetic acid does not produce a low frequency absorption may also be interpreted as meaning that the group interacting with the CO is not a glutamic acid or aspartic acid side chain. The pK of the transition (about 8) is reasonable for an N-terminal amino group, but could also be due to histidine, lysine, or a cysteine sulfhydryl group. Finally, the infrared observations of half-bandwidths suggest that the heme pockets of the three heme enzymes are wider than that of hemoglobin. The bandwidths of the en zymes are all substantially larger than that of HbCO. This indicates that the enzyme heme pockets have a more random molecular environment and are, therefore, more open to the aqueous solvent than is the case with hemoglobin. The half bandwidth measurements still reflect a high degree of struc ture because they are significantly narrower than those found for denatured HbCO. These observations are consistent with the requirement that the enzymes have a wide heme 59 pocket to bind a substrate molecule in close proximity to the heme as well as oxygen or a peroxide at the heme itself. Hemoglobin, however, requires only a narrow, rather non polar heme environment to carry oxygen reversibly. SECTION II. HEMOGLOBIN CONFORMATION AND INTERACTION WITH ANIONS

I. INTRODUCTION The sulfhydryl groups of proteins in general and hemo globin in particular have been the subject of continuing research for many years because they provide a chemical probe into structure-function relationships for native pro teins. The six sulfydryl groups of human hemoglobin occur in three pairs, only one of which is particularly reactive to SH reagents under normal conditions. The reactive pair, at position 393 is located close to the interface an(* very close to the porphyrin. The other two pair, at posi-* tions al04 and 3112 are located within the ci^^l interface and only become significantly reactive under dissociating conditions. Early investigations of the reactive 393 cysteine resi dues were oriented towards determining the relation of thes SH groups to the functional properties of hemoglobin. Riggs and Wolbach (60) found that the reaction of hemoglo bin with p-chloromercuribenzoate (PMB) at the 39 3 SH groups caused profound changes in oxygen affinity, heme-heme in teraction, and the Bohr effect. A similar effect was found with N-ethylmaleimide (NEM) (61). From these data, it was 60 61 suggested that the 39 3 SH groups play an important role in regulating the functional properties of hemoglobin and, furthermore, that they were even the source of the Bohr, effect protons (62). However, Benesch and Benesch (63) con cluded that the reactive SH groups were not directly in volved in the Bohr effect and later work with a variety of SH reagents (64) showed that the 39 3 SH groups were not directly related to heme-heme interaction either. Additional work by Riggs (61) and Benesch and Benesch (65) indicated that the reactivity of the 393 groups was very dependent upon ligand binding, the rate of reaction being much less for deoxyhemoglobin. This was attributed to conformation differences between the oxy and deoxy forms, which was later verified by the X-ray structure work of Perutz, et al. (66). Further investigations by Guidotti (67) showed slight differences in conformation depending upon the type of ligand bound, based on differences in SH reactivity. Evidence also exists that binding of DPG (68) or I HP (69) to deoxy lib further decreases SH reactivity. This was interpreted as stabilization of the deoxy confor mation by these anions; however, the structural significance of this is unclear. Ogawa and McConnell (70) attached spin labels to the 393 cysteine SH groups and found differences in the elec tron spin resonance spectra between oxy and deoxy Hb, which were also taken as evidence for a conformation change near 62 these SH groups. Additional experiments (71) showed that, with certain spin labels, isosbestic points were not ob tained upon progressive oxygenation, indicating intermedi ate conformations in the transition from the deoxy to the oxy structure. Recent work by Huestis and Raftery (72), using a label containing fluorine at the 39 3 sulfydryls 19 in conjunction with F NMR, indicated differences in the conformations of a variety of liganded hemoglobin deriva tives and a large difference between these and deoxy or metheraoglobin. Furthermore, the latter two hemoglobin species were found (15) to have a pH dependent NMR signal which could not be entirely accounted for in terms of pres ent knowledge of the Bohr effect and cooperati.vity. How ever, as Huestis and Raftery (15) have mentioned, the inter* pretation of the effects of conformational changes by use of a magnetic resonance technique with a probe situated so close to the induced magnetic field of the porphyrin is open to question. The other two pairs of cysteine residues in human hemoglobin, at the ctl04 and 3112 positions, are buried in the contact and have been less thoroughly investigated because of their inaccessibility to SH reagents. Perutz (73) has indicated that they have no special role, although he later suggested (71) that the al04 SH group was inter acting with the 3127 glutamine side chain in horse oxyhemo globin. The contact moves only slightly during oxygenation and Perutz has not indicated that any signifi cant changes occur in the bonding structure at this inter face as he has for the contact ^ . In this study, a technique was developed for direct spectroscopic observation of protein sulfhydryl groups. Applied to hemoglobin, evidence was obtained showing that each type of SH group experiences a unique environment. One of these was tentatively identified. They were found to have the expected differences between.the oxy and deoxy states, but methemoglobin was found to be different from either of these. Furthermore, while binding of DPG or IHP to deoxy Hb does not change the set of environment of SH groups, binding to oxy Hb induces a set of SH environment very similar to those of methemoglobin. : 64 II. MATERIALS AND METHODS A. Materials 1. Chemicals Bis (2-hydroxyethy1)iminotris(hydroxymethyl)methane (BISTRIS) and inositol hexaphosphate (IHP) were purchased from Sigma. Potassium cyanide, potassium ferricyanide, and sucrose were purchased from J. T. Baker Chemical Com pany while sodium chloride was obtained from Matheson, Coleman and Bell. Pentacyclohexylammonium 2,3-diphosphoglycerate (DPG) was purchased from Calbiochera. Benzalde- hyde was purchased from Fisher Scientific and diethyl ether was obtained from Allied Chemical Company. L.-cysteine was purchased from Mann Research Laboratories. Argon gas was purchased from Burdette Oxygen Company. Dowex 50W-X8 cation exchange resin, 200-400 mesh, was obtained from Biorad Laboratories and Sephadex G-25, coarse, was pur chased from Pharmacia. Unless otherwise indicated, all chemicals were reagent, analytical, or chemically pure grade and were used without further purification. Glass redistilled water was used for all experiments.

2. Proteins A stock solution of human hemoglobin was prepared from blood freshly collected in heparin, drawn from a nor mal non-smoking subject. Crystalline horse, pig, and cow hemoglobins were purchased from Pentex. Cow hemoglobin 6 5 was also prepared from fresh blood collected in sodium citrate from a Holstein heifer and donated by Dr. G. J. Kociba (Department of Veterinary Clinical Sciences, Ohio State University). Cytochrome p~450camphOr was prepared from Pseudomonas putida in the laboratory of Dr. I. C. Gunsalus and was furnished to us as a lyophilized powder by Dr. Karl Dus (Department of Chemistry, Univer sity of Illinois).

