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CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957 (1958)

Chapter: STRUCTURE OF HEMOGLOBIN

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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 20
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
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Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 23
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 24
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 25
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 26
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 27
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 28
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 29
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 30
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 31
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 32
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 33
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 34
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 35
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 36
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 37
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 38
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 39
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 40
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 41
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 42
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 43
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 44
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 45
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 46
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 47
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 48
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 49
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 50
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 51
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 52
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 53
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 54
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 55
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 56
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 57
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 58
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 59
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 60
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 61
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 62
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 63
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
×
Page 64
Suggested Citation:"STRUCTURE OF HEMOGLOBIN." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Page 65

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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,

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 known—namely 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

CONCEPTS OF HEMOGLOBIN STRUCTURE—EDSALL 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'Brien—that 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 · · . . . . · . . .

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 excluded—namely 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,

CONCEPTS OF HEMOGLOBIN STRUCTURE—EDSALL 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

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.

CONCEPTS OF HEMOGLOBIN STRUCTURE—EDSALL 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

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.

CONCEPTS Ol4' HEMOGLOBIN STRUCTURE—EDSALL 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.

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 interactions—the 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.

CONCEPTS OF HEMOGLOBIN STRUCTURE—EDSALL 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

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.

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.

INDIVIDUAL VELOCITY CONSTANTS IN THE CHAIN OF REACTIONS OF SHEEP HEMOGLOBIN WITH DISSOLVED GASES S. AINSWORTTI, Q. H. GIBSON AND lo. J. W. ROUGHTON* Dr. Edsall: you, sir, opened with what I think was a major contribution in spite of what you modestly said about our paper being the first major con- tribution. You began with a reference to the Barcroft Memorial Meeting ore Hemoglobin at Cambridge in 1948, a meeting which was greatly strength- ened by the team of visitors from this country. I am sure I express the feelings of the Cambridge folk, who are now here at the invitation of their Americans colleagues, in saying how grateful we are for this opportunity of a return trip. I look forward to this particular conference being just as memorable as the Barcroft Conference in 1948. There is one matter about that con- ference to which I might refer. You recall that a book1 was published des- cribing the proceedings. It is interesting to note that the publishers printed 2000 copies, and according to the most recent advices, 1970 copies have now been sold. It is not always the case, I am afraid, that experiment and calcu- lstion agree within one and a half per cent. That is, in matters concerned with hemoglobin. I will now get straight ahead with my main topic: the individual velocity constants in the chain of the four reactions of hemoglobin with oxygen, carbon rr~onoxide and nitric oxide. At Cambridge in 1948 wee only had very sparse k'~ Hb4 + X = Hb4X kl Karl l'l ——K1 _ Lo kl Hb4X + X=TIb4X, ~ - K. k Hb4~., + X = Hb4X3 ; K3 k: kt4 Hb4X3 + X = Hb4X4 k4 X = 0, or CO 11 l, Lo Zl3 L3 lo k'4 1~4 K4 _ L4 k4 1~ FIG. 1.—Intermediate compound hypothesis of hemoglobin reactions. * This paper was presented by Dr. Roughton.

VELOCITY CONSTANTS AINSWORTH, GIBSON AND ROUGHTON 15 knowledge as to the values of any of these individual constants. But since then we have learned quite a bit, largely resulting from the stimulus of that conference. We have reasonably good values for the four equilibrium constants of the reaction of oxygen with sheep hemoglobin through the work of Drs. Otis and Lyster and myself,-' and also of horse and human hemoglobin through the work of Dr. Lyster.3 It will be noted in figure ~ that in the case of the oxygen reaction we use K symbols, in the case of carbon monoxide L symbols, and in the case of nitric oxide, not shown in the figure we use ~ symbols. Over sixty years ago, Haldane and Lorrain Smith first observed that light dissociated CO from hemoglobin. Many others extended this work to CO home compounds in general. After the photo dissociation the CO recombined in the dark, and, as a matter of fact, this was the method by which Hartridge and Roughton over thirty years ago succeeded in measuring the kinetics of the first hemoglobin light reaction. But with the short intense lights obtained in flash photolysis, far more can be done, as Gibson4 has shown. The layout of his apparatus is quite simple. The solution to be examined is placed in a cell through which passes the observation light, rendered approx- imately monochromatic by a Alter, and then falls on the photocell and is recorded photographically by an oscillograph, after amplification. Then a brilliant flash for about two tenths of a millisecond is provided from the flash source, bringing about photodecomposition of the compound in the cell. f r Ll ~ ~ 1 Solution. Photocell. Oscillograph. FIG. 2. Block diagram of flash photolysis apparatus. Are there any photochemical after-effects of these flashes? Controls with myoglobin show-cd there were not; when CO myoglobin was photo-dissociated and the CO and myoglobin allowed to recombine the reaction followed a simple bimolecular course. The function plotted vertically in figure 3 against time gives a straight line, the slope of which is proportional to the velocity constant. If the same reaction is studied by one of the flow methods the same velocity constant is obtained. Furthermore, it does not matter whether all the carbon monoxide is dis- sociated from the myoglobin by the flash or only some. In each case a line of the same slope is obtained (see figure 4~. The velocity constant is independent of the amount of photo dissociation as would be expected in the case of a

16 PART I. STRUCTURE OF HEMOGLOBIN I1 1 to — 1 pH 71 x/ 19°C. At FLOW x/ , / / a: FLASH ~ i: BIB = TOTAL Mgb = TOTAL CO ~ If = COMgbatt 0 2 4 6 8 10 12 14 16 18 20 22 24 TIME in MILLISECONDS FIG. 3. Recombination of CO with myoglobin. simple bimolecular reaction with one atom of iron per myoglobin molecule. Very different, however, is the state of affairs when different amounts of CO are initially split off from hemoglobin by the flash (see figure 5~. If the Rash is such as to split off the whole of the carbon monoxide, then there is only a relatively slow rate of recombination compared with that which occurs when a small fraction of the carbon monoxide is split offal The slope of the -0 1 1 30 >0 _ l .. . . . I. ~ em. o ~- 0 s 10 15 20 25 30 ; - TIME in MILLISECONDS FIG. 4. (left) Recombination of CO with myoglobin with 90 ;e rut z so O 70§ 60 te 50 40 0 20 0 TIME (MILLISECONDS) absence of interaction. FIG. 5. (right) Recombination of CO with hemoglobin, showing interaction.

VELOCITY CONSTANTS AINSWORTH, GIBSON AND ROUGHTON 17 line in the latter case, i.e. the upper line, is of the order of 10 to 20 times greater than that of the louver line. This does supply an extraordinarily clear- cut and dramatic proof that it is indeed true in the case of sheep hemoglobin that the fourth reaction of combination of CO with hemoglobin goes vastly faster than the first one. 100 80 - c~ z 60 40 20 \Hb4 (CO)4 \ Mb4(C0)2 O ~ 100 80 60 40 20 0 % COHb otter LIGHT FrG. 6. Chart illustrating intermediate compound hypothesis. Concentration of in- termediate compounds as related to total reaction. Figure 6 shows the theoretical basis of the difference. According to the in termediate compound hypothesis, the dissociation will take in four steps forming successively compounds containing three, two, one and no molecules of combined CO. In calculating the quantity of each intermediate form when the amount of light applied is not enough to displace all the combined CO, it is assumed that the quantum yield is the same whichever intermediate compound is being irradiated. On that basis, the calculated amounts of the various intermediates at various total amounts of photolysis are as shown in figure 6. When only 10 to 157O of the CO is photolysed the main inter- mediate will be Hb4(CO)3. It is in that way that the value of 1~4 can be isolated. 1 - —~ _ Similarly, (especially if one starts not with fully saturated CO hemo- globin but with a mixture containing 10 per cent of CO hemoglobin and the rest reduced hemoglobin and one irradiates such a mixture), the compound present is almost exclusively Hb4, so that now the velocity constant 1'~ can be isolated. The correction for the later reactions can be reduced to 3 per cent or less. These applications of flash photolysis have thus provided very telling methods of dissecting out two of the individual velocity constants of the chain of the carbon monoxide reactions with hemoglobin. Figure 7 shows the effects of pH on these two velocity constants. The first curve, i.e. the full line, has a scale of values of about one eighth the scale of values of the last one. The scales are chosen so that the values of the two constants coincide at neutral pH. The discrepancy in the effects of pH on the two constants is very apparent. In the case of 1'~, the curare could be inter- preted in terms of the two heme-linked pK's, which Dr. Edsall discussed. In the case of 1~4, there is no obvious effect of pH on the acid side of neutrality.

PART I. STRUCTURE OF HEMOGLOBIN 12. . 10 8 _ XIO{-IS 6 4 _ 2 0 , 4 5 ,'t4 ,' ~ ' \ 'Y A' 7 6 4 (4 xlo6 3 2 1 1 1 1 1 1 '0 6 7 8 9 10 11 pH FIG. 7. Effect of varying pH on values of l'1 and 1'4 (M—1 S—1) for sheep hemoglobin by flash photolysis. (Ainsworth and Gibson, 1957). :F~igure 7 thus provides a striking contradiction to the old creed that pH affects all the constants of the chain of reactions evenly. Later, another and sharper example of that type of contradiction will be shown. The next two figures demonstrate the effects of PCMB and the reversal of the PC~B effect by the addition of glutathione on the two velocity con- stants. The lower full curve in figure g is the same as that for 1', in the previous figure, but the upper curve is what is obtained when PCMB is added. The minimum is shifted to ~ rather different pH and there is a considerable increase in the actual value of lo of the order of 3-to-4-fold. The addition of glutathione, how-ever, restores the normal result. This parallels rather significantly some of the observations of Dr. Riggs on the effects of PCMB on the equilibrium of oxygen with hemoglobin. With 1~4 however, a different effect is observed (see figure 9~. PCMB, 40 36 32 28 10 - 5 I 1 16 12 8 4 to a\ bus olo Or t5 0 / ~ o} WITH PCMB ° WITH Path GSH In, o - <O TV 5 6 7 9 10 11 pH FIG. 8. Effect of pH on l'1 with additions of PCMB and of PCM13 plus GSH.

