Cover Image

Not for Sale



View/Hide Left Panel
Click for next page ( 2


The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement



Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.

OCR for page 1
PART I. STRUCTURE OF HEMOGLOBIN CURRENT CONCEPTS OF STRUCTURE OF HEMOGLOBIN JOHN T. EDSALL First, may I say that this promises to be the most international conference on hemoglobin, and the broadest in its scope, since that which was held in Cambridge, England, in June, 1948, in memory of Sir Joseph Barcroft. The proceedings of that conference were published the following year.) I had the good fortune of being present at the Barcroft Memorial Conference an occasion that Forte of us who were there is ever likely to forget and I am glad to note that several of the contributors to that conference are at this one also. I feel very humble in attempting to offer comments on the structure of hemoglobin here, since I have not myself worked on the subject at all. How- ever, I am deeply concerned with problems of protein structure in general, and no protein offers problems more fascinating than those presented by hemoglobin. The heme proteins are substances of vast antiquity in nature. They contain three fundamental constituents iron, protein and porphyrin. Iron has been with us since the beginning of time. As to the amino acids, which compose the protein, we have good evidence now from the work of Millers and Abelson3 that they could have originated, and probably did originate, at a very early stage of the earth's history. Whatever the exact composition of the earth's primeval atmosphere, the reactions that would have occurred in that atmos- phere under the influence of ultraviolet light and electric storms would have led to the formation of at least some of the amino acids. As to the porphyrins, there is ample evidence from deposits of great antiquity of their presence in nature many hundreds of millions of years ago, at least. Thus these constituents of the heme proteins were probably present, and began to interact, long before even the most primitive living organisms appeared on earth. The heme proteins that are in and around us today represent the product of a very long period of evolution and natural selection, and are highly adapted to the performance of specific functions. Among the heme proteins, eve shall confine the discussion to hemoglobins, leaving aside the catalases, peroxidases, and myoglobins; although the latter are so closely related to the hemoglobins that they will enter the discussion from time to time. This conference will concentrate almost entirely on mamma- lian hemoglobins, containing four heme groups per molecule, with chief emphasis on human hemoglobin. EIere I shall try to sketch a general picture of the characteristic features of these molecules, as they are known today,

OCR for page 1
2 PART I. STRUCTURE OF HEMOGLOBIN with emphasis on some aspects of structure that will not be covered in detail by later speakers. The hemoglobins have a four-fold structure, in that they contain not only four heme groups, but also apparently four peptide chains per molecule. Dr. Schroeder will have more to say about the latter point at this conference. Moreover, all of the four groups at the amino terminal ends of the peptide chains are valine residues. This however is not proof that the four peptide chains are all alike. Indeed, the latest evidence from the work of Rhinesmith, Schroeder and Pauling indicates that they fall into two different classes, two in each class. This fits in nicely with many lines of evidence from other di- rections that the hemoglobin molecule is made up of two equivalent subunits; the most decisive evidence on this point comes from the x-ray crystallographic studies of Dr. Perutz. I need not say much here about the shape of the molecule, because Perutz and Dintzis will go into that question far more deeply, on the basis of their x-ray studies. In these studies a long, hard road had to be traveled before it was possible to obtain even a rough picture of tee approximate shape of the molecule. It was indeed clear many years ago, from studies on hemoglobin in solution, that the hemoglobin molecule must be a pretty compact structure, not far from spherical in shape. Sedimentation, diffusion and viscosity meas- urements all fitted in with this, and so did Oncley's work on dielectric dis- persion.4~5 According to Bragg and Perutz,6 the best approximate model is a structure resembling a spheroid, with axes 71 and 54 A in the ~ plane of the crystal, and with a greatest thickness of 52 A along the b axis, perpendicular to the ~ plane. However there is a fairly deep dimple, or hollow, around the center, along this axis. The outlines of the surface are certainly irregular, with bumps and hollows; any simple model, such as an ellipsoid of revolution, is certainly an oversimplification. The picture of the molecule so far obtained, however, is fuzzy and of low resolution; for many purposes the earlier model of Bragg and Perutz, of an ellipsoid of revolution 70 ~ 55 ~ 55 A, is still a useful rough approximation. The sulfhydryl groups have been approximately located; the four which can be detected in the undenatured molecule are arranged in two pairs, 30 A apart, and about 5 A from the surface of the molecule.7 Nobody, I think, knows where the heme groups are located in the molecule. Dr. Perutz has some important evidence as to the orientation of the heme groups in rela- tion to the molecular axes, but I will leave it to him to speak of that later on. Since we shall hear about the amino acid analysis of hemoglobin later from Dr. Stein, I will not try to discuss that here, except to remind you of oIle very striking feature, which has long been knownnamely the large number of histidine residues, approximately 35 per hemoglobin molecule. No other pro- tein, I think, contains so large a proportion of histidine residues; and we may suspect that their presence has some deep structural significance. In one