B. Methods 1. Preparation of Potassium 2,3-diphosphoglycerate The cyclohexylammonium salt of DPG was dissolved in a minimum amount of water. This was extracted with an equal volume of benzaldehyde. Following centrifugation, the aqueous phase was removed from between the two organic phases and was extracted twice with diethyl ether. This was passed through a filter containing an excess of air- dried Dowex 50W-X8 in the potassium form which had been washed to neutral pH with water. The DPG solution was then lyophilized and redissolved in a minimum amount of water. This was adjusted to pH 7.1 with KOII. The DPG concentration, determined by the method of Keitt (74), was 0•22M.

2• Preparation of Human Hemoglobin Derivatives Human hemoglobin was prepared by addition of 1 volume of water and 0.5 volumes of toluene to fresh, saline-washed 66 red cells. The resulting clear hemolysate was stripped of organic phosphates by passage through a column of Sephadex G-25 previously equilibrated with 0.1M NaCl, according to the technique of Benesch, et al. (75) and was concentrated by pressure ultrafiltration. BISTRIS (pH 7.1) was added to a concentration of 0.05M. The hemoglobin solution was deoxygenated by several cycles of evacuation and equilibra tion with Argon. The final solution was 18mM in heme, de termined as cyanmethemoglobin, and was 0.05M in BISTRIS and 0.1M in chloride at pH 7.1. Deoxy Hb-DPG was prepared by addition of 2 equivalents per Hb tetramer of DPG to the deoxy Hb in a rubber capped syringe filled with Argon. Oxy Hb-DPG was formed by allow ing the sample to reoxygenate in the syringe. Deoxy Hb-IHP and oxy Hb-IHP were prepared as above but in the presence of 4 equivalents of IHP (pH 7.1) per Hb tetramer. Human methemoglobin (met Hb) was prepared by hemolys is of red cells in 2 volumes of water. After removal of stroma by centrifugation, the hemolysate was oxidized with a slight excess of K^FefCtOg. Ferrocyanide and organic phosphates were removed as described above. The solution was first concentrated by pressure ultrafiltration and then by dialysis against saturated sucrose for 4 hr. The con centrated met Hb solution was dialyzed against 1000 vol umes of water overnight. The final concentration of this 67 solution was 17mM (heme) and contained no extraneous salts. Met Hb-IHP was prepared by addition of 4 equivalents of IHP per Hb tetramer. A second preparation of met Hb (14mM) was used to prepare cyanmethemoglobin (HbCN) by addition of 2 equivalents per heme of KCN.

3. Preparation of Animal Hemoglobins Commercial samples of horse, pig and cow hemoglobins were dissolved in water and centrifuged to remove insoluble material. These samples were entirely autoxidized upon re ceipt. The concentrations of the solutions were 9.2, 7.5, and lOmM in heme respectively. Cow hemoglobin was also prepared by addition of 1 vol ume of water and 0.5 volumes of toluene to fresh, saline- washed red cells. The resultant hemolysate was about 10 mM in heme and was used without further purification.

4. Preparation of Cytochrome p~^60camp^or

The lyophilized powder was dissolved in water con taining 6mM camphor. This was reduced with ^ 2 8 2 0 ^ powder under a carbon monoxide atmosphere. The heme concentra tion was estimated at 3mM.

5. Determination of Infrared and Visible Spectra Infrared spectra were taken on a Digilab FTS-14 in terferometer equipped with a liquid-nitrogen-cooled In- dium-Antimonide detector. Most spectra were taken in cells with CaF2 windows and a path length of 0.2 mm. Spectra of HbCN, P-450, and cysteine were taken in cells with a path length of 0.1mm. All spectra were taken at 5° C ex cept those for HbCN, P-450, and cysteine, which were taken at ambient temperature (28° C). Curvature of the baselines of infrared spectra proved to be a significant problem in this study (see Figure 15) because of lack of a convenient reference solution which contained no sulfhydryl groups. Baseline curvature was minimized by the choice of low con centrations of aqueous n-butylammonium chloride as a ref erence. Visible absorption spectra were taken on a Perkin- Elmer 4000A spectrophotometer in the infrared cells.

6. Quantitation of Infrared Data Infrared data were quantitated by curve-fitting the absorption envelope to the sum of Lorentzian functions as described in the General Introduction, following subtrac tion of the baseline. Absorption envelopes were fitted to the minimum number of Lorentzian functions required to obtain a reasonable fit. Consequently, although hemoglobin absorption spectra are considered to contain three SH ab sorptions, in most cases good fits were obtained by assum ing that only two absorptions were present, with one of these being understood to be the sum of absorption bands due to two pairs of cysteine residues. The spectrum of oxy Hb required the sum of three different Lorentzian nS ein Al pcr eertodv. water. vs. ratioed were spectra All region. SH in cyanmethemoglobin (center), and cytochrome p"^50cainpilor (b°p) cytochrome and (center), cyanmethemoglobin

ABSORBANCE Fig. 15. Infrared spectra of aqueous cysteine (bottom^ (bottom^ cysteine aqueous of spectra Infrared 15. Fig. 0.02 04 .0 0 03 .0 0 0.01 0 0 5 2 O ---- FREQUENCY HbCN 2540 (cm-') 2580 P-450, CYSTEINE cam 0 2 6 2 69 functions to obtain a.reasonable fit. Areas of individual abosrptions were calculated from

Area - (it/2) ( A ^ x Av1/2) which describes the area under a Lorentzian function (2). All infrared frequencies are corrected to vacuum. III. RESULTS A. Aqueous Cysteine The infrared spectrum of aqueous cysteine (0.05M, pH 5.5) (Figure 15) shows a low intensity absorption at 2576 cm 1 (Table 7) with a very broad half-bandwidth. This is attributed to the SH stretching vibration, which is well- known (76) to occur between 2500 and 2600 c m ” '*'. Parker (77) observed vSH of aqueous cysteine at 2538 cm”1, but ,the very high concentration used undoubtedly produced strong hydrogen bonding of the SH groups to other cysteine molecules. This is known to occur at concentrations great er than 0.211 for a variety of other SH compounds (76). The wide half-bandwidth of the cysteine SH absorption (Table 7) is characteristic of a species well solvated by a polar solvent, e.g., aqueous azide ion has a half bandwidth of about 25 cm 1 (52). The apparent area ex tinction coefficient given for cysteine (Table 7) must be considered a limiting value because it is not known wheth er any significant oxidation of the SH groups occurred.