VELOCITY CONSTANTS AINSWORTH, GIBSON AND ROUGHTON 19 9~ 81 7 1 0 - `;.1 ~ .L 3 2 rat · ) WITH PUB WITH `~B+GSH · /^ ·~/ 1,0 11 FIG. 9. Effect of pH on 14 with additions of PCMB and of PCMB plus GSH. OH instead of increasing the velocity constant, now decreases it and the effect of pH almost disappears. The effect of PC~B is again reversible by glutathione. In a paper recently published in the B Series of the Royal Society of London,6 we have analysed the complete course of the kinetics of carbon monoxide and hemoglobin in terms of the four velocity constants, lit, lid, 1~3 and 1~4 in accordance with the equations: Hb4 + CO -~ Mb4CO, dt s q 1', U s I' qr — I' qs HO ~ CO ~ + CO - ` Hb4 ~ CO ~ I, dt Hb4(CO), + CO -~ Hb4(CO)3, dt ~q Hi v q 1~4 W dv Hb4(CO):3 + CO ~ Hb4(CO)4, dt l Mu I ~qv Having two of the constants independently has made this possible. But even so we have had to resort to automatic electronic calculation on a large scale and to the assistance of Dr. Daniels and Nor. East of the Cambridge Statistical Laboratory. Fortunately, in this case the reverse constants of dissociation are so small that it is only over about the last five per cent of the reaction that they have any positive importance. By avoiding that range, we can effectively work with only the four combination velocity constants. Figure 10 shows how good is the agreement between calculation and experiment. Actually the agreement is within about 0.2 per cent between

20 PART I. STRUCTURE OF HEMOGLOBIN 80 60 -6952,2-521-4, 13-3476 . 14 13904 :,,Hb4 X—(CO) I 40 ~ ,, 0 ,, 20 o + 037a ~ ~3 - "~ olO`~ %~, --4--X- /Hb4X2 ,~b&~4 ' ~ ,,-- :~_ ;:— -___ aft"" ;~~ 10 20 30 40 50 60 70 80 90 100 TIME IN MllLl£CS. (CALC~D -art) f ~1 % XHb [Hb44 +2 [Hb4X23 + 3 [Hb4Xi ~ 4 [Hb4X4] K)O ~ [Hb4] + ~b4XJ +[Hb4X23 + ~b4X3] + [Hb4X43 ) FIG. 10. Comparisons of calculated and observed results. what one would expect on the basis of the four velocity constants and what one observed in regard to the total amount of CO hemoglobin formed at various times from the beginning of the reaction. The full line curve in the top panel displays the observed course of the combination. The dotted curves show the calculated course of the several intermediates. In the bottom panel, the discrepancy between observed results and calculated results are plotted against the extent of the reaction. The breadth of the panel is 0.3 per cent, so that the worst discrepancy is only about 0.2 per cent. Figure 11 shows the varying effects of pH on these various velocity con- stants. The speckled rectangles are for pH 7.1 and the clear rectangles for pH 9.~. When one passes from the first to the second constant, the speckled rectangle gets a little smaller, whereas the clear one gets quite a bit bigger. At the third constant, the speckled rectangle takes a leap up and the clear

VELOCITY CONSTANTS AINSWORTH, GIBSON AND ROUGHTON 21 FIG. }1. Effect of pH on l 1' l ~' l 3 nd l' 7 1 ~3 9 1 7ln =1 1 Sty '2 9 l ~ _ . ~ ~4 one takes a leap down, and finally they are pretty close together on the last constant. Figure 11 thus demonstrates very clearly the differential effect of pH when the individual constants are dissected out and the effect of pH on each of them is studied. TABLE I Fitted ratios Experim ent - 1 1, lO 1'.3 3.5 1 1 C.V.'s (c, 0.3) 1 ., 1 3 Original 7/19 7/22 9/24 9/26 9/27 9/17 9/16 4 4 4 4 4 4 4 4 4.2 5.4 5.8 6.3 7.5 6.6 5.9 1.8 1.7 1.7 2.2 2.3 2.5 2.4 2.3 6.4 15.2 17.9 6.2 15.0 10.8 6.2 1 1.6 10.2 4.6 8.0 8.4 4.7 8.2 2.8 5.5 8.0 2.6 6.9 10.9 2.9 7.2 12.6 3.0 MEAN 4 : 5.6 : 2.1 6% 11.2C/o 7.3% Table I shows the values and standard errors of the first three constants for 8 different sheep samples, the first constant being taken as proportional to four. The standard errors are of the order of five to ten per cent, which is about as good as could be expected in this kind of work. Now, to turn a little to the other side of the story, the individual velocities of dissociation. Gibson and Roughton~ have shown (see figure 12) that the in dividual velocity constants of dissociation, as regards the first molecule

22 PART 1 STR[CTURE OF HE~OOLOBIX 12 . 8 4 L°glOV o 4 \ V ~ zz RT T ^~=~z_~ ' ~O) VT = vel const.ot no. =[li~s/~ ~ = Z 71SZ8 .. E = E~~y o' A~t~n R =~s ~"ont (~*colJ HzO+~C~4 \ H~+H~ \ O VT+IO / VT 0~ c~1~0 . . \ . IO~ 2~ 30,~0 4~0 ENE~ of ~TI~ION (colori's) FIG. 12.-Rel~tloD of actlvatloD ener~y to velocity constants. coming oR, do fall very nicely into line ~>h classic~1 kinetic theory, the logarithm of the velocIty const~nt bein~ linea[ly rel~ted to the ener~y ol activation. In accorJ~nce ~ith Dr. U auro~z's teaching' the d~sociatlon DETER~I\~IOX ~F ki ~XD ^~4 1/ r ~here At Constant p CO R~te of ~ispl~cement of C~ by CO 7 4(I + k4pO~ \ //4 P CO )4 [~2 Combincd ~ith H b] ~4 Nb4Oa = Nb4O6 I 02 k74 /4 Hb3CO)+ ~ Hh(CO)~ + CO //4 1 . ~ 4/k4 _ versus nC~ ~1VCS . ~ >- ~ ~ k 4// ~ FIG. I3.

VELOCITY CONSTANTS—NINSWORTH, GI:13SO>I AND ROUGHTON 23 reactions have been written in the form 8,0 + FIb4OS, etc. There is over a millionfold variation in rate between kit, the velocity constant for first oxygen coming off, and Jo, the velocity constant for first nitric oxide coming off. Nevertheless there is a good agreement with simple classical theory. We now turn to some of the improvements we have recently made in the determination of these dissociation velocities constants which it is hoped may be helpful when we come to the study of abnormal hemoglobins. Figure 13 illustrates the method used by Gibson and RoughtonS two years ago for obtaining k4. A solution of carbon monoxide was mixed with oxy- hemoglobin, which had been equilibrated with various pressures of oxygen ranging from a tenth of an atmosphere to a whole atmosphere, the carbon monoxide pressure being constant. The theory given in figure 13 then shows that if the inverse of the rate of displacement is plotted against the oxygen pressure, a straight line is obtained, of which the intercept on the vertical is equal to 4/k4. Figure 14 shows an application of the principle. The method, however, r 07 06 as 04 03 02 0-1 / } ~4 0-095 ream. Mb 0-5 1 norm. CO pH 91, 18 SAC. 0 0-1 02 03 04 05 06 07 08 m.rnol. O2 / LITRE. FIG. 1 5. EEect of CO on 0`, dis- . . SOCla~lOn. ~ < 100 80 6 O cat o o 40 o 20 O _ 0 20 40 60 80 100 TIME in MILLISEC5 r IG. 14. Displacement of GO from Hb by CO. \~ pH90,Temp.21°C. ~ \~' OF - Cm x ~ ~o~4 0~2 °/o Na2S204 Monoxide after 0108 m M H b Jmixturc l

~4 PART I. STRUCTURE OF HEMOGLOBIN is laborious since it requires as many as six separate kinetic experiments. The determination of k4 has now been greatly shortened by the following device. solution of oxyhemoglobin is mixed with sodium dithionite (9670 pure trade name "Manox") saturated with carbon monoxide at one atmosphere. What happens ? The Hb4OS dissociates into O and Hb4Oe. The O is mopped up by the dithionite and the Hb~O6 by the carbon monoxide, these two reactions proceeding so fast that the overall rate of oxygen dissociation is conditioned throughout by the speed of the unimolecular reaction Hb4OS~ Hb4O6 + O.,, i.e. by kit. The upper curve in figure 15 is an example of such ar, experiment. The lower curve in figure 15 shows the result obtained when oxyhemo- globin is mixed with sodium dithionite without the carbon monoxide. Figure 16 gives the calculation of the unimolecular "constant" of oxyhemoglobin in the two cases. In the former, the value of the constant does not change as the reactions progress: the actual value of the constant in this particular case was equal to 11, in agreement with the value obtained by the more laborious extrapolation method previously used. In the latter case, however, the calcu- lated value of the "constant" rises progressively, since, in absence of CO, the reaction Hb~OS ~ Hb~O6 + O.,, is succeeded by the reaction Hb4Oc ~ ~ b~O4 + O., and further intermediate reactions of the same type. The velocity constants of some of these later reactions are greater than k4 and as their influence comes more and more into play the calculated value of the overall dissociation constant correspondingly increases. The determination of lo, the velocity constant of the first carbon monoxide ~1 z30 o 920 cr 0 1 0 ~1 I-) o I I I I 1 `_, O 20 40 60 80 100 PER CENT EXTENT OF REACT I ON I at _ ~07~ ~ ~ (O2Hb+Na252O4+co~i-~44/4 DH 9 0. 21°C. FIG. 16. Effect of CO on oxy- gen dissociation constants.