OCR for page 1
CONCEPTS OF HEMOGLOBIN STRUCTUREEDSALL 3 respect, at least, their biological significance is obvious; since the pK values of such groups lie near 7, their presence in such large numbers makes hemo- globin one of the best of all protein buffers in the physiological pH range. Some of these groups, as Wyman8 in particular has inferred, may play a very special role in the maintenance of the heme-globin linkage. That, however, is still a somewhat controversial point, to which we return later. . . . The sulphydryl groups of hemoglobin are evidently of crucial importance. There are at least four such groups which are readily reactive in the native protein, and the work of Ingram9 and of Benesch, Lardy and Beneschi sug- gests that there are others which become uncovered on denaturation. The notable work of Riggs-- who is here today and can discuss the matter further has shown that the blocking of the sulfhydryl groups (by mercurials, for instance), has far-reaching effects on the oxygen amenity of the hemoglobin molecule and on the interactions between the heme groups. Steinhardt and Zaiseri3 have shown that hemoglobin contains approxi- mately 36 negatively charged groups, and apparently an equal number of positively charged groups, which in the native molecule are non-titratable with acids and bases, but become titratable upon acid, and perhaps also upon alka- line, denaturation. They have presented evidence that the process of denatura- tion is art all or none affair; there is either a native molecule in which none of these groups can be titrated with acids or bases, or a denatured molecule in which all 36 pairs have become detached from their moorings and are avail- able for the uptake of protons. The transition state between the native and the denatured molecule is, therefore, presumably such a brief and transient affair that it cannot be distinguished as a separate entity. This denaturation is apparently reversible, at least in me/hemoglobin, under the conditions de- Ened by Steinhardt and Zaiser. Tanford44 has made a recent interesting contribution to this problem, by showing that the intrinsic viscosity of ferrihemoglobin rises from 3.5 cc/gram in the native protein to 13.5 cc/gram in a solution after exposure to pH 3.5 for 10 to 15 minutes. This indicates a marked expansion of the molecule in acid solution, similar to that which has been observed in acid solutions of serum albumin. It is also known for instance from the work of Field and O'Brienthat hemoglobin below pH 6 dissociates into half molecules, the dissociation being nearly complete below pH 4. Over a short period of time the dissociation is reversible. Tanford concludes that most, and possibly all, of the increased acid binding in denatured as compared with native hemo- globin at acid pH may be due to the decrease of electrostatic repulsion between the charged groups when the molecule expands on denaturation. When the charges are close together, as in the compact native molecule, it becomes in- creasingly difficult to add more protons, as the net positive charge on the molecule increases. On expansion and dissociation of the molecule, the charges move further apart, the repulsion for approaching protons diminishes, and . . . . . . .