B. Hemoglobin The infrared spectrum of cyanmethemoglobin (HbCN) (Fig ure 15) represents the first direct spectroscopic observa tion of native protein sulfydryl groups in aqueous solu tion. These overlapping absorption bands are attributed to the SH stretching absorption of cysteine residues Table 7. Frequency (vSH) and Half-Bandwidth (Av^2) Parameters Giving Best Fits to Infrared Sulfhydryl Spectra3 and Sulfhydryl Group Extinction Coefficients*3

VSH AVl/2 emM VSH Avl/2 emM VSH Avl/2 emM alO4/093 (Hb) 0112 (Hb)

Human deoxy Hb 2557.1 14.7 1.92 2563.8 12.5 1.29 deoxy Hb-DPG 2557.1 14.7 1.85 2563.8 12.5 1.32 deoxy Hb-IHP 2557.1 14.7 1.69 2563.8 12.5 1.18 oxy Hb 2552.1 12.5 1.81 2557.6 12.5 1.74 2565.1 12.5 1.24 oxy Hb-DPG 2552.6 14.5 1.73 2565.8 12.7 1.30 oxy Hb-IHP 2552.8 14.5 1.77 2565.8 12.7 1.25 met Hb 2552.8 14.5 1.73 2565.8 12.7 1.32 met Hb-IHP 2554.3 14.7 1.73 2566.8 12.5 1.21 HbCN 2553.1 12.5 1.59 2565.1 11.5 0.99 Horse met Hb 2555.6 14.2 1.96 Pig met Hb 2554.9 13.5 1.72

P-450 2562.2 23.6 (2.26) Cysteine 2576.2 22.6 (0.70)

l. Estimated accuracy of vgH and Av^y2 *-s *1*° cm**^

b. Calculated from the area under the computed Lorentzian function as described in the Methods section. 73 because virtually no other fundamental absorptions occur in this region. The band intensity is dependent upon pro tein concentration and the possibility that it might be due to some other protein absorption is excluded because the infrared spectrum of cow hemoglobin (Figure 16) shows no absorptions in this region. The abosrption envelope for HbCN was resolved into two bands (Table 7) by curve-fitting. The larger, lower frequency absorption was suspected of containing two un resolved absorptions because human hemoglobin is known to contain three types of sulfhydryl groups. Confirmation of this and a tentative assignment of one of the absorptions was possible from the examination of animal hemoglobins. Both horse (78, 79) and pig (80) hemoglobins are known to lack only the 8112 cysteine residues, of the three types present in human hemoglobin. The other two pairs of cys teine SH groups are expected to have environments very similar to the corresponding SH groups in human hemoglobin. The infrared spectra of these animal hemoglobins (Figure 16) show a single, almost symmetrical band comparable to the 2553 cm"1, absorption of human HbCN. This strongly suggests that the small absorption at 2565 cm ^ in human hemoglobin is due to the 6112 cysteine residues. Bovine hemoglobins A and B are the only hemoglobin types found in cows of northern European origin (81) and both types are known to lack both the ctl04 and $112 methemoglobin vs. methemoglobin ABSORBANCE Fig. 16, Infrared spectra of horse, pig, and cow cow and pig, horse, of spectra Infrared 16, Fig. 0.04 0.06 0.02 2500 b ' 5 aqueous n-butyl ammonium chloride. ammonium n-butyl aqueous E S R O H metHb Y C N E U Q E R F 0 4 5 2 COW metHb PIG metHb 2580 (crrr1) 2620 74 7 5 cysteines (83), having only the (393 cysteine residues. However, neither the commercial preparation of bovine met Hb or fresh oxy Hb showed the presence of any SH absorp tion. This suggests that the 89 3 SH groups of cow hemo globin are blocked, although there is no independent evi- • dence for this. It was, therefore, not possible to make further assignments of the SH absorptions of human hemo globin. Infrared spectra of human deoxy and oxy Hb (Figure.17 and Table 7) show clearly that the SH groups experience different environments between these two derivatives and that both are different from HbCN. This suggests that the conformations of these three hemoglobin species, as seen by the SH groups, are different. Binding of DPG or IHP to deoxy Hb (Figures 17 and 18) shows no changes in the absorption envelope from deoxy Hb in the absence of these anions. This probably reflects the stereochemical complementarity of the anions to the deoxy Hb anion binding site. Binding of either of these anions to oxy Hb, however, produces a striking change in the envelope (Figures 17 and 18), causing it to become very similar to that of met Hb (Figure 18). This is almost en tirely caused by a movement of the al04/893 absorption to lower frequency. Binding of IHP to met Hb (Figure 18) pro duces further changes in the SH absorptions, shifting all bands to slightly higher frequency. 76

oxy Hb-DPG

0.16

oxyHb

0.12

Ui o z

0.04 deoxyHb

25 00 2 5 4 0 26202580 FREQUENCY (cm-')

Fig. 17. Infrared spectra of human hemoglobin deriva tives vs. 7.5% aqueous n-butyl ammonium chloride. Top: oxy Hb-DPG, Upper center: deoxy Hb-DPG, Lower center: oxy Hb, Bottom: deoxy Hb. 77

0.16 metHb-IHP

0.12 metHb iu o z < m ce 0.06 t< o oxyHb-IHP

0.04

deoxyHb-IHP

2500 2540 2580 2620 FREQUENCY (cm-')

Fig. 18. Infrared spectra of human hemoglobin deriva tives vs. 7.5% aqueous n-butyl ammonium chloride. Top: met Hb-IHP, Upper center: met Hb, Lower center: oxy Hb- IHP, Bottom: deoxy Hb-IHP. 78 A final interesting feature of this is that the ratio of the calculated area of the al04/89 3 absorption to that of the 8112 band (Table 8) is consistently greater than 2 for all hemoglobin derivatives. This was initially ascribed to experimental error. However, calculation of area extinc tion coefficients per SH group (Table 7) for each type of SH group, including those for horse and pig met Hb, sug gests that there is a significant difference (about 25%) in the extinction coefficients for the SH groups contributing to the two absorptions. The physical significance of this is unclear and sufficient data is not available to estab lish whether the variation is linear with frequency, as has been observed for metal carbonyl CO absorptions (83).