VELOCITY CONSTANTS—AINSWORTH, GIBSON AND ROUGHTON 25 coming off, was first made over 20 years ago,9 by mixing very dilute CO hemoglobin with buffer containing various amounts of dissolved oxygen, following the rate of replacement and using a similar extrapolation procedure as in the first method of determining k4. Instead of using dissolved oxygen as a replacer, we now use dissolved nitric oxide. Nitric oxide has about 1500 times the affinity for hemoglobin that carbon monoxide has.~° 300 200 s 100 o 4 (1' [; [CO]N - (4 ~ oL4 [N°]; 0 100 300 500 700 CO PRESSURE in mm. Hg (offer mixing) FIG. 17. Determination of 1'4 by use of the high affinity of NO for hemo- globin. Figure 17 shows a plot similar to figure 14 for a series of experiments in which CO hemoglobin in equilibrium with various pressures of CO was mixed with a buiEer containing some dissolved nitric oxide. Again a straight line is obtained but there is, in this case, an experimentally observed point on the vertical axis itself.l1 Such a point is obtained when a 100~7O CO hemo- globin solution in equilibrium with five millimeters of carbon monoxide is mixed with nitric oxide in solution. Since this point coincides with that gotten by the extrapolation of other points, the determination of l4 can be made in 3 single experiment. Table II shows how well the values of 14 agree whether TABLE II x 0~ NO 14 Hb4(CO)4 =~_ Hb4(C0)3 + CO Hb4(CO), + X ~ Hb4(CO)3X etc. Sheep Hb: pH 9.1, Temp. 23.5°C. l4 0.042 0.044 one uses oxygen or nitric oxide as the replacer. It is, eve think, a very crucial

;~6 PART I. STRUCTURE OF HEMOGLOBIN test of the whole theory upon which these types of measurements and the deductions from them are based. The determination of j4, the velocity constant for the rate of dissociation of the first nitric oxide from hemoglobin, can be carried out by simple classical methods. The reaction is so slow that if a dilute solution of NO hemoglobin is rotated in a tonometer containing carbon monoxide at one atmosphere pressure and rotated for several hours, the time course of reaction and the value of the unimolecular constant can be arrived at from spectroscopic determinations at 3-hourly intervals. The whole procedure is very simple. 9 1 8 o 17 o 16 15 20, ' \ j4,ooooos2 \ pH 91,16 SAC. \D ~. l O 2 4 6 8 TIME in HOURS. We are now applying constants to the different them to abnormal types fractions. It is, however, a rather striking fact that, although these different hemoglobins show much different electrophoretic and solubility patterns, yet the reactions with the small ligand molecules so far have not revealed anything like such great differences. In the case of human sickle cell hemo- globin, for example, both the oxygen equilibrium curve and the kinetics of dissociation of oxyhemoglobin are more or less the same. It will be very interesting to see whether this extends to various other types of human hemo- globin. Finally, a word as to the major kinetic problems in front of us at the moment. We are now trying to make a complete kinetic analysis of the reaction of oxygen with hemoglobin, involving eight velocity constants 100 90 80 70 60 OZ FIG. 18. Determination of j4, the O velocity constant for rate of dissocia- 50 ~ tion of first NO from Hb. Note reaction time is in hours. 40 these various methods for the individual velocity types of sheep blood and hope later on to extend of human hemoglobin and to various hemoglobin ~ ~ . ~ 1_ 1 _ _ _ ~ _ 1~ ~ _ ~ _ _ ~ ~ ~ ~ ~ _ _ _ tour combination, tour reversible ones. l o nave any chance or success in this problem, we must know independently at least six of the eight velocity constants. We have four of them at the moment, and I am pretty well sure that we may have five and possibly six. Then it will be a matter of going on to the electronic calculator. But what troubles will be in store for us, who can say? All I can do in closing is to ask for your prayers.

VELOCITY CONSTANTS—AINS~rORTH, GIBSON ID ROL-GHTON 27 REF`EREN CES 1. Haemoglobin: a symposium based on a conference in memory of Sir Joseph Barcroft; F. J. W. Roughton and J. C. Kendrew, editors. Butterworths, London, 1949. 2. Roughton, F`. J. W.' Otis, A. B., and Lyster, R. L. l.: The determination of the individual equilibrium constants of the four intermediate reactions between oxygen and sheep haemoglobin, Proc. Roy. Soc., Series B. 144: 29, 1955. 3. Lyster, R. L. J.: Ph.D. thesis, Cambridge University, 1955. 4. Gibson, Q. H.: An apparatus for flash photolysis and its application to the re- actions of myoglobin with gases, J. Physiol. 134: 112, 1956. 5. Gibson, Q. H.: The direct determination of the velocity constant of the reaction Hb4(CO)3 + CO ~ Hb4(CO)4, J. Physiol. 134: 123, 1956. 6. Gibson, Q. H. and Roughton, F`. J. W.: The determination of the velocity constants of the four successive reactions of carbon monoxide with sheep haemoglobin, Proc. Roy. Soc., Series B. 146: 206, 1957. 7. Gibson, Q. H. and Roughton. F. J. W.: The kinetics of haemoglobin and haem compounds as models for enzyme action, Faraday Society Discussions 20: 195, 1955. 8. Gibson, Q. H. and Roughton, F`. J. W.: The kinetics of dissociation of the first oxygen molecule from fully saturated oxyhaemoglobin in sheep blood solution, Proc. :lloy. Soc., Series B. 143: 310, 1955. 9. Roughton, F. J. W.: The kinetics of haemoglobin VI. The competition of carbon monoxide and oxygen for haemoglobin, Proc. Roy. Soc., Series B. 115: 473, 1934. 10. Gibson, Q. H. and Roughton, F`. J. W.: The kinetics and equilibria of the reactions of nitric oxide with sheep haemoglobin, J. Physiol., 136: 507, 1957. 11. Gibson, Q. H. and Roughton, F`. J. W.: The kinetics of dissociation of the first ligand molecule from fully saturated carboxyhaemoglobin and nitric oxide haemoglobin in sheep blood solution, Proc. Roy. Soc., Series B. 147: 44, 1957. DISCUSSION liar Ed.;all In tile early days when T was ~ strident in (~ambrirl~e Fn~l~n~l —~.t, —, ~ , in 1924 and 192S, Professors Hartridge and Roughton were carrying out the first of their studies on the reaction velocities of hemoglobin with oxygen and carbon monoxide. The progress that has been made is certainly phenom- enal. In those days the idea of detecting individual velocity constants was regarded as completely out of the range of possibility. We have some time free for discussion and there are many directions the discussion could take. Dr. [Walter Hughes: I have just one brief comment which really refers to the work of Dr. Riggs. He has measured the effect of mercurials on the dissociation constant for oxygen. I have done the reverse and measured the effects of carbon monoxide and oxygen on the association constants for methylmercury. I find a difference between the two. This is perhaps more subtle than Dr. Riggs' difference in the sense that I think most people consider carbon monoxide and oxygen to form very similar complexes when combined with hemoglobin. Dr. Riggs measured the difference between reduced hemoglobin and oxyhemoglobin. In the case of bovine hemoglobin

28 PART I. STRUCTURE OF HEMOGLOBIN I find that methylmercury is bound about three times more tightly to oxy- hemoglobin than to carbonmonoxy hemoglobin. Dr. Roz~ghtor': May I say a word about Dr. Hughes' contribution? If ' understood him right, it was the competitive equilibrium between carbon monoxide and oxygen in hemoglobin that he measured. Dr. Hughes: It was not measured that way. These were equilibrium con- stants between methylmercury and carbonmonoxy hemoglobin on the one hand and with oxyhemoglobin on the other hand. I found the equilibrium constant for methylmercury for oxyhemoglobin was three times that for carbonmonoxy hemoglobin. I want at some time to have a discussion on sulfhydryl groups. I think there are only two sulfhydryl groups in bovine hemoglobin. Within the spread of my data (equilibrium constants on individual measurements vary by 20 per cent) the affinity constants for both seemed to be the same. Thus there does not seem to be any interaction between the two. Dr. Roughton: It does not disturb me that you find a difference between CO hemoglobin and oxyhemoglobin. It is an old creed that these two com- pounds are physico-chemically similar, but this creed must now go, for the more intimately one studies them, the more frequent are the differences that one finds. Perhaps the most dramatic instance thereof is that the velocity of combination of the first oxygen molecule with hemoglobin is independent of temperature, whereas the velocity constant for the combination of the first carbon monoxide molecule with hemoglobin has a very clearcut dependence on temperature. Dr. Edsall: Or. Riggs was, I think, the first person to observe the effect of sulfhydryl groups on oxygen dissociation. As his work has already been referred to, I would like to call on Dr. Austen Riggs. or Rim.: I believe mv original measurements) of the oxygen equilibrium I, ,~ ... . , O ~ ~ of hemoglobin in the presence of p-chloromercuribenzoate ~ PCMB ~ shed some light on the affinity of mercurials for hemoglobin. These experiments indicate that at low degrees of oxygenation PCMB greatly increases the oxygen affinity, whereas at high oxygenation levels PCMB decreases the oxygen affinity. This effect is indicated most clearly if we plot the difference in the degree of oxygenation in the presence and absence of PCMB versus the oxygen pressure. (Fig. 1~. In such a plot we see that there exist two maximum shifts: first a large increase in oxygen affinity, then a decrease, faith the peaks at about 25 and 75 per cent oxygenation. Since this appears to be a completely reversible reaction, this gives us some information about the relative affinity with which PC~B is bound. Thus, if PCMB drives oxygen off, then oxygen must decrease the affinity of hemoglobin for PCMB. This appears to be the situation at high oxygen saturations. At low oxy- genation levels PCMB increases the oxygen affinity, so that it appears that 2 j per cent oxygenated hemoglobin has a higher affinity for PCMB than . .