OCR for page 1
4 PART I. STRUCTURE OF HEMOGLOBIN more protons are therefore bound. We still do not know enough to say whether this is sufficient to explain the effect of acid denaturation on the titration curve. It may still be necessary to assume that a large number of acid and basic groups are actually inaccessible in the native molecule, for steric reasons, or because of hydrogen bonding. This was the general hypothesis pro- posed by Steinhardt and Zaiser. Tanford's observation does not necessarily invalidate the hypothesis, but it certainly does introduce a new factor which must be considered ire searching for the true explanation. Hemoglobin is certainly the prime example known to us of a molecule chemically organized to perform a biological function with a high degree of efficiency. In this case the function is clearly defined, although other possible functions of hemoglobin are not excludednamely to transport oxygen, and assist in the transport of carbon dioxide, in the respiratory cycle. To this end, a molecule has been evolved which displays remarkable interactions between the four heme groups which bind the oxygen, and between these groups and the heme-linked acid groups which change their strength on oxygenation. The general relations which describe such linked functions have been developed with great insight and rigor by- Wyman,S whose review remains the most comprehensive statement of the general principles involved. This is no place for a comprehensive discussion, but a few remarks may be offered. We consider a molecule (lobs) containing four binding sites, to each of which a ligand X may be attached. For our present purposes, X is oxygen of perhaps carbon monoxide. The problem is much simplified if we may consider that all four sites are equivalent if no X has been bound, so that the chance of picking up X is the same for any of the four. Is this justifiable for hemoglobin? Certainly there is no rigorous proof of the equivalence of the four hemes in hemoglobin; indeed there is now clear evidence, as we have mentioned above, that the molecule is not made up of four identical subunits, but only of two, each containing two hemes. The two peptide chains of each subunit are different, so that the environment of the hemes cannot be quite the same. Yet, for a working hypothesis, we may follow Wyman in assuming the hemes to be intrinsically all alike in their amenity for X. There are, in any case, apart from unbound Hb4, four classes of complexes: Hb4X, Hb4X2, Hb4X3, fIb4X4. Setting the activity of X equal to its partial pressure p, we have the four association constants: k (Hb4X) 1 , (Hb45)p k2 ((Hb ~X) ); k3 ~ Hb4~4 ~ (Hb4X3)p (Hb4X3) ( Hb4X2 ) P . (1) The constant k1 is four times as great as the intrinsic constant for the binding of X by one heme group, for Hb4 can bind X in any of four places,

OCR for page 1
CONCEPTS OF HEMOGLOBIN STRUCTUREEDSALL while Hb~X can give up X from only one. Similar statistical reasoning leads t<' the definition of a set of intrinsic constants x', are, X3, ^~4, which are related to the k's by the equations: kl - 4 ; ~~' Ok. Ok: 3 ~4 = 4k4 (2) If all the hemes were equivalent and independent in their interactions with X, then we should have ^~1 /_ -~3 ~4- If the binding of one ~ molecule makes the binding of the next more difficult, the system shows negative interactions, as is almost invariably the case for acid-base equilibria, Andrea ~ x3 ~ ice ~ hi. If the system shows positive interactions, the bind- ing of one X facilitates the binding of the next, and art > x3 > ^~2 > Act. It has long been known that the interaction of hemoglobin with oxygen (or carbon monoxide ~ involves strong positive interactions; WymanS gave a searching discussion of the data available ten years ago. Recently Roughton, Otis and Mystery and Lysteri' have carried out measurements on the hemoglobin- oxygen equilibrium (X 0~) of far greater accuracy than any previously attained. They devoted particular care to very precise measurements near zero combined oxygen, and near 100 per cent saturation of the hemoglobin with oxygen; it is in these two ranges that high accuracy is essential if reliable values of kit (or ail) and of k4 (or X4) are to be obtained. The binding of X may be expressed by the quantity v, the mean number of X molecules bound per mole of Hb4 present. For hemoglobin, of course, `, can vary from zero to 4, which corresponds to 100 per cent saturation. In terms of p, the activity or partial pressure of X, and of the k's defined in the equation ( 1), we obtain k, p + 2k~k~ p' + 3klk2k3 p3 + 4kik2k3k4 pi By `, v 1 + Kit p + k~k2p ~ + Kit k~k3p ~ + k, k~k31~4p4 If we make use of (2), and express ~ in terms of the it's, this becomes: 4 trap + 3x'~p2 + 3~ 3<,~3p3 + x1~2~^~4p4] <4y + Chap + 0~K,,p- + ~X~^~> Hap' ~ ^~/'2^~3^~4P In our own laboratory we have studied certain systems very different from the hemoglobin-oxygen system, and much simpler: they consist of solutions of the metallic ions zinc, or cupric copper, and a ligand which is imidazole or 4-methylimidazole.iS~4 9 Formally the equations defining these systems are identical with equations (3) and (4), if we replace the hemoglobin molecule by a copper or zinc ion with its four co-ordination positions, and replace 0., or CO by an imidazole molecule (Im), taking the activity of Im as approxi- mately equal to its molar concentration, which replaces p in equations Aid, (3) and (4~. Following a suggestion of Professor George Scatchard, we have found that the evaluation of the association constants, and the recognition of positive