C. Cytochrome g-450eamphor

The infrared spectrum of P-450 (Figure 15) shows a broad band at somewhat higher frequency (Table 7) than was generally observed for the major hemoglobin absorption. This is consistent with the report that this enzyme has 6 sulfhydryl groups (84) and suggests that they all have a similar, but very randomized environment, such as would be expected if they were exposed to the aqueous solvent. 79

Table 8.. Ratio of Area of al04/893 SH Absorption to Area of 8112 Absorption for Hemoglobin Derivatives3

deoxy Hb 2.96 deoxy Hb-DPG 2.81 deoxy Hb-IHP 2.85 oxy Hb 2.86 oxy Hb-DPG 2.63 oxy Hb-IHP 2.81 met Hb 2.62 met Hb-IHP. 2.85 HbCN 3.17

a. Areas calculated from area under Lorentzian func tion as described in Methods section. 80

IV. DISCUSSION The direct spectroscopic observation of sulfhydryl groups represents a major advance in structure analysis of native proteins. Consideration of extensive data on the SH stretching frequency of thiophenols by David and Hallam (85) indicates that the major factors affecting are hydrogen bonding and local dielectric constant. Polar interactions are extensively involved in the maintenance of protein structure and in the binding of substrates to enzymes. Consequently, observation of changes in these forces, as seen by sulfhydryl groups, is expected to be a unique probe into protein conformation, as is demonstrated here for hemoglobin, and into the mechanisms of enzymes with SH groups at their active sites, such as glyceralde- hyde 3-phosphate dehydrogenase or papain. These data indicate that hemoglobin may assume three general types of conformations: 1) the deoxy structure, whether organic phosphate is bound or not, 2) the oxy struc ture, which is unique, and 3) a modified oxy structure, occurring upon anion binding and which is remarkably simi lar to that of met Hb. The differences in conformation ob served here for oxy and deoxy Hb are consistent with a variety of previous observations, including magnetic reson ance studies (70, 72), SH reactivity studies (62, 63), and X-ray evidence (7, 66). The similarity between met Hb and deoxy Hb and the dissimilarity of met Hb and HbCN 81 observed by Huestis and Raftery (72), however, appear to be due to ring current effects because the infrared spectra of deoxy and met Hb are clearly different while those of met Hb and HbCN are very similar. The presence of two oxy conformations has not been revealed by any other technique except possibly the SH re activity studies which showed a decrease in SH reactiv ity upon anion binding (68, 69). This has been interpreted as a shift towards the deoxy conformation. However, the SH absorption frequencies of the modified oxy conformation are both shifted further from those of deoxy Hb in the same direction as they were for oxy Hb, i.e., the al04/39 3 band moves to still lower frequency while the 8112 band moves to still higher frequency. Therefore, decreases in SH reactivity caused by anion binding to oxy Hb cannot be interpreted in terms of stabilization of the deoxy conform ation. The fact that the oxy conformation is unique raises questions regarding the X-ray structure determined for oxy Hb. This was actually determined for met Hb because oxy Hb oxidizes very readily in an X-ray beam (86). Perutz and TenEyck (86), however, suggest that there is no signifi cant difference between the structure of oxy and met Hb, based on a Fourier difference synthesis between met Hb and HbCO. They further argue that the oxy/met hemoglobin struc ture is the same in solution as in the crystal, based on work with salt free crystals. This question is of funda mental importance to any interpretation of the X-ray struc tures in terms of hemoglobin function. The infrared observations bear directly on this point. The environments experiened by SH groups at the al04 and 393 positions of oxy Hb are clearly different from those seen by the corresponding SH groups of met Hb in solution. Furthermore, because these SH groups are rather far removed from each other, the conformational differences between oxy and met Hb in solution may be assumed to be of a fairly ex tensive nature. This, however does not suggest that the X-ray structure of oxy/met hemoglobin (7) is not valid, because it was de termined under entirely different circumstances, i.e., oxy Hb in the presence of a high anion concentration, result- ^ ing from the concentrated ammonium sulfate solution used for crystallization. It is apparent from the spectrum of oxy Hb-DPG (Figure 17) that DPG interacts strongly with oxy Hb and influences the conformation at the SH groups. Evi- « dence is presented elsewhere in this dissertation that small anions, such as chloride and phosphate, act in an identical manner, although at much higher concentrations. Therefore, the conditions for the oxy Hb-DPG spectrum are more comparable to those under which the X-ray structure was determined and it can be seen (Table 7) that the SH absorptions of oxy Hb-DPG and met Hb are, in fact, nearly 83 identical. The fact that Perutz and TenEycJc (86) observed no differences between salt free and normal crystals sug gests that crystal lattice forces may be more important in determining hemoglobin structure than was previously sus pected. Additionally, it is proposed that this modified oxy structure is a physiologically significant conformation. The conditions used for the oxy Hb-DPG experiment are simi lar to those existing in the erythrocyte, except for tem perature, and, therefore, the oxy Hb-DPG complex may be a significant fraction of the total oxyhemoglobin in the red cell. The changes in the structure of the a-^i interface occurring upon oxygenation, as determined by infrared spec tra of the SH groups, are consistent with those observed by Perutz (7). Comparing deoxy Hb and oxy Hb-DPG, the total shift of the ctl04/89 3 absorption is 5 cm Both components of the absorption must move approximately this amount because no change occurs in the apparent bandwidth of this absorption. This indicates that the al04 SH groups are involved in.a stronger interaction with the 8127 gluta mines in oxy Hb-DPG than in deoxy Hb. Perutz (7) states:. The relative displacements of atoms in the contact is only O about 1 A.” A decrease in the distance between the c*104 and 8127 side chains of this magnitude could easily pro

duce the observed frequency shift. The $ 1 1 2 absorption, 84 on the other hand, only moves 2 cm ^ and suggests that in teraction of this SH group with its neighbors is very lim ited, consistent with the observations of Perutz (73). The frequency shifts for oxy Hb, in the absence of DPG, cannot presently be defined because the two components of the al04/89 3 absorption do not shift equally. However, the conclusion that the conformation change from the deoxy to the oxy (in the absence of anions) structure is not as large as that observed by Perutz (7) seems to be justified because no component of the absorption envelope of oxy Hb is shifted as much as for oxy Hb-DPG. The situation with the 893 SH groups is more difficult to interpret. The 89 3 absorption, although it cannot be unequivocally identified, has neither the frequency nor the bandwidth characteristic of aqueous cysteine. The X-ray structure of oxy Hb (7) shows that this SH group is freely accessible to water. The low frequency and narrow bandwidth, however, suggest that it is possibly interact ing with a neighboring side chain, perhaps on the H helix, and is in an environment approximately as structured as those of the other SH groups. For deoxy Hb, X-ray evidence shows the 89 3 SII groups behind the H helix and relatively inaccessible to solvent. In this case, the frequency is higher, suggesting that interaction of the 89 3 groups with neighboring side chains has become weaker, probably because of the movements of the F and H helices relative to each other. PART II. HEMOGLOBIN LITTLE ROCK: ANOMALOUS EF FECTS OF AN AMINO ACID SUBSTITUTION AT THE 2,3-DIPHOSPHOGLYCERATE BINDING SITE