DISCUSSION - , . . . -15 - +~0 - 25./. HE O1 l ,,/~ v ~ ~ O % H b O2 O- l - _ ~ ~ / ·_ ~ / 4. - 5 _ ~ / ~ ~ ~ / ~ 10 ~ /o _ _ \ o/ 75 % Hb O 1 ~ ' I ~ 1 ' -t.0 0.0 t O io9 Oxygen Pressure 29 FIG. 1. The effect of the mer- cu ri al, p-chloromercu ribenzoate, upon the amount of oxygen bound by hemoglobin, plotted as the increase or decrease in per cent saturation of human hemoglobin with oven versus · ,, , 4, the logarithm of the oxen en ~ Hi. pressure expressed in milli- meters of mercury. The arrows hate the per cent saturation of normal hemoglobin in the absence of mercurial at the in- dicated oxygen pressures. . , . does reduced hemoglobin. These maximum changes occur in the same positions as do the changes in the dielectric constant increment obtained in the meas- urements by Takashima and Lumry upon which Dr. Edsall commented at the beginning of the conference. I believe that there is an important relation- ship here. REFEREN CE 1. Riggs, A.: Sulfhydryl groups and the interaction between the hemes in hemoglobin, J. Gen. Physiol. 36: 1 (September) 1952. Dr. Edsall: There is orate thing that struck me as I was listening to Pro- fessor Roughton's paper. I do not know whether or not this is a possible explanation of the phenomena, but I was profoundly struck by the great difference in pH effect on the different velocity constants, particularly the contrast between the 1'~ and the Z,4. This certainly suggests that there is a different kind of coupling with the heme-linked acid groups in these two cases On the other hand, if, as most of us have generally assumed, all of the heme groups are initially equivalent before any reaction has occurred, it is a little difficult for me to see offhand why this should be so. I wonder if it is possible that the fourth group is structurally different from all the rest. and may perhaps be actually completely unavailable for reaction with oxygen or CO initially, so that there has to be a structural rearrangement in the molecule to permit that group to be capable of reaction at all. But once it is opened up and is available, then it has a higher oxygen affinity than any of the others. I have not tried to think through all the implications of this view.

30 PART I. STRUCTURE OF HEMOGLOBIN Probably Professor Roughton has considered this possibility already and may have some reason for knocking down my argument. Dr. Roughto?~: Dr. Edsall, I have, it is true, worried about your sug- gestion over the years and have tried to make quantitative deductions from it. The results have not been too promising. The difficulty is this. When any three ligand molecules combine with hemoglobin carbon monoxide, oxygen or nitric oxide—then for some reason a change in the configuration occurs which makes iron atoms behave independently. We do, indeed, now have a very considerable body of evidence which indicates that when either three or four ligand molecules are com- bined with hemoglobin, the hemoglobin then behaves like myoglobin, the iron atoms all behaving; in the same play and independently of one another This makes it seem unlikely that one out of the four iron atoms was, before combination, different from the others. What we must really seek is the mechanism of how the iron atoms become independent of one another at the particular stage when three or four of the iron atoms are combined ravish ligands. No explanation is so far available. Dr. Jacinto Steinhardt: I was very much impressed by one of the findings o ~ Professor Roughton but I want first to be sure I have understood it correctly. As I understood it, under one set of experimental procedures if part of the gas is removed from the hemoglobin, or if all of it is removed, the velocity of recombination is the same in both cases. However, with another procedure, which I believe in this case was flash photolysis, the initial velocities are very different in the two cases. I would like Professor Roughton to repeat what the differences were, and further, whether the differences in velocity persist until all the gas is recombined, or whether as more and more gas goes back on, the velocities become the same (i.e., they differ only for a particular amount taken off). Dr. Roughton: I am not sure I got your second point, but to the first point the answer is clear. The experiments in which the velocity of recombination of CO spas independent of the amount of gas dissociated by the light were or1 myoglobin and not on hemoglobin. In myoglobin there is only one carbon monoxide to come off, and you cannot in that case have the situation which was depicted in the slide for hemoglobin with its four intermedites, that is, different concentrations of the four intermediates corresponding to varying degrees of flash photolysis. Dr. Steinhardf: You have clarified that perfectly. The second point remains. If, let us say, half of the carbon monoxide is removed from the protein, and then the rate of its recombination is measured, you get one result. If you take all of it off, you get a lower velocity constant. Dr. Roughton: There you have to hammer through the mathematics, preferably by means of automatic electronic computation. When you do that,

DISCUSSION 31 you find that the rate of the whole process is governed over such an enormous part of it by the earlier, slower velocity constants that, until the reaction has proceeded to well over 90 per cent of completion, it does not matter whether the last constant is 20 times greater than the first or a different number of times greater than the first. It is not rate-limiting at all. So, although your question is theoretically valid, yet in practice it is only possible to pick up the differences in very high ranges, e.g., over 93 per cent completion, but these are technically very difficult to study with sufficient accuracy. Dr. Steinhardt: It might be worth remarking that if you denature, let us say, half of CO hemoglobin or ferrohemoglobin, the rate at which it re- generates is considerably faster than if you denature 9j per cent of it in pre- cisely the same environment. This difference is in the velocity constant, i.e., it persists until regeneration is complete. Dr. Riggs: I would like to ask Dr. Roughton about the experiments with sodium dithionite. I have never been able to obtain the original hemoglobin with which I started by treating hemoglobin with dithionite, even with exceedingly small amounts. The spectrum is changed, and this seems to be an irreversible phenomenon. I wonder if he has any comments about this? Dr. Ro2~phton: In the first place, we use the purest dithionite we can get, and we take the greatest possible care to protect it in all stages from oxygen. We also take care to keep the dissolved oxygen content of the hemoglobin with which it is going to react as small as is compatible with nearly com- plete oxygenation of the hemoglobin. Furthermore, we do not trust the dithionite beyond a few tenths of a second. But in kinetic experiments, under these carefully controlled con- ditions, one seems to get pretty good agreement at different wave lengths. If you do not take that care, you will get quite a different answer; that is, if you use one part of the spectrum rather than another to examine the course of your reaction. I quite agree with you that after some little time has elapsed, you have a very, very poor hope indeed of getting back your original hemoglobin. Your sad experience is probably of hemo~lc~hin exnnsed to dithionite for longer than two tenths of a second. Dr. Rices: Yes. At, Dr. Edsall: In other words, dithionite for equilibrium measurement is not a very good thing to use. It is only suitable when you work in ranges of a few tenths of a second. Dr. Ro?tyhton: Even then you have to watch it all the time. Dr. Reinhold Benesch: I would like to draw attention to the possibility of using an enzyme mixture, namely glucose oxidase and catalase, for re- covering oxygen from oxyhemoglobin. This can be done very fast, and in a continuous way, almost to 90 per cent to hemoglobin. This might be useful in measurements of the kind that have just been discussed, because an arti- fact such as impure dithionite should not occur. It is possible depending,

32 PART I. STRUCTURE OF HEMOGLOBIN of course, on the amount of the enzyme—to vary the rate of reduction of oxyhemoglobin to reduced hemoglobin. It is possible to do this inside the intact red cell as long as the solution is sufficiently buffered against the gluconic acid which accumulates, since the enzyme, which is a very active one, sucks the oxygen out of the red cell without affecting its integrity. Dr. Edsall: From what tissue does this enzyme come? Dr. Benesch: It is available commercially. It contains the glucose oxidase and some catalase. In some cases the commercial preparation is deficient in catalase and then it is necessary to add it. Polarographically it is very easy to tell the proportion of catalase to glucose oxidase. I would like to make one further remark in connection with your presenta- tion, Dr. Edsall, with regard to the very cogent arguments that imidazole groups are responsible for the heme-linked ionizations. I think you will agree that one of the major arguments which Wyman brought forward to link the imidazole groups to this phenomenon was that he determined the difference in dissociation constants of these groups in hemoglobin at different temperatures. The heats of ionization derived from these data agreed very well with the imidazole groups, namely, between six and seven kilocalories. I would like to draw attention to our finding some time ago, which has also been confirmed independently by Cecil in Oxford, that the heat of dissocia- tion of the sulfhydryl group falls within the same range six to seven kilocalories. Furthermore, pK's such as the second heme-linked group, 6.8 to 7.9 and so on, would be perfectly conceivable for some sulfhydryl groups, depending on what groups were dissociating in the neighborhood of the sulfhydryl group. For example, if some very positive groups were located in the neighborhood of such a sulfhydryl group, the pK of the sulfhydryl group could fall within this range. Dr. Edsall: The possibility that the heme-linked acid groups might be sulfhydryl groups has occurred to a number of people. I know Dr. Riggs has discussed it with me once or twice. I think this is something that we cer- tainly have to bear in mind. Dr. falter Hughes: It seems very unlikely to me that the sulihydryl groups are directly linked to heme since they vary in number from species to species. In bovine hemoglobin, I find only half as many sulfhydryls as hemes, and myoglobin has no -SH groups. I would expect this variation to be reflected in different oxygen equilibria for different species and even in different absorption spectra, whereas no such differences have been observed.