OCR for page 1
6 PART I. STRUCTURE OF HEMOGLOBIN of negative interactions between the binding sites to which the ligands are attached, are much facilitated by plotting the experimental data in terms of a function Q: a, Q (4 imp (I) Since ~ and p are the quantities directly determined by experiment, Q is readily evaluated from the experimental data. In terms of the constants Gil to x~ inclusive, Q may readily be shown from (4) to be: act (1 + 3x p + 3~/~3p- + An: DIP 1 + 3~f~p -A 3~;c_p- + scud.. Cape (6) It is readily seen that, as p arid v approach zero, Q approaches at, and as p becomes very large (v~ 4) Q approaches ^~4. Thus a plot of Q (or log Q) against v gives x~ (or log x') as the extrapolated intercept on the ordinate axis on the left, and x4 (or log /4) as the extrapolated intercept on the ordinate axis on the right.* If all the birding sites are equivalent and inde- pendent, the curve is a horizontal straight line. Positive interactions are shown by a positive, negative interactions by a negative slope. Thus the copper-imidazole systems show strong negative interactions, while the inter- actions in the zinc-imidazole systems are strongly positive.iS~~9 We have taken some of the beautiful data on hemoglobin-oxyger~ inter- actions from Lyster's thesis, kindly made available to us by Professor Rough- ton and Dr. Lyster, and have represented them in this fashion. Figure 1 shows a plot of v against log p, for some of Lyster's data, and Figures 2A and B show corresponding plots of log Q against v. The extrapolation of the experimental points at the ends of the curve to obtain log x~ and log act is extremely easy, thanks to the superb quality of the experimental data. Figures 2A and 213 immediately exhibit the very strongly positive nature of the interactioIls; x~ is approximately 100 times as great as %1, whereas for a molecule with equivalent and independent binding sites the two should be identical. The evaluation of the association constants can of course be carried out in other ways; the most rigorous and objective is probably that of Roughton, Otis and Oyster, which is too elaborate to be discussed here. There are distinct species differences in the character of the interactions between the hemes. In sheep hemoglobins the binding of one or two oxygen molecules to a hemoglobin molecule definitely but only moderately enhances the avidity of the unbound hemes for oxygen; but where three hemes are * The evaluation of K`, and K3 from the data is somewhat more complex. See refer- ences 18 and 19 and also the discussion in reference 16. This plot has been extensively employed by Wyman (8) in his classical review, using as ordinate the fractional saturation with oxygen, which of course is simply equal to v/4.