I. INTRODUCTION Hemoglobin Little Rock (HbLR) is a new high oxygen af finity variant which is unique in having normal Bohr effect and heme-heme interaction (87). In the present work, these observations are extended and presented in detail. The amino acid replacement of HbLR is identified as 3143 (H21) His -*■ Gin, corresponding to a site which has been identif ied by X-ray structure analysis (21) and chemical evidence (20) as being responsible in normal hemoglobin (HbA) for binding 2,3-diphosphoglycerate (DPG). DPG is the major allosteric effector of hemoglobin oxygen affinity, having been reported (75) to bind much more strongly to deoxy than to oxyhemoglobin, thus stabilizing the deoxy conformation and lowering oxygen affinity. If the only effect of sub stitution of glutamine for histidine at this site were to reduce DPG binding, HbLR and HbA should have similar oxy gen affinities in the absence of DPG. However, the pre liminary results (87) showed that HbLR retains a high af finity, relative to HbA, even at very low concentrations of DPG. 86 87 In response to our preliminary report (87), Perutz (88) suggested that the anomalous high oxygen affinity may be explained by the formation of hydrogen bonds between the abnormal glutamine residues and the 3139 (H17) asparagine residues in the opposing 3 chains, in only the oxy confor mation of HbLR. These inter-3 chain hydrogen bonds are unique to HbLR and would have the net effect of stabiliz ing the oxy conformation by several kcal/mole, thus pro ducing a high oxygen affinity. The results to be described are entirely consistent with this proposal. These studies include a quantitative analysis of in teraction between hemoglobin and DPG in terms of Wyman's (25) treatment of linked functions and extend the inter pretation by Antonini, et al. (24), of anion effects on hemoglobin oxygen affinity. DPG and other anions are shown to be formally analogous; the differences being quantita tive rather than qualitative. 88 II. MATERIALS AND METHODS Hemoglobin Little Rock hemolysates were prepared for chromatography by freezing and thawing fresh, saline- washed red cells in an equal volume of water. Solutions were stored over liquid nitrogen as the carbon monoxide derivatives until use. A small portion of HbLR hemolysate was prepared by addition of 2 volumes of water and 0.5 vol umes of toluene to fresh red cells for the determination of the oxygen equilibrium of the unseparated hemoglobins. DPG was purchased from Calbiochem as the cyclohexylammonium salt. It was converted into the potassium salt by passing a saturated solution through a- large excess of dry X Dowex 50W-X8 in the K form which had previously been washed to neutral pH with water. The resultant concen trated DPG solution was adjusted to pH 7.1 v/ith KOH. The DPG concentration was determined by the method of Keitt (74). Purified HbLR and HbA were prepared chromatographically as described in the legend to Figure 1. The hemoglobin fractions were concentrated by precipitation from 50-55% ammonium sulfate at pH 8.0. The precipitate was redis solved in, and dialyzed exhaustively against water. It was then dialyzed against 0.05M BISTRIS buffer (pH.7.1), resulting in solutions about 3mM in heme. These were stored until use as described above. No detectable denaturation or methemoblogin formation was produced by • 89 this procedure for either HbLR or HbA. Electrophoresis was performed on the concentrated hemoglobin fractions at pH 6.0 in 1% agar gels prepared from Difco Bacto-Agar using 0.05M potassium citrate as the supporting electrolyte. A disc electrophoresis appar atus was used at 5° C with 6 x 150 mm tubes which were fire polished sufficiently on the lower end to hold the gel in place. The gel meniscus was removed with a razor blade. Ten pi samples, about 3% in hemoglobin and 30% in sucrose, were layered onto the gels under the buffer and run at 12 ma per tube for 2 to 3 hr. Gels were stained overnight in 1% Coomassie Blue in 1M acetic acid and de stained 4 hr in 50% aqueous ethanol. Photographic re sults of a typical electrophoretic separation are shown in Figure 2. Chromatographically purified HbLR was esti mated by densitometry to contain only traces of HbA, while HbA solutions contained 5 to 10% HbLR. The effect of DPG on oxygen equilibria of the iso lated hemoglobins was determined at 20° in 0.05M BISTRIS buffer (pH 7.1) and 0.1M in total chloride, by the tech nique of Benesch, et al. (89), with a Cary Model 15 spec trophotometer equippped for temperature control of the — 5 tonometer. Hemoglobin solutions were 5 x 10 M in heme. Denaturation was minimized by applying to the tonometer a film of Beckman Desicote, which was renewed before each experiment. * Methemoglobin formation was usually less than 90 5%, as determined by the method of Benesch, et al (89), using extinction coefficients for methemoglobin appropri ate to pH 7.1, Removal of carbon monoxide from the hemo globin was accomplished by equilibration of the tonometer with pure oxygen for 10 min in an ice-water bath under illumination by a 500 W floodlamp. Oxygen was removed by 2 to 5 cycles of evacuation, flushing with nitrogen, and equilibration for 5 min in a 20° water bath under illumina tion. A minimum time of 10 min was found to be required for equilibration after each air addition. All pH measure ments were at 20°. Effects of other ions on the oxygen equilibria of the hemoglobins were examined in the same manner as for DPG except for buffer composition. Data for each experiment were-plotted according to Hill's equation (90) and each curve was defined by the log p0£ (log Pgg) and slope of the line (n) at 50% saturation, as determined by linear regression analysis. 91 III. RESULTS A. Effect of DPG It is immediately apparent from the DPG titration curves (Figure 21) that, while HbLR interacts.with DPG less effectively than HbA, the oxygen affinity of HbLR is much greater than that of HbA at all DPG concentrations. The midpoint of the titration curve (K^^) (Table 9) is the simplest empirical measure of DPG interaction and in dicates that DPG is about four times less effective in interacting with HbLR than HbA. To obtain a better quantitative description of the behavior of HbLR in the presence of DPG, the data were submitted to a curve-fitting analysis based on the re lation :

O 1 KD 1 (KD + DPG> 109 P50 ~ 109 Pso ~ T 109 Kg + 4 109 (Kg + DPG)

in which KQ and Kg are the dissociation constants for DPG from deoxy and oxyhemoglobin, respectively. Log p^^ is determined at the specified DPG concentration and log p°Q is equal to the log p^0 in the absence of DPG. This equa tion follows directly from Wyman*s treatment of linked functions (25) and is derived from simple equilibrium con siderations in Appendix A. Values of KD and KQ calculated from equation (1) which give the best graphic fit to the experimental data and to 92 that given by Benesch, et al. (91), for fresh HbA are pre sented in Table 9. It is apparent that DPG binds to deoxy HbLR less strongly than to deoxy. HbA by a factor of about 3. However, it is bound less strongly to oxy HbLR than to oxy HbA by a factor of.nearly 7. This is reflected in the

Kq /Kd ratios (Table 9), which are a measure of the differ ence in strength of binding of DPG to the two hemoglobin conformations and, consequently, indicate the ability of DPG to lower oxygen affinity. The slopes of the curves are also consistent with a

larger K q /Kd ratio for HbLR. The slope at any point on the curve has been treated (25) as the difference in the number of ions bound per heme to deoxy and oxyhemoglobin molecules. For the tetramer, the slopes taken at the mid points for both HbLR and HbA approach 1. However, the slope for HbLR is slightly, but significantly, larger than that for HbA, indicating a larger difference in binding of DPG to deoxy HbLR, relative to the oxy form, than the cor responding difference in binding to the two forms of HbA.