A SURVEY OF THE EVIDENCE FOR AND AGAINST A CREVICE CONFIGURATION FOR THE HEME IN HEMOGLOBIN* PHILIP GEORGE AND R. L. J. LYSTER; I?'tro~nct~on. For many purposes the reactions of hemoglobin and methe- moglobin can be quite adequately represented by the traditional formulae Hb + O — HbO~ HA + OH- = HbOH HA + CN- — ~bCN However, in more detailed discussions of the factors which govern the reac- tivity of the heme iron, a consideration of the way the heme is bound to the protein becomes essential. The fact that heme is also the prosthetic group of peroxidase and catalase, aIld of cytochrome c, too—although in a slightly modified form emphasizes the dominant role of the heme-protein linkage in determining specificity. This is further borne out by other considerations. Hemoglobin among all the hemoproteins shows most clearly differences in reactivity, depending not only on the species of origin, but also on its physical state as this is altered from that of a fresh lysed-cell preparation to that of a many-times recrystallized sample. Several as yet undefined changes in the conformation of the molecule probably accompany this transition. Not only is the primary reaction of the iron altered (for instance the oxygen amenity is usually increased by dialysis, crystallization, etc.), but the secondary processes that affect the primary reaction are also modified. Although the magnitude of the Bohr effect is scarcely altered, heme-heme interaction is less, or is lost entirely.) This is especially true of the reactions of recrystallized methemo- globin; in some studies it has been found that n I, showing interaction to be completely absent.'~3~4 Hence, the process of heme-heme interaction is to be regarded as resulting from a far more critical and specific structural feature of the molecule. In the case of muscle hemoglobin, myoglobin, it may be that the recrystallized con- dition represents a final conformational state wh ich can only be further changed by denaturation itself. The smaller Bohr effect observable in the metmyog;lobin-fluoride reaction reappears unchanged with the hemoprotein reconstituted from hemin and apomyoglobin.5 The binding of the heme to the globin has usually been discussed from two points of view: either to suggest a mechanism for heme-heme inter- action or the Bohr effect, or to account for the remarkable property of ~ This review has been based in part upon research, now in progress, that is sup- ported by grants from the National Science Foundation ( G23 09 ) and the National Institutes of Health (RG4850). ~r This paper was presented by Dr. George.

34 PART I. STRUCTURE OF HEMOGLOBIN reversible oxygenation by considering specific bonding to a particular amino- acid residue, notably histidine. However, some observations and quantitative data bear on the more general question of the configuration of the heme, quite apart from the chemical nature of the group or groups in the protein to which the iron atom is attached, and quite apart from the role these may play be ~ ~ ~ 1 — — _ _ ~ _ ~ in determining the interaction effects. This kind of evidence will first reviewed under three headings that represent extreme structural types: a) heme bonded only by its side-chains: no Ye-protein bond b) a surface configuration: one Fe-protein bond c) a crevice configuration: two Fe-protein bonds. Then a comparison will be made between pH effects that can originate in the ionization of a heme-linked group, and in the fission of a crevice bond. In some respects these extreme types are not mutually exclusive, and in particular certain structures may be regarded as intermediate between ~ and c. For example, while there may be one actual iron-protein bond, another part of the protein molecule may nevertheless shield the heme from direct attack by ligand molecules, introducing steric hindrance as an important factor in complex formation. Such a structure could also be considered as a crevice configuration. However, for classifying the evidence, the extreme structural types defined above provide ~ very convenient frame of reference. No Iron-Protein Bonds. This structure would require firm bonding of the heme group to the protein by the side-chains of the heme. Only the pro- pionic acid groups need be considered, since the very ready formation of hematoheme with hemoglobin excludes the possibility of linkage by the reaction of the vinyl groups, as in cytochrome c.6 Esterification or amide formation with groups on the protein is unlikely in view of the very rapid removal of the heme that occurs in strong acid. A more likely possibility is the formation of salt bridges between the negatively-charged carboxyl groups and positively-charged groups on the protein. One consequence of this structure would be weaker binding of heme in solutions acid to the pK of the carboxyl groups; and this would accour~t for the easy removal of heme in solutions at pH 3.7 However, this reaction probably involves several groups other than those directly linked to the heme, in view of Zaiser and Steinhardt's finding that in this pH region, 36 extra basic groups per molecular weight of 68,000 become titratable.S Were it not for the tact that both heme and hemin exist as polymeric species in aqueous solution, a comparison of their absorption spectra with those of hemoglobin and methemoglobin would provide valuable evidence as to the plausibility of this structure. The differences (which are especially marked between hemin and me/hemoglobin, and which permit the spectro- photometric titration of apohemoglobin with heme and hematin) could how- ever be attributed entirely to the monomeric state of the prosthetic group ashen it is attached to the protein. It is interesting that, in certain solvents,

CREVICE CONFIGURATION GEORGE AND LYSTER 35 especially alcohol, hemin gives an absorption spectrum that resembles methe- moglobin very closely.9 The difficulty of accounting for the specificity of hemoglobin that this configuration would entail might be overcome by supposing that the iron atom becomes simultaneously attached to the protein when a ligand is bound. One Iron-Protein Bond. In this structure the iron in hemoglobin is regarded as forming a classical octahedral complex with four bonds in a plane to the pyrrole N-atoms, a fifth bond to the protein on one side of the porphyrin ring, and a sixth bond on the other side to a coordinated water molecule: id N e.g., Globin Fe—HERO / I At N The magnetic susceptibility data,10 which establish that hemoglobin and methemoglobin can form octahedral complexes, taken in conjunction with the recent theoretical developments in ligand field theory which account for the stability of transition metal compounds in general,~~~-~~3 make the in- clusion of a water molecule to complete the octahedral coordination shell an extremely reasonable assumption. This hydrate structure was first discussed in some detail by Hauro~vitz in 1935,14 and, seeking direct experimental confirmation, he later extended Zeynek's observations on the drying of hemoglobin.45~~6~i' He showed that with oxyhemoglobin scarcely any change in the absorption spectrum occurs, and ever at very low pressures (0.1 mm Hg) the oxygen cannot be removed. In contrast, unoxygenated hemoglobin on drying is converted into a com- pound named anhydro-hemoglobin characterized by a hemochromogen-like spectrum. Although not stated explicitly, it appears from the description of the experiments that anhydro-hemoglobin is unable to combine with oxygen unless re-hydrated. Haurowitz pointed out that the formation of anhydro- hemoglobin may be due either to an intramolecular change, with the iron atom forming a bond to nitrogen within the same molecule, or to an inter- molecular reaction with the iron exchanging the water for a N-atom on a neighboring molecule. The latter polymerization mechanism was preferred. In the presence of a large excess of glucose no hemochromogen-like spectrum was observed in accord with the expectation that, through its multiple polar groups, the glucose would become attached to the surface of the hemoglobin and thus prevent the mutual association forming the polymeric nemochrom- ogen. Keilin and Hartree extended these studies by showing that, on drying, methemoglobin underwent a similar change giving the corresponding para- hematin while the fluoride, abide and cyanide complexes could be dried without change, thus resembling oxyLemcglobin.:S

36 PART I. STRUCTURE OF HEMOGLOBIN These observations appear to demonstrate the presence of a water molecule attached to the iron in both hemoglobin and me/hemoglobin; but this con- clusion would only be valid if it could be shown that an alteration in the number of solvent water molecules, hydrating the protein as a whole, played no part in bringing about the change in structure around the iron atom. If salvation effects were significant, then on the basis of these observations alone no choice could be made between the hydrate structure, a structure in which another unidentified labile group is attached to the iron, and a structure in which the sixth coordination position is unoccupied. At present there is some evidence to suggest that the water does play an essential role beside that of a potential ligand for the iron. lIaurowitz noted that by changing the partial pressure of the water the conversion of hemoglobin into anhydro-hemoglobin could be repeatedly shifted from left to right Hb.H2O ~ ~ anhydro-Hb + H2O while anhydro-hemoglobin apparently only forms oxyhemoglobin when both oxygen and water are present. Now it is understandable that dried oxyhemo- g~obin resists deoxygenation, since calculation shows that its formation is probably accompanied by a very favorable free energy change. But, unless water also enters into the reaction in the capacity of an unspecific solvating agent, it is difficult to understand why oxygen should not be able to react with anhydro-hemoglobin just as water is presumed to do according to the hydrate hypothesis. Further experiments on anhydro-hemoglobin with oxygen and other ligands, such as CO and NO for which the iron has a greater affinity, are really needed to bring out more clearly the role of the water. There is another aspect of these reactions that has received less attention. If it is accepted that the heme is joined to globin by one strong bond, then the fact that anhydro-hemoglobin has a hemochromogen structure suggests very strongly that the bond is to a N-atom. The other possibility—namely, that a strong bond to some other atom breaks and two new F`e-N bonds form appears far less likely in view of the ready reversal of the equilibrium with changes in water vapor pressure. An important feature of the hydrate structure is that it leads directly to a simple and reasonable mechanism for the conversion of acidic methemoglobin to the alkaline form. As the following equations show, the ionization of proton from the coordinated water molecule is equivalent to its replacement by a hydroxyl ion. Fe+H.,O = FeOH + H+ :lTe ~ H.',O + OH - ~eOH + H ,O Keilin and Hartree observed that in freezing to liquid-air temperatures, alkaline methemoglobin reverts to the acidic form. They lateriS considered this to be direct evidence for the hydrate structure, but an effect of this kind would be shown by any endothermic ionization, as demonstrated by Keilin and Hartree in their earlier experiments. Hence it cannot serve to identify