OCR for page 1
CONCEPTS OF HEMOGLOBIN STRUCTUREEDSALL so - _ . .. . 3.5 _ 3.0 2.5 - y 2.0 1.5 1.0 O- -2.0 _- ,. HUMAN Hb; pH 7.0, 19 C HUMAN Hb; pH 9.1, 19 C p - HORSE HE (4.6'~); / pH 7.0, 19*C / (0.6 M. PHOSPHATE) _ / - -1.5 -1.0 -0.5 0 +0S +1.0 +1.5 ~2.0 LOG. P02 C0MBINATI0N 0F HEM0GL0B1NS WITH 0XYGEIY 1 1 1 FIG. 1.The binding of oxygen to human and horse hemoglobin at 19 C. From the measurements of Lyster. combined with oxygen, the affinity of the remaining unbound site is enormously enhanced, lC4 becoming over 280 times as great as Hi. For horse and human hemoglobin, on the other hand, the enhancement of affinity goes up in a more nearly regular and progressive fashion for each oxygen that is bound; though here again the biggest change seems to come at the last step.)' Certainly the binding of oxygen to any one of the hemes sets up far- reaching repercussions elsewhere in the molecule; and these appear to be particularly great after three of the heme groups are occupied by oxygens. Interactions of such intensity have suggested to more than one inquirer in recent years the conception that oxygenation (or combination with carbon monoxide, or oxidation to ferrihemoglobin ~ must lead to a rather drastic rearrangement of the total configuration of the molecule as a whole. Such thoughts were perhaps first expressed by St. George and Pauling,~ and from a rather different point of view by Wyman and Allen.2i Since Dr. Philip George, a little later in this symposium, will be dealing, with some of the ideas put forth by St. George and Pauling, I shall pass them over and say

OCR for page 1
8 PART I. STRUCTURE OF HEMOGLOBIN + 0,5 0.0 A CC4BlNAllON OF HUMAN HEMOGLOBIN WITH OXYGE N ~0.5 _ V-1.0 _ 0 1. s -2.0 _ 1 1 1 // pH 7.0 t06M phosphote,, '9.C JO / B +1.0 O.S _ Cot _ ~ <3 O So -0.5 - / -1.0 . 1 1 COMBINATION OF HUMAN HEMO0LOBIN WITH OXYGEN I' i' ,: pH 9 1 (02 M book), t9.C J 0 1 2 3 4 0 1 2 3 4 FIG. 2. A. The data of figure 1, for human hemoglobin at pH 7, plotted according to equation S. B. The data of figure I, for human hemoglobin at plI 9.1, plotted . according to equation 5. more about the proposals of Wyman and Allen, and some subsequent devel- opments. There are striking and obvious changes in many physical properties of hemoglobin when it combines with oxygen or carbon monoxide. The changes in absorption spectra are too well known to need comment; likewise the change from the paramagnetic reduced hemoglobin ~ ferrohemoglobin ~ to the diamagnetic oxy- or carbonmonoxyhemoglobin, discovered by Pauling and Coryell, is most dramatic. However, these alterations in physical proper- ties need not imply any general change in the molecular configuration of hemoglobin. The observed changes in solubility, however, seem hard to account for except in terms of some more general change in the molecule. These changes are indeed highly individual for the hemoglobins of different species. Horse oxyhemoglobin crystallizes quite readily from dilute salt solu- tions near pH 7 or a little below; on deoxygenation the solubility increases manyfold, and the crystals dissolve, as I learned soon after I joined Dr. Edwin CohIt's laboratory just over thirty years ago. Later on, when human hemo- globin was studied in that laboratory, it was found that the solubility relations are reversed. Dr. Alan Batchelder indeed, about 1941, worked out a method of crystallizing nearly salt-free human ferrohemoglobin simply by starting with a very concentrated oxyhemoglobin solution and pumping oh the oxygen. The crystals were very good crystals, but the yield was not especially good since adult human hemoglobin, even in the ferro form, is pretty soluble.