• A final feature deserving comment is the low log p^Q and Hill's n of HbA, which was isolated from the same col umn as HbLR. A DPG titration of fresh, "stripped" HbA, prepared by the method of Benesch, et al. (75), gave re sults in agreement with those given by Benesch, et al. i (91). An experiment to test the effect of possible resid ual carbon monoxide on the experimental system indicated 93

Table 9. DPG Titration Curve Parameters and Calculated Dissociation Constants for HbLR and HbAa

Isolated Isolated Fresh HbLR HbA HbA (pH 7.1) (pH 7.1) (pH 7.3)

Kmid 6.7 x 10“4 1.5 x 10~4 1.1 x 10~4 log p 50 at Kmid 0.21 0.69 0.85 slope 0.91 0.82 0.84 4.5 x 10“5 1.5 x 10”5 1.0 x 10"5 k d 1.0 x 10"2 1.5 x 10~3 1.25 x 10~3 Ko - VKD 220 100 125 Alog P50 0.59 0.50 0.52

a. Calculated by curve-fitting of equation (1) to experimental data. b. Calculated for data of Benesch, et al. (11). that this effect was negligible. A curve-fitting proced ure which assumed a sum of Hill equations for various mix tures of HbA and HbLR showed that a mixture of HbA and 10 % HbLR could account for the decrease in Hill's n to 2.4 but would not greatly affect the log P^q - Therefore, we con clude that the HbA must have been slightly modified by the conditions of isolation. This effect is not unknown, e.g., correction for pH and temperature of the data for "stripped" HbA in reference (20) gives log p^Q values very similar to those presented here for chromatographically purified HbA. Whether this effect also occurred with the purified HbLR is not known; however, it is reasonable that HbLR isolated under the same conditions may have been stabil ized by the additional hydrogen bonds postulated by Perutz (88).

B. Effect of Chloride and Phosphate The effects of chloride and phosphate ions on the log

P^q of isolated HbLR and HbA are shown in Figures 22 and 23, respectively. The curves for isolated HbA appear identical to those given by Antonini, et al. (24), allow ing for a displacement on the log p^g axis. It is again apparent that HbLR has a much higher oxygen affinity than HbA. The values given in Table 10 do not show a significantly smaller interaction of HbLR with these anions than for HbA and the slopes of the curves, taken at the 95 Table 10. Chloride and Phosphate Titration Curve Param eters and Calculated Dissociation Constants for HbLR and HbAa

Chloride Phosphate • • HbLR HbA HbLR HbA

A. Calculated by curve fitting of equation (2) to experimental data. 96 midpoints, and the total changes in oxygen affinity

(Alog P cjq) are identical within experimental error. These data were curve-fitted to an equation of simi lar form to equation (1 ), but expanded to provide for a second oxygen linked anion binding site.:

kdkd . 1 , kd + A |[ ^ + A ' log p5 0 = log p°q - 7 log - ^ - 7 + 7 log I KoKo ’ \ K° + A /\ko + a (2) in which KD and Kq are dissociation constants of the anion (A) from one binding site of deoxy and oxyhemoglobin, re- • 1 spectively, and and K q are the corresponding constants for the second site. This equation is identical to that derived by Wyman (25) for the Bohr effect and is also de rived in Appendix A. A second binding site for small an ions is indicated by the slopes of the curves, which are approximately equal to 2 on a tetramer basis. This is in terpreted to mean that, at the midpoint, there is a dif ference of two bound anions per tetramer between the de oxy and oxy conformations. This agrees with the findings of Chiancone, et al. (92), that hemoglobin has two chlor ide binding sites from nuclear quadrupole resonance stud- 35 ies of the interaction of Cl- with hemoglobin. In or der to calculate apparent binding constants (Table 10), equation (2 ) was simplified by the assumption that the two sites had equal affinities for the anions. The limited 97 range of the data did not justify resolution of the four individual constants. The approximate correctness of this assumption is indicated by the symmetry of the curves be cause, if the sets of dissociation constants were greatly different, the curves would appear biphasic. These calcu lated dissociation constants indicate that chloride and phosphate bind to HbLR about as effectively as to HbA. Furthermore, there is no apparent difference between HbLR and HbA in the relative strength of binding to the oxy 2 and deoxy forms. In this case, (Kq /K^J is the basis for comparison. The similarity of HbLR and HbA in interactions with small anions suggests that the 3143 histidine is not im portant in binding these anions. This is consistent with Arnone's (21) finding of an anion situated near the 382 lysine residue in a re-examination of previous X-ray data.

Binding of small anions to both 8 chains at this site ac counts for the observation of two binding sites per tetra mer and, because both 882 lysine side chains are expected to have very similar dissociation constants for small an ions, it also explains the symmetry of the titration curves.

C. Oxygen Equilibrium of Unseparated HbLR and HbA The oxygen equilibrium of a fresh, unseparated HbLR hemolysate was also examined. The Hill plot for this is shown in Figure 24. This plot is distinctly biphasic and 98 was analyzed by curve-fitting to a sum of Hill equations for two possible situations: Case A, in which there were equal concentrations of two non-interacting hemoglobin A A A IjR species present, <*2^2 an(^ a 2^2 ' anc* Case -*-n which three species were present in statistical equilibrium,

0 ^8 2 ' a2^2R/ an<* t*ie hybrid, The values of log PgQ which provided the best fits to the experimental data are shown in Table 11. All n values were assumed to be 2.9. Both cases gave very reasonable fits to the data and could not be distinguished either graphically or by analysis of the assumed log p^g values.