CREVICE CONFIGURATION GEORGE AND LYSTER 37 the chemical nature of the ionizing group. That the methemoglobin and rretmyoglobin ionizations are endothermic was later established by George and Hanania, the values of AH being 5.8 + 0.7 and 3.9 A- 0.5 kcals/mole respectively.4~~0 A suggestion that has received considerable attention is that while the heme groups are attached by only one iron-protein bond, the groups are sufficiently deeply buried in the body of the protein that combination of a limed with the iron requires a dilation of the nrotein.-i This the of steric ~ ~ - 1~ r- - ------ - ---- -a r - ~ ' ~ ~ ~ · . · ' 1 ~ T' '1 · ~ 1 . ~ . . ~ - h~ndrance has been criticized bY merlin on the grounds that the iron atoms of hemoglobin are known to bind firmly some rather large molecules;~'' for example, nitrosobenzene appears to be bound about as strongly as carbon rr~onoxide. Moreover with 4-methyl imidazole, Kendrew and Parrish found such a small change in the unit cell of metmyoglobin that they suggested that the heme group is more likely to be located on the surface.''3 Certain oxidizing and reducing agents have also been instanced as large molecules having apparently easy access to the heme group. But close approach to the metal atom itself is not essential for electron transfer, as is shown in reactions in- volving the octahedral coordination con~plexes of iron, ruthenium, and osmium with dipyridyl and phenanthroline.'4 A similar example is found in the reaction of ferrocytochrome c and the second intermediate compound of cytochromec peroxidase.-, All these reactions are very fast indeed, their velocity constants being greater than 10;3 1. mole-i sec.-i Evidence of a more direct kind for this type of steric hindrance divas sought by St. George and Pauling by studying the binding of a series of aliphatic isocyanides with ferroheme and hemoglobin.'') Their results, together with those of Lein and Pauling's later Fort on myoglobin,~6 are given in Table I. TABLE I LOGARITHM OF EQUII IBRIUM CONSTANTS FOR THE COMBINATION OF ALKY! ~ SOCYANIDES WITH HEME AND HEMoPRoTErss AT PH 6.8 AND 25 C.-1 -I; iso- tert.- ethyl n-propyl propyl butyl Ferroheme 9.43 9.14 9.04 Horse Myoglobin 5.30 5.04 4.15 3.00 Bovine Hemoglobin 3.9 6 — 3.48 1.70 Human Hemoglobin 4.11 3.52 1.80 The most interesting feature is the sharp drop in the affinity of hemoglobin for the tertiary butyl isocyanide, which is not reflected in the results with ferroheme. This sharp drop was ascribed to steric hindrance. If this inter- pretation is correct, the problem now arises whether steric hindrance affects the binding of only those ligands equal (or larger) in size to tertiary butyl isocyanide, or whether it is always present as a factor in determining ligand affinities, however small the ligand. If the latter alternative is adopted, it becomes necessary to postulate for the iso-propyl isocyanide some sort of

38 PART I. STRUCTURE OF HEMOGLOBINT steric assistance, such as a complementary hollow in the appropriate part of the protein near the heme group. This difficulty is further emphasised by a consideration of Lein and Pauling's data for myoglobin and approximate estimates of the maximum lengths of the ligand molecules, based on the ap- piopriate covalent radii, which are given in Table II. In its fully extended TABLE II APPROXIMATE MAXIMUM LENGTHS IN A NTGSTROM U NITS OF AL KYL I SOCYAN IDES ethyl 7.7 iso-propyl 7.7 n-propyl 9.0 ter;.-butyl 7.7 form, n-propyl isocyanide would project furthest of all from the plane of the heme group, yet its affinity is only half that of ethyl isocyanide. On the other hand, in its coiled configuration, it would project to the same extent as tertiary butyl isocyanide, but it would appear necessary to postulate an additional complementary hollow to contain the extra methyl group. The difficulties involved in comparing a series of primary, secondary and tertiary alkyl ligands may be illustrated by considering Table III. These TABLE III BINDING OF BASES By' SILVER IONS AT 25 C. - ~ - - S- - S3 log K /\G AH /\S Amine: Ag ratio 1 :1 (kcals/mole) (kcals/mole) (e.u.) Ammonia 3.32 —4.52 —6.9 —8.0 Ethylamine 3.37 —4.6 —6.73 —7.15 Iso-propylamine 3.3 8 —4.61 —6.73 —7.1 Benzylamine 3.29 —4.49 —6.73 —7.5 Amine: Ag ratio 2 : ~ ~ Ammonia 7.23 —9.86 —13.8 —13.3 Methylamine 6.79 —9.26 —11.5 —7.5 Ethylamine 7.30 —9.96 —13.5 —11.8 Iso-propylamine 7.24 —9.87 —13.5 —12.0 Benzylamine 7.14 —9.74 —13.5 —12.5 Dimethylamine 5.37 —7.32 —9.7 —8.0 Diethylamine 6.38 —8.70 —10.65 —6.5 results are from the work of Bj errum, 'I Bruehlman and Verhoek,9S and Fyfe,99 on the binding of amines by silver ions. Silver can bind either one or two molecules of base; in either case, if the base is a primary amine, the thermodynamic data are almost identical for all the amines listed. However, for the formation of the 2:1 complete with secondary amines, there is a difference of a factor of ten between diethvlamine and dimethylamine for the affinity, though the acid dissociation constants of these two amines differ by less than a factor of two. Thermodynamically, ~ H for diethylamine is more favorable to the extent of 0.S kcals/mole, and AS also more favorable to the extent of 1.5 e.u. Similar thermodynamic data for the reaction of

CREVICE CONFIGURATION GEORGE ANTD LO STER 39 hemoglobin and myoglobin with the isocyanides would be of the greatest interest. The apparent existence of steric interaction of tertiary butyl isocyanide with the protein in hemoglobin led St. George and Pauling to propose that the phenomenon of heme-heme interaction also had a steric origin.') How- ever, it appears from their curves of percentage formation versus ligand con- centration that heme-heme interaction almost disappears in the on crucial case of tertiary brutal isocvanide: it is difficult to see who a large litany should . . ~ . , , , . j ~ c, Produce the hemoglobin to show less heme-heme interaction than a small ligand like oxygen. Characteristic of this hypothesis that heme-heme interaction has a steric origin is the trend in thermodynamic data to be expected from such a mechanism. If the first ligand molecule to combine reduced the steric hind- rance for the second, and so on, it would be predicted that in comparing data for the first and last oxygen molecules the heats of reaction should show a trend to a more favorable value, since more er~ergy would be required to dilate the molecule for the entry of the first; whereas the entropy change should show a trend to a less favorable value, because the entry of the last oxygen molecule would be accompanied by a smaller increase in the freedom of the molecule in the neighborhood of the heme. Experimentally, exactly the opposite trend is shown in the only system for which direct experimental results are available. The data of Roughton, Otis and Lyster for the reaction of oxygen and sheep hemoglobin at pH 9.1 are given in Table INT.:~° The first TABLE IV THERMCDYNTAMIC D ETA FOR THE OXYGENATION OF SHEEP HEMOGLOBIN AT pH 9.1 AND 25 C.:~° 1st Oxygen 2nd Oxygen 3 rd Oxygen 4th Oxygen (I./^'nole) 1.1 X 104 5.S X 104 1.2 ~ 10;' 3.9 X 106 (locals /mole) —12.1 + 0.8 2.5 —4.2 + 3.7 —5.1 + 3.3 AS (e.~.) —22.2 + 3 —4.4 + 8 +9.2 + 12 - 13.0 + 11 molecule to combine has a large and favorable heat of reaction and art un- favorable entropy change, while the affinity of the last molecule to combine is high despite a low heat of reaction, because of a large and favorable entropy change.3i This trend is in direct conflict with the mechanism for heme- heme interaction suggested by St. George and Pauling. However, the only data as yet available for the carbon monoxide reaction indicate no such trend.3> The entropy changes for the first and last carbon monoxide molecules are very similar, and the heme-heme interaction originates entirely in the change in ASH from 7.1 kcals/mole to 10.5 kcals/mole, in the direction opposite to that for oxygen. It would thus appear that heme-heme interaction may not only be a oronertv of the hemoglobin molecule, but also have a different 1 1 ~

40 PART I. STRUCTURE OF HEMOGLOBIN thermodynamic origin, depending on the chemical nature of the ligand. If this is borne out by future quantitative measurements and for two ligands like oxygen and carbon monoxide that have such similar thermodynamic properties there are such enormous differences in ~ H and ~ S then it may be necessary to question whether in fact simple steric effects like those considered so far play any significant part in this intricate phenomenon. Two Iron-Protein Bonds. Conant, in his Harvey Lecture of 1933, first considered various structural possibilities in which iron was bound to the protein on both sides of the heme group.:33 Wyman suggested that the imi- dazole residues of histidine, one of these possibilities, were likely to be the groups concerned, on the basis of his finding that the heat of ionization for both the heme-linked acid groups of hemoglobin was 6.5 kcals/mole;34 this value is characteristic for histidine. Assuming that imidazoles are involved, Coryell and Pauling have given a detailed picture of the reactions in terms of resonating structures.3;, The dissociation constants of these heme-linked ion- izing groups are listed in Table V. TABLE V HEME-~NKED IONIZATIONS OF HORSE HEMOGLOBIN AT 25 C. 30 Pi:,. pK~ l\Iethemoglobin 5.75 6.68 Oxyhemoglobin 5.75 6.68 Hemoglobin 5.25 7.93 Figure 1 shows one of the possible resonating structures proposed by Coryell and Pauling; they assign pK, to the glyoxalinium nitrogen of the distal group and pi to the imino nitrogen of the proximal group. The structure shown differs from Conant's original suggestion in having a water molecule co- ordinated to the iron atom; this removes the objection that hemoglobin does Proximal HN~1 Fe+(H20) G Iobin Distal N~NH FIG. 1. Diagram of a possible res- onating structure proposed by Coryell and Pauling. not have an absorption spectrum like histidine hemochromogen. A weak link between the iron atom and the distal group, however, is postulated. George and Hanania found spectroscopic evidence for ionizations with similar pK', in methemoglobin solutions of low ionic strength.4 These ionizations became undetectable at higher ionic strengths; and from this effect of neutral salt, it was possible to argue that while pK~ could be attributed to such a group as that shown in figure 1, pK~ could not, if their pK~ is the same as Wyman's