OCR for page 1
CONCEPTS Ol4' HEMOGLOBIN STRUCTUREEDSALL 9 Largely because of that, and of certain technical problems, and because there was too much else to do in wartime, the method was never published. The decrease in solubility on deoxygenation, however, was certainly very striking. On a deeper level, and reflecting presumably the same molecular alterations, are the marked differences in crystal structure that Perutz has observed between ferro-, oxy- and ferrihemoglobins. Wyman and Allen laid particular stress on the differences in the standard entropy changes (~ S) associated with the successive steps in oxygenation. These entropy changes are of course given, if the standard free energies ~ ~ F ~ and heats of reaction ~ ~ H ~ are known, by the fundamental thermodynamic equation: ~ S (~ H ~ F~/T - (~H + RT in k)/T (7) Here, T of course is the absolute temperature, and k is the equilibrium constant of the reaction under study. The value of H may be determined either by direct thermal measurements, or from the temperature coefficient of in k. Layman and Allen assumed from the available data that ~ H was the same for each of the four steps in oxygenation described by the four k values in the equation (1~. Then, because of the positive interactions pre- viously discussed, k4 for instance is much greater than k', it follows from equation (7) that ~ S is decidedly greater (i.e. more positive:) for the in- sertion of the fourth oxygen into a hemoglobin molecule than for the insertion of the first. The assumption that ~ H is the same for all the successive steps in oxy- genation, however, is not borne out by the very careful measurements of Roughton et alit on sheep hemoglobin at pH 9.1. The reaction is exothermic (~ H negative) at every step, but ~ H progressively decreases from o H 15.7 + 0.8 kcal/mole, for the equilibrium described by kit, to H 8.7 + 3.3 kcal/mole for the process described by k4. Making use of the values for kit ant k4, Roughton et al. calculate for the standard entronv changes in the first and last steps: 0 0 ~ S 2, S = 43 cat. per degree per mole (8) This large and positive entropy difference is in the same direction, although smaller in magnitude, as that associated with protein denaturation. It sug- gests a change from a more ordered to a more disordered type of structure in the hemoglobin molecule, at least at certain stages in the oxygenation process, which is much more drastic than could readily be accounted for by local changes of bond character in the heme groups and closely adjoining amino acid residues. The nature of the changes is certainly not clear as yet; some alternative possibilities have been weighed by Wyman and Allen, in a subtle and careful analysis. The revised ~ H values of Roughton et al.

OCR for page 1
10 PART I. STRUCTURE OF HEMOGLOBIN actually serve to strengthen the original argument of Wyman and Allen about the importance of the entropy changes. Recent studies of dielectric increments and dielectric dispersion by Taka- shima'~ and Takashima and Lumry~3 strongly reinforce the view that some rather far-reaching change in molecular configuration is associated with the oxygenation process. Figure 3 shows the dielectric increment and the relaxation 70 50 r,X 108 30 sc 10 b J ~ RELAXATION ~ - DIEI~CTRIC INCREMENT 8.19 ~ _ 1 1 1 1 1 1 1 1 19 0 1 2 3 4 5 6 7 8 9 oxy O2 PART lAL PRE SSU RE FIG. 3. Variation in dielectric increment and mean dielectric relaxation time with oxygenation for horse hemoglobin at 15 C. Under the experimental conditions the protein is saturated with oxygen at about 10 mm. pressure. The points at extreme right were obtained in air. (From Takashima and Lumry, J. Am. Chem. Soc., in press). time of horse hemoglobin as a function of the degree of oxygenation. The dielectric increment passes through a high maximum, then through a minimum, followed by a second, somewhat lower maximum, finally descending, as saturation is approached, to a rather low value close to that of Oncley4 for horse carbonmonoxyhemoglobin. The dielectric dispersion curve is very similar to the increment curve. The full implications of these results are not yet apparent, but they practically compel the interpretation that marked changes ir1 the molecular shape of hemoglobin, or its charge distribution, or both, are occurring during the formation of the various intermediate complexes between hemoglobin and oxygen. Since there must be a mixture of such intermediates at incomplete total saturation with oxygen the completely oxygenated and completely deoxygenated forms being favored because of the strong positive heme-heme interactionsthe analysis of the observed dielectric data is by no means simple. Further work in this field will be awaited with the greatest Interest. Finally a few remarks on the heme-linked acid groups. There are several of these, but two per heme group are particularly associated with oxygenation.