Table 11. Assumed Log p^g Values for HbLR Hemolysate

Case A Case B

HbA 0.78 0.90

HbLR 0.25 0.10 hybrid ----- 0.52 99

1.4 scale

7.2 z > = o ~

0.6 6.8 1.0 HbLR HbA

6.4 met Hb m 0.5

6.0

0 2 3 4 5

ELUANT VOLUME (liters)

Fig. 19. Chromatographic separation of HbLR and HbA on carboxymethylcellulose. Ten ml of carboxyhemoglobin, lOmM in heme, were applied to a 2.5 x 100 cm collumn con taining 600 ml wet resin equilibrated with 0.01M potassi um phosphate buffer, pH 6.0. The column was eluted with a linear pH and ionic strength gradient from the equilibra tion buffer to 0.02M dipotassium phosphate. Eluant frac

tions were analyzed f o r HbCO content at 540 nm and for pH and conductivity. The methemoglobin fraction was composed of HbLR and HbA in approximately equal proportions. 100

Pig. 20. Photographic results of agar gel electro phoresis of: .1) HbLR hemolysate, 2) chromatographically isolated HbLR, 3) chromatographically isolated HbA, and 4) HbA hemolysate. 101

3.0 c CO

x 2.0 1.0

0.8

0.6 H b A o m CL o 0.4 o

0.2 H b L R

-5 .0 -4.0 -3.0 2.0 LO G [0PG]

Fig. 21. Effect of DPG on the log p5Q and Hill's n of isolated HbLR ( -f ) and HbA ( o ). Arrows on the or dinate indicate log p^g values in the absence of DPG, while that on the abscissa shows the hemoglobin concentration. Other arrows mark the midpoints of the titration curves. Smooth curves for log p^g data were computed from equation 1 using constants in Table 9. 102

0.8

0.4

o io H b A a .

-0 .4 H b L R

- 0.8

-4 .0 -3 .0 - 2.0 - 1.0 0 L O G [CHLORIDE]

Fig. 22. Effect of chloride on the log p5Q of isolated HbLR and HbA. Arrows indicate midpoints of the titration . curves. Smooth curves were computed from equation (2) us ing constants in Table 10. 103

0.8

0.4

H b A

-0.4

0.8

-4.0 -3.0 - 2.0 1.0 0 LOG (PHOSPHATE]

Fig. 23. Effect of potassium phosphate on the log p_. of isolated HbLR and HbA. Arrows indicate calculated 50 midpoints of the titration curves. Smooth curves were com puted from equation (2) using constants in Table 10. yae ae n .Mptsimpopaebfe (H 7.15). (pH buffer phosphate potassium 0.1M in taken lysate j O «j? LOG Fig. 24. Oxygen equilibrium of unseparated HbLK herao- HbLK unseparated of equilibrium Oxygen 24. Fig. 0.5 -0 - -i.5 .5 0 1.0 1.0 4 O . 08 1.2 0.8 0.4 O .4 0 - t O pO? LOG • • 104 IV. DISCUSSION It is clear that the very high oxygen affinity of HbLR, relative to HbA, cannot be explained in terms of reduced DPG binding. Fetal hemoglobin (HbF) also lacks histidine at the 3143 site, having serine instead, and also exhibits reduced DPG binding (20). A straightforward comparison of HbLR and HbF is not possible because of lack of complete titration data for HbF; however, the oxygen affinity of HbF is not greatly different from that of HbA (20) while HbLR consistently has an oxygen affinity about three times that of HbA. Therefore, there are structural consequences of substitution of a large polar residue at the 3143 site which do not occur with substitution of a small, polar residue, and which go beyond a simple re duction in DPG binding. Perutz's (88) proposal, in response to our prelimin ary results (87), of two additional inter-chain hydrogen bonds in the oxy conformation of HbLR, between the 3143 glutamine and 3139 asparagine side chains provides a unique explanation for the increased oxygen affinity. In accord

ance with this proposal, the high KQ/KD ratio for DPG interacting with-HbLR, indicating a greater reduction in DPG binding to the oxy than the deoxy conformation, sug gests a perturbation of the oxy HbLR DPG binding site not present at the HbA site. Binding of smaller anions, such as chloride or phosphate, will not be sensitive to 106 stereochemical changes at a polycationic site and this is reflected m the similarity of the (Kg/Kp) 2 ratios and dis sociation constants for these anions. The increase in Hill’s n with increasing DPG concen tration can also be interpreted in terms of Perutz's pro posal. A glutamine side chain, being non-ionic but polar, will tend to become less soluble under conditions of high local ionic strength, such as would occur if a highly charged DPG molecule were held in a fixed position near it. This will make hydrogen bond formation more favorable. These hydrogen bonds contribute to the free energy of in teraction because they are not present in the deoxy con formation. Consequently, DPG, in stabilizing these ab normal hydrogen bonds, will increase the free energy of interaction of the system, of which Hill's n is a function. HbLR has a greater total change of oxygen affinity than HbA under the influence of DPG in spite of the fact that it binds DPG less effectively than HbA. This be comes reasonable upon consideration that hemoglobin oxygen affinity under the influence of an allosteric effector i’s entirely dependent upon the difference in free energy be-r tween the oxy and deoxy. states. Therefore, from simple thermodynamic considerations, modulation of oxygen affin ity is a sole function of the difference in strength of binding of the effector to the two conformations. This is also a requirement of Wyman's (25) linkage relations, which 107 are solidly based on mass action principles. The total