CREVICE CONFIGURATION—GEORGE AND LYSTER phi. This difficulty persists in any structure where a proton acceptor is in- volved in bonding to the iron. Apart from this difficulty, the identification of pK~ and pK2 with imidazole groups may be criticized in two ways. First, there seems to be no obvious reason why the heat of ionization of the imino group of the proximal ring in figure 1 should have the same heat of ionization as that found in free his- tidine. Secondly, the recent studies by Roughton and co-workers have shown that the individual equilibrium constants of the oxygenation reactions 3' and the velocity constants for the combination of CO 35 are not uniformly affected by pH changes. In view of this, it should not be assumed too readily that Wyman's value for the average heat of ionization is actually that of each heme-linked acid group. The recent work of Corwin and Reyes, however, provides circumstantial evidence in favor of the imidazole hypothesis.;3'3 These workers prepared imi- dazole ferrohemochromogen and found that, in the solid state, it was able to combine reversibly with oxygen. l his property was not shared by the similar pyridine ferrohemo<:hromogen. III this renotinn In 1~re~lim~hl~r rlicnln~ e~ ., ~ . . .- . --I --a A--- ret ~~: ---or- ar ~m~ctazote group bound directly to tile iron atom, as in the formulations given by (:onant. Such displacement reactions, regardless of the chemical nature of the groups involved, are characteristic of all structures in which the iron is joined to the protein by two bonds, and will show certain general features to which we now turn. Crevices in General. The general features of reactions involving crevice structures will be considered from two points of view; first, the trend in thermodynamic data to be expected from such a reaction, and secondly, the effect such ~ structure has on the pH dependence of the affinity for a ligand. In considering thermodynamic data a useful comparison with cytochrome c can be made, since a crevice configuration has beers fully established for this hemoprotein. George and Hanania have compared the thermodynamic data for binding of cyanide ions by ferricytochrome c ant by ferrimyoglobin ;~0 the figures are given in Table VI. Though the affinities are quite similar, the T H ERA ODY N.N M IC D ATA Ferricytochrome c Ferrimyoglobin difference TABLE VI FOR BINDING OF CYANIDE AT 25 C.~O /\C~° WHO ( locals /mole ) ~ - ~ —8.3 —11.4 +3.1 ( kcals / mole ) +1.1 —18.6 +19.7 AS° (e.u.) +31.3 —24 —55.3 heats and entropies of reaction differ in a very significant fashion. While the heat change is favorable for ferrimyoglobin complex formation, it is ad- verse for the ferricytochrome c reaction; exactly the reverse holds for the entropy change, so that, but for the favorable entropy change ferricytochrome ~ cyanide would not in fact be formed. The adverse AH for ferricytochromec

PART I. STRUCTURE OF HEMOGLOBIN can be attributed to energy lost in breaking the Fe-N bond and other links that may be concerned in forming the crevice in the parent compound. The favorable entropy change may be attributed to the freeing of the polypeptide chain carrying this basic group. This freeing may be compared to a process of partial denaturation. The reversible heat denaturation of proteins is char- acterized by unfavorable heat changes and large and favorable entropy changes; for example, Anson and Mirsky found for trypsin that the heat of reversible thermal denaturation was + 68 kcals/mole, and the entropy change +213 e.u.4i From the figures in Table VI, it seems reasonable to conclude that, if the above interpretation is correct, crevice opening plays little or no role in the formation of the ferrimyoglobin complex. No thermodynamic data are avail- able for the corresponding reaction with hemoglobin, but it is known that the corresponding data for the ionizations of ferrihemoglobin and ferrimyo- giobin, and for the formation of their fluoride complexes, are very similar indeed. Thus it seems that in these particular recrystallized preparations the reactions of the ferric form of myoglobin and hemoglobin involve no crevice structures. Furthermore it may be noted that with the ferrihemoglobin no heme-heme interaction divas observed. The effect of a crevice structure on the pH dependence of the affinity for a ligand can be readily deduced. It is natural to assume that the crevice- J forming group is basic since this property is characteristic of all ligands that bind to metal atoms. Hence when the crevice bond Fe-Y is broken as a consequence of the iron atom combining with a ligand L, the group Y will associate with a proton in solutions where the pH is less than the pK of the group YH+, and remain in its conjugate basic form Y in solutions where the pH is less than this pK value. i.e., pH ~ pi-: pH > pi-: Fe Y + L + H ~: < > FeL YH + Fat e Y + L ~ > FeL Y These are generalized equations. With ferro- and ferrihemoglobin, the iron atom probably carries a net charge of zero and +1 respectively (the other two ionic charges being balanced by negative charges on two of the pyrrole N-atoms of the porphyrin ) and the ligands are usually neutral molecules and anionic species respectively. The crevice-forming group has a charge of zero arbitrarily assigned to it: similar equations can be written with the group as Y-, giving the conjugate acid YH. Since combination with the ligand breaks the crevice bond, it is convenient to consider a hydrate structure of . . . . .... . the hemoprote~n In equ~l~0r~um with the crevice configuration, , ~ Fe(H~O) Y ~ Fe Y + H,O

CREVICE CONFIGURATION—GEORGE AND LYSTER 43 and the formation of the complex via the reaction of the ligand L with the hydrate structure, -- KI, A__ F`e ~ HERO ~ Y + L ~ ~ lTeL Y + H '0 where the equilibrium constants are KCr and KI. respectively. The equilib- rium reaction giving the complex is then directly comparable with the formu- lation usually employed when no crevice is being considered, i.e., Fe(H.,O) + L ~ ~ FeL + H O The solution of the appropriate equations based upon the above mechanism gives the follow-in" expression for the observed equilibrium constant, evalu- ated as [complex] Free hemoprotein] x ~ligand] (H+ + KHY) Kobs = Kin x ~ H + + Ker. KITN- ~ This may be compared with the corresponding expression reflecting the in- uence of a heme-linked ionization on the affinity of the iron for a ligand, Kobs — KL x ~ H + Kp) Denoting the heme-linked group by H ~ X, then Kit, refers to the equilibrium constant for complex formation with the group in its conjugate acid form i.e., H +X.F~e ~ H..0 ~ + L (Kr) H ~ X.FeL + HERO (Kp) and Kr and Kit, are the ionization constants of the group in the reactant and product respectively. The two expressions are identical in so far as they give the same variation of Kobs with pH (H+ + Ka) i . e., }(obs tax where Ka and Kb are simply the experimentally determined constants. Ac- cording to the crevice mechanism Ka would be identified with KAY, and Kb with Ken Kuy; whereas according to the heme-linked ionization mechanism Ka would be identified with Kp, and Kb with Kr A consideration of the relative magnitudes of Kit and Kb shows that it is nevertheless possible in certain circumstances to distinguish between the two mechanisms. For a significant fraction of the hemoprotein to be present in the crevice configuration the equilibrium constant KCr must be greater than unity. This implies that only when Kb > Ka is the crevice mechanism applicable: those cases for which Kb < Ka must be attributed to the ionization of a heme-

44 PART I. STRUCTURE OF HEMOGLOBIN linked group. If the condition Kb > Ka holds then when data are only avail- able for one single reaction it is impossible to distinguish between the two mechanisms. However, provided that data are available for several reactions of the same hemoprotein, then, according to the crevice mechanism, Ka (ider~- tified with Kim) should to a first appro~cimatior~ be independent of the nature of the ligand, whereas according to the heme-linked ionization mechanism Kit (identified with Kp) would be expected to vary from one ligand to an- other, since the ionizing group H+X is understood to be linked in some way to the iron atom, i.e. H+~.FeL. According to both mechanisms Kb would be independent of the ligand, since it is identified respectively with K`r Kim and Kr, the ionization constant of the group in the parent hemoprotein. Since an ionizing group forming a crevice in this way results in the affinity of the iron for a ligand being dependent on pH it could still be described as a heme- linked group. The ionization is entirely normal in the complex and is affected in the parent hemoprotein only by virtue of its conjugate base forming a co- ordination bond to the iron above a threshold pH value (given by KCr.KH~-~. Nevertheless it is better to regard such a group as a special case because of the inter-relationship imposed on K;, and Kb. Turning now to hemoglobin, and the data in Table V, it is at once clear that pK~ cannot be explained by the crevice mechanism. This is the ionization responsible for the main Bohr effect, and as can be seen, the pK charge on oxygenation is in the wrong direction. However, it is clear that SKI, from the scant: available data, does meet the required condition, Kb > A. Never- theless, if a crevice is formed, it is a weak one with KCr ~ 3; this is readily calculated from the change in pK since K~/Ka—K<.r. This particular ion- ization is the one responsible for the reverse Bohr effect in acid solutions, and as yet there is no good evidence for a similar group in myoglobin.4:3 The other criterion can be illustrated by considering the pK values~for myoglobin derivatives in Table VII. Although Kit, ~ Kit, there is a different pK value for each complex, which thus again rules out the possibility of a crevice mechanism. TABLE VII ACIDITY OF THE IIEME LINKED IONIZATION FOR VARIOUS MYOGLOBIN DERIVATIVES4:] pie: 1'(°~) Acidic Ferrimyoglobin 6.1 20 Ferrimyoglobin Fluoride 6.5 20 F`errimyoglobin Cyanide 7.1 20 lTerromyoglobin 6.6 30 Oxymyoglobin 6.3 27 The One-Equivalent 7.5 20 Higher Oxidation State of Ferrimyoglobin* * The most satisfactory structure to account for the chemical reactions of this compound is that of a complex; "ferry!" ion.