OCR for page 1
CONCEPTS OF HEMOGLOBIN STRUCTUREEDSALL 11 One, according to Wyman,S becomes a weaker acid on oxygenation of the neighboring heme, its pK increasing from 5.25 to 5.73. We may note that these pK values fall in the pH range in which hemoglobin begins to dissociate reversibly into half-molecules.~5 This factor, unknown at the time of Wyman's studies, may compel reconsideration of some of the structural interpretations suggested in the past. In any case, this group, although of great chemical interest, is of little physiological importance. The other group, which is crucial for the biological function of hemoglobin, has a pK value which decreases, on oxygenation, from 7.93 to 6.68 at 25,* thereby serving power- fully to assist the release of carbon dioxide in the lungs, and in the other phase of the respiratory cycle to assist its uptake from the tissues. Wyman, arid Coryell and Pauling,'' inferred that both these heme-linked groups were imidazole groups of histidine residues. This view has been attacked, for instance by Haurowitz and Harding and I know that Professor Roughton also strongly doubts the imidazole hypothesis. Personally, after much re- flection, I still consider the question open. I should like to elaborate on this problem further here, but any real discussion would take far too long. It is worth noting, however, that Theorell2` has made use of the now precisely known sequence of the amino acid residues adjoining the heme group in cytochrome c, to construct a model in which the imidazole group of the histi- dine residue in the peptide chain fits nicely on to the iron atom of the heme, if it is assumed that the peptide chain is coiled in an a-helix. This of course does not in itself prove the existence of an iron-imidazole link in cytochrome c, and in any case the heme-protein linkage in hemoglobin is very different in many ways from that in cytochrome c. Nevertheless this observation is highly suggestive. Finally it may be remarked that Wyman and pollens have suggested that the variations of pK values of the heme-linked groups with oxygenation may reflect alterations in the large-scale framework of the hemoglobin molecule, such as have been discussed above, rather than locally-transmitted electronic effects due to the binding or release of oxygen by the heme iron. This raises many questions: for instance, should not a large-scale change of molecular configuration affect the pK values of many acidic groups in the hemoglobin molecule, rather than specifically affecting just two groups per heme? But I have gone on long enough; it is best perhaps to end with a question rather than an affirmation, and turn to the reports of those who have new experimental work to present. Acknowledgment: I am greatly indebted to Professor Roughton and Dr. Lyster for providing me with the data from which figures ~ and 2 have been constructed, and to Drs. Takashima and Lumry for providing figure 3. * These are Wyman's values. Regarding the interpretation of the titration data, see an important recent note by Alberty.24