change in oxygen affinity is proportional to -log (Kd /Kq ), assuming a single binding site (equation (1)). Thus, a convenient means of comparing different hemoglobin spec ies in their interactions with DPG is the KQ/KD ratio. A larger ratio indicates a larger difference in binding be tween the oxy and deoxy conformations and, consequently, a larger total change in oxygen affinity. Therefore, any re duction in DPG binding, as long as it is equal for the two conformations, will not affect the total change in log P5q # but only the concentration of DPG required to reach a given log P j q - In the case of HbLR, the reduction in DPG binding is greater for the oxy than the deoxy conformation, Kq /Kd is larger, and the total change in log p^Q (Alog P^q ) is somewhat greater than for HbA. For chloride and phosphate, the situation is somewhat more complex, because of multiple binding (24, 92). The same mechanisms are operative, however, since these anions appear to obey the linkage relations explicitly, except at very high concentrations (> 111) where secondary effects such as subunit dissociation or cation binding occur (24). Consequently, because DPG and other anions can be for mally treated by linkage relations, differences between these, types of anions in their interaction with hemoglobin are not due to fundamentally different mechanisms, but, on the contrary, are simply due to differences in the numbers bound and the strength of binding at a particular site. The strength of binding is a particularly important question. Because hemoglobin anion binding sites are polycationic in nature (21), polyvalent anions will bind more strongly and lower oxygen affinity more effectively than monovalent anions. The limit would be the case where hemo globin and anion are stereochemically complementary. This appears to be fulfilled with DPG. Anions such as inositol hexaphosphate (IHP), an even more potent modulator of oxy gen affinity than DPG, and pyridoxal phosphate would be exceptions to this scheme. In the former case, IHP has been shown to operate on oxyhemoglobin (22, 27) in some presently unknown manner and pyridoxal phosphate is known to bond covalently to both oxy and deoxyhemoglobin (93). This is consistent with Gray and Gibson*s (22) division of phosphates into three categories. The DPG binding site for oxyhemoglobin has not been characterized. It is reasonable that it is a polycationic i site which lacks much of the stereochemical complementarity possessed by the deoxyhemoglobin binding site. The de creased complementarity accounts for the increase in the dissociation constant, relative to deoxyhemoglobin, by several orders of magnitude. There is no special require ment that it be at the same location as in deoxyhemoglobin, but this possibility appears most likely. The oxyhemo globin binding site is required to be either oxygen linked in the deoxy conformation or negatively oxygen linked in the oxy conformation. Otherwise, the presence of such a site would not be revealed by a linked function analysis. Because it is well established that deoxyhemoglobin has only one oxygen linked site at physiological pH and ionic strength (21, 76) and because DPG does not exert any ef fect on the oxygen equilibrium (13, 27) or kinetics (22) of fully oxygenated hemoglobin, as would be expected if the site were negatively linked, the suggestion that the oxy and deoxyhemoglobin DPG binding sites differ only in stereochemistry seems quite reasonable. However, DPG is still capable of binding very strong ly at the oxyhemoglobin binding site, i.e., K q is a small number, relative to Kq values for phosphate or chloride. Therefore we conclude that it is not correct to state that DPG does not bind significantly to oxyhemoglobin under physiological conditions (95) . It can be shown that, in dilute solution, i.e., 1.25 x 10 — 5 M hemoglobin tetramer and DPG, only about 1% of oxyhemoglobin molecules bind DPG. This is calculated assuming a dissociation constant _ 3 of 1.5 x 10 M, which is five times greater than that used by Benesch, et al. (95). Even a tenfold increase in the DPG concentration will only double this figure. However, in a normal erythrocyte, the concentrations of both hemo globin and DPG are approximately 100 times greater than those used experimentally and are comparable with the value 110 of the dissociation constant. Consequently, the oxyhemoglobin-DPG complex is a physiologically significant state, although DPG binding to deoxyhemoglobin remains as the effective regulator of oxygen affinity because, under the same conditions, essentially all deoxyhemoglobin molecules bind DPG. APPENDIX A

The equilibria of hemoglobin (Hb) with oxygen in the presence of two anions, A and B, may be expressed by the following schematic diagram:

BHb 4 r ABHb

* AHb

> ABHbO 8

* AHbO

This model specifies random binding of oxygen and the two anions and assumes that the oxygenation reactions may

* be expressed by overall apparent dissociation constants (K.f K., K^r K ). Hemoglobin subunits are assumed to be functionally equivalent and consideration of partially oxygenated intermediates is not necessary. The individual equilibrium expressions necessary to define this sytem are:

, [HbOn] [A] v - [Hb] [A] v _ O______(2) a [AHb] (1) a [AIIb0o] 8 ' 111 112

V - [Hb] [B] , , ■ _ [ H b 0 8 ] tBI ... b [B0E] (3) ’Sj ---- [BHboJ'8 -- (4>

K = [AHb], tB3 /c\ K' = [AHb°8] [B] ab [ABHbJ ' ' ab lABHbOg] ^

[Hb] [O,]4 Ki ■ ' THbOgJ <7>

At 50% oxygen saturation, the following condition appliest

[Hb] + [AHb] + [BHb] + [ABHb] _ , [HbOg] + [AHbOg] + [BHbOg] + [ABHbOg] ~

Substituting equations (1), (3), and (5) into the numerar- tor and equations {2), (4), and. (6) into the denominator, the following is obtained:

[Hb] + [Hb] + [Hb^ tB] + [Hb] [A] [B] K u K , u LUbOgj LAj [HbOg] [B] [HbOgJ [A] [B] [Hb0o]8 + j -r j------* r ------1----- r

*’aK- « b Ka Kab

Then, by multiplying through and factoring,

i t i tBb» W a b (KA K,b- + V a h ^ 1 + KaKabtBl + V AllB]) ^ Ca V KaVabtKX*i» + V a b tA1 + KlKib[Bl + K ^ H B ] ) (8) Prom equation (7), = (pgg)o 4 at 50% oxygen saturation in the absence of anions. Because the system is defined as 1 1 3 being at 50% oxygen saturation, may be related to the other equilibria using the following considerations:

Ki _ tp50* = tp50*4 _ [Hb] (po2)4 (po2)4 (p50)4 [Hb0sl

Substitution of this into equation (8) gives:

4 , , , fso\ = ‘‘a V a b (KaKbKab + KbKab[A1 + KaKabtB] + V A] [BU

Pfo ^ V a b + X a b ^ 1 + KaKabtBl + V A)[Bl) which may be expressed logarithmically as:

log P50 = log P°o +

l^ X v C b 'W a b + V ab'^ + KaKab[Bl + VA)[BI) x i 0 g ------1— i — 1------1— 1------1— 1------1------W a b ^KaKbKab + KbKab[A] + KaKabtB) + V Al[B>>

This is the general equation expressing the relationship between oxygen affinity and interaction with two anions. It may be simplified by the assumption that binding of the first anion does not affect binding of the second, i.e.,

^ = Kab- Then'

, o . 1, KX (KaKb + V A] + Ka [Bl + tAl[B]) log p50 = log p 50 + 3-log ------1— i--- -r na^^a^ + + Ka[B] + [A] [B] ) 114 Furthermore, if the anions A and B are identical, the gen eral equation may be reduced tos

Ka A\ Kb + A log P50 = log p°0 - Jlog + |logl . K + A Ka*b a *b

which is identical to equation ( 2 ) given in Section II,. Part II. If there is only one anion binding site, such as for DPG, or, if the anion binds equally to one site of both V oxy and deoxy Hb, the ratio becomes unity and the equation reduces to:

o 1 Ka 1 Ka + [A] log p50 = log p50 - x log— r + t I ^ - i------50 K K + [A] a a which is identical to equation (1) in Section II, Part II. The graphic slope of this equation at any point may be de termined from; 4

3 lope = [Al (Ka + Ka> (K a + [A]) (k a' + [A])

4 and the anion concentration at the midpoint ls:

Kmrd ■ , = v/KV a K*a BIBLIOGRAPHY

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