OCR for page 1
12 5. PART I. STRUCTURE OF HEMOGLOBIN REF`EREN CES 1. Haemoglobin: a symposium based on a conference held at Cambridge in June 1948 in memory of Sir Joseph Barcroft; F`. J. W. Roughton and J. C. Kendrew, editors. Butterworths Scientific Publications, London, and Interscience Publishers, New York, 1949. xii + 317 pp. 2. Miller, S. L.: Production of some organic compounds under possible primitive earth conditions, J. Am. Chem. Soc. 77: 2351, 1955. 3. Abelson, P. H.: Amino acids formed in primitive atmospheres, Science 124: 935, 1956. 4. Oncley, J. L.: Studies on the dielectric properties of protein solutions. I. Carboxy- hemoglobin, J. Am. Chem. Soc. 60: 1115, 1938. Edsall, J. T.: The size, shape and hydration of protein molecules, in "The Pro- teins" ( H. Neurath and K. Bailey, editors ) Vol. I 13, Chapter 7, Academic Press, New York, 1953. See in particular pages 646, 654, and 720. 6. Bragg, Sir Lawrence, and Perutz, M. F.: The structure of haemoglobin. VI. Fourier projections on the 010 plane, Proc. Roy. Soc. (London) Series A, 225: 315, 1954. 7. Kendrew, J. C., and Perutz, M. F.: X-Ray studies of compounds of biological interest, Annual Rev. of Biochem. 26: 327, 1957. See especially pp. 353-356. 8. Wyman, J.: Heme proteins, Advances in Protein Chem. 4: 407, 1948. 9. Ingram, V. M.: Sulfhydryl groups in hemoglobins, Biochem. J. 59: 653, l9SS. 10. Benesch, R. E., Lardy, H. A, and Benesch, R.: The sulfhydryl groups of crystal- line proteins. I. Some albumins, enzymes, and hemoglobins, J. Biol. Chem. 216: 663, 1955. 11. Riggs, A. F.: Sulihydryl groups and the interactions between the hemes in hemo- globin, J. Gen. Physiol. 35: 23,1951. 12. Riggs, A. F`., and Wolbach, R. A.: Sulfhydryl groups and the structure of hemo- globin, J. Gen. Physiol. 39: 585, 1956. 13. Steinhardt, J., and Zaiser, E. M.: Hydrogen ion equilibria in native and denatured proteins, Advances in Protein Chem. 10: 151, 1955. 14. Tanford, C.: The acid denaturation of ferrihemoglobin, J. Am. Chem. Soc. 79: 3931, 1957. ~ 15. F`ield, E. O., and O'Brien, J. R. P.: Dissociation of human hemoglobin at low pH, Biochem. J. 60: 656,1955. 16. Roughton, F. J. W., Otis, A. B., and Lyster, R. L. J.: The determination of the individual equilibrium constants of the four intermediate reactions between oxygen and sheep haemoglobin, Proc. Roy. Soc. (London), Series B. 144: 29, 1955. 17. Lyster, :R. L. J.: Ph.D. thesis, Cambridge University, 1955. 18. Edsall, J. T., Felsenfeld, G., Goodman, D. S., and Gurd, F`. R. N.: The association of imidazole with the ions of zinc and cupric copper, J. Am. Chem. Soc. 76: 3054, 1954. 19. Nozaki, Y., Gurd, F. R N., Chen, R. F., and Edsall, J. T.: The association of 4- methylimidazole with the ions of cupric copper and zinc; with some observa- tions on 2, 4-dimethylimidazole, J. Am. Chem. Soc. 79: 2123, 1957. 20. St. George, R. C. C., and Pauling, L.: The combining power of hemoglobin for alkyl isocyanides and the nature of the heme-heme interactions in hemoglobin, Science 114: 629, 1951. 21. Wyman, J., and Allen, D. W.: The problem of the heme interactions in hemoglobin and the nature of the Bohr effect, J. Polymer Sci., 7: 499, 1951.

OCR for page 1
CONCEPTS OF HEMOGLOBIN STRUCTURE EDSALL 13 22. Tal;ashima, S.: Dielectric properties of hemoglobin. I. Studies at 1 megacycle, J. Am. Chem. Soc. 78: 541, 1956. 23. Takashima, S., and Lumry, R.: Paper submitted to the J. Am. Chem. Soc. See also Lumry and Takashima: Dielectric dispersion studies on the oxygenation of horse hemoglobin, Abstracts of 131st meeting, American Chemical Society, Miami, Florida, April 7-12, 1957, p. 50 C. 24. Alberty, R. A.: The ionization constants of the heme-linked groups of hemoglobin, J. Am. Chem. Soc. 77: 4522, 1955. 25. Coryell, C. D., and Pauling, L.: A structural interpretation of the acidity of groups associated with the hemes of hemoglobin and hemoglobin derivatives, J. Biol. Chem. 132: 769, 1940. 26. Haurowitz, F`., and Hardin, R. L.: Respiratory proteins, in 'The Proteins" ( H. Neurath and K. Bailey, editors) Vol. II A, Chapter 14, Academic Press, New York, 1954. 27. Theorell, H.: Relations between prosthetic groups, coenzymes and enzymes, in "Currents in Biochemical Research 1956" (D. E. Green, editor), p. 275. Inter- science Publishers, New York, 1956.