CREVICE CONFIGURATION GEORGE AND LYSTER 45 The criteria discussed above are of a general nature, and cannot alone lead to any identification of the chemical groups involved. For this, accurate thermodynamic data would be very helpful. Nevertheless a difficulty arises in the wide variability in the hemoglobin from different species, the vari- ability being manifested not only in the aidinitv for oven, but also in the -- -d ~ ~ D--- magnitude and pH range of the Bohr effect.45~40 To -~ BIAS Air Ai ~ ;~^ . . . . . . . 11 LllC ~l ~1 Ly ~1 1~VUl~lUl~ oxygenation requires a bond from the iron to a particular group, such as imi- dazole, then the variability suggests that the configuration of adjacent parts of the protein has a considerable influence on its reactions. In this case, the thermodynamic functions for its ionization will not be the same for the group in free solution, and it 'will be necessary to call upon other criteria for its . . ,~ . 1C .elltl~CatlOIl. Conclusions. At present, x-ray analysis, amino-acid sequence studies, and direct physical and chemical methods for determining protein structure have not yet progressed far enough to establish how the heme is bound to globin. Hence this can only be discussed in the light of the very extensive but more indirect chemical and physicochemical evidence. Nevertheless, the discussion of such evidence has a value in itself. In a more general manner it prepares the way for the further problems that will arise when the chemical structure is fully known, namely the correlation between chemical reactivity and specific structural elements. The crevice configuration that has been examined is just such a structural element that could have a profound influence on the reactivity of the iron. On the other hand, it is quite possible that a crevice, if it were a shallow one, would have no effect on the reactivity, and so be indistinguishable in this respect from a surface configuration. Depending on the type of crevice, it is not entirely certain whether the direct methods could substantiate its presence or absence in aqueous solution. If the iron is joined to the protein by bonds on both sides of the heme, no doubt would arise. However, if there is only one Fe-protein bond and part of the protein is merely in the vicinity of the heme on the other side, then it is quite conceivable that the extent of this shielding could differ in the crystalline state and in aqueous solution. That some kind of conformational changes in the protein occur in solution is suggested by experiments of many lacings, and there is little doubt that, with accompanying changes in solution and neutral salt interaction, they are an extremely important factor in de- termining the full biochemical reactivity of the heme. The precise nature, however, of such changes remains obscure. Unlike cytochrome c, where the hemochromogen-like spectrum of its ferrous form provides direct evidence for a crevice configuration,46 all the evidence with regard to hemoglobin is indirect. It is certain that a hemochromogen structure is not involved, and very unlikely that the iron is joined to the globin by bonds on both sides of the porphyrin ring. Whether or not part of

~6 PART I. STRUCTURE OF HEMOGLOBIN the protein shields the heme that is otherwise held by one iron-protein bond (leaving aside side-chain interactions) is more difficult to decide. The entree of large ligands might be sterically hindered: the evidence is somewhat con- flicting. There is little reason though to believe that the combination of small ligands is subject to any steric hindrance. This structure with one iron-protein bond is the most satisfactory, and although there is no unequivocal experi- mental evidence that the sixth coordination position is occupied by a water molecule in the absence of an added ligand, theoretical considerations support this as a very reasonable assumption. REFEREl\-i CES Hi. 1. 3. Altschul, A. M. and lIogness, T. R.: The hemoglobin-oxygen equilibrium, J. Biol. Chem. 129: 315, 1939. Scheler, W., and Jung, F`.: Hamoglobinstudien. IV. Uber die Temperaturabhang;ig- keit der Reaktionsgleichgewichte einiger Methamoglobinverbindungen, Biochem. Z., 325: 515, 1954. Havemann, R., and Hirse, H.: Dissoziation und Hydrolyse den Methamoglobin- rhodanids und Methamoglobincyanats, Z. Physikal. Chem., 204: 68, 1955. 4. George, P., and Hanania, G.: A spectrophotometric study of ionizations in met- haemoglobin. Biochem. J., 55: 236, 1953. 5. 6. 7. 8. Raiser, E. M., and Steinhardt, J.: Kinetic evidence on the mechanism of the acid denaturation of horse CO hemoglobin, J. Am. Chem. Soc., 73: 5568, 1951. S. Adler, A. D., and George, P.: Unpublished observations. 10. Pauling, L., and Coryell, C. D.: The magnetic properties and structure of hemo- globin' oxyhemoglobin, and carbonmonoxyhemoglobin, Proc. Nat. Acad. Sci., 22: 210, 1936. Orgel, L. E.: The en ects of crystal fields on the properties of transition-metal ions, J. Chem. Soc., 4756, 1952. 1~. Griffith, l. S.: On the stabilities of transition metal complexes, J. Inorg. and Nucl. Chem. 2: 1, 1956. 13. George, P.: Crystal field theory of complex formation in ions in the first transition Series-- Experimental thermodynamic data, Rec. Trav. Chim. des Pays-gas, 75: 131, 1956. 14. Haurowitz, F`.: Ober Globin und seine hamaffine Gruppe, Z. Physiol. Chem., 232: 146, 1935. 15. V. Zeynek, R.: Nowing Lekarske Poznan, 3S: 10, 1926. 16. Haurowitz, F`.: The Bond between Haem and Globin, in Haemoglobin: a sym- posium based on the Barcroft Memorial Conference, Butterworths, London, 1949. 17. Haurowitz, F.: Hemoglobin, anhydrohemoglobin, and oxyhemoglobin, J. Biol. Chem., 193: 443, 1951. 18. Keilin, D., and Hartree, E. F`.: Effect of drying upon the absorption spectra of haernoglobin and its derivatives, Nature, 170: 161, 1952. 1C'. Keilin, D., and Hartree, E. F.: Effect of low temperature on the absorption spectra of haemoproteins, Nature, 161: 254, 1949. 20. George, P., and Hanania, G.: The ionization of acidic metmyoglobin, Biochem. J., 65: 756, 1957. George, P., and O'Hagan, J.: Unpublished observations. Davenport, H. E.: Reductive cleavage of cytochrome c, Nature, 169: 75, 1952. Lewis, U. J.: Acid cleavage of heme proteins, J. Biol. Chem. 206: 109, 1954. -

CREVICE CONFIGURATION GEORGE AND LYSTER 47 21. 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. Keilin, D.: Position of haems in the haemoglobin molecule, Nature, 171: 922, 1953. Of myoglobin and the 22. 23. Kendrew, J. C., and Parrish, R. G.: Imidazole complexes position of the haem group, Nature, 175: 206, 1955. 24. George, P., and Irvine, D. H.: Electron-transfer reactions between metallic ions, co-ordination complexes, and haemoglobin, J. Chem. Soc., 587, 1954. 25. George, P.: The chemical nature of the second hydrogen peroxide compound formed by cytochrome c peroxidase and horseradish peroxidase, Biochem. J. 54: 267, 1953. . Lein, A., and Pauling, L.: The combining power of myoglobin for alkyl iso- cyanides and the structure of the myoglobin molecule, Proc. Natl. Acad. Sci., 42: 51, 1956. Bjerrum, J.: Metal ammine formation in aqueous solutions, P. Haase, Copenhagen, 1941. 28. Bruehlman, R. J., and Verhoek, F. H.: The basic strengths of amines as measured by the stabilities of their complexes with silver ions, J. Am. Chem. Soc., 70: 1401, 1948. 29. Eyfe, W. S.: Complex-ion formation. Part III. The entropies of reaction of the silver and hydrogen ions with some aliphatic amines, J. Chem. Soc. 1347, 1955. 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., Series B. 144: 29, 1955. 31. George, P.: Discussions of the Faraday Society, 20: 291, The physical chemist.y of enzymes, 1955. 32. Roughton, F~. J. W., and George, P.: Discussions of the Faraday Society, 20: 288, The physical chemistry of enzymes, 1955. 33. Conant, J. B.: The oxidation of hemoglobin and other respiratory pigments, The Harvey Lectures, 28: 159, 1933. 34. Wyman, J.: The heat of oxygenation of hemoglobin, J. Biol. Chem., 127: 581, 1939. 3 5. 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. 36. Wyman, J., and Ingalls, E. N.: Interrelationships in the reactions of horse hemo- globin, J. Biol. Chem., 139: 877, 1941. Paul, NV., and Roughton, F. J. W.: The equilibrium between oxygen and sheep haemoglobin at very low percentage saturations, J. Physiol., 111: 23, 1951. Gibson, Q. H., and Roughton, F. J. W.: The determination of the velocity con- stants of the four successive reactions of carbon monoxide with sheep haemo- globin, Proc. Roy. Soc., Series B. 146: 206, 1957. Corwin, A. lI., and Reyes, Z.: Preparation and properties of imidazole ferro- and ferriprotoporphyrin complexes, J. Am. Chem. Soc., 78: 2437, 1956. 40. George, P., and Hanania, G.: Thermodynamic data for myoglobin, haemoglobin and cytochrome c reactions, and the position of the haem groups, Nature, 175: 1034, 1955. 41. Anson, M. L., and Mirsky, A. E.: The equilibrium between active native trypsin and inactive denatured trypsin, J. Gen. Physiol., 17: 393, 1934. George, P., and Hanania, G.: Effect of haem-linked and other ionizations on haemoprotein reactions, Nature, 174: 33, 1954 43. George, P., and Hanania, G.: The haem-linked ionizing group in myoglobin, Dis- 37.

48 PART I. STRUCTURE OF HEMOGLOBIN cussions of the Faraday Society, 20: 216, The physical chemistry of enzymes, 1955. 44. George, P., and Irvine, D. H.: A possible structure for the higher oxidation state of metmyoglobin, Biochem. J., 60: 596, 1955. 45. Riggs, A.: The metamorphosis of hemoglobin in the bullfrog, J. Ger,. Physiol., ]5: 23, 1951. 46. Wald, G., and Riggs, A.: The hemoglobin of the sea lamprey, petromyzon mari- nus, J. Gen. Physiol., 35: 45, 1951. 47. Keilin, D.: On cytochrome, a respiratory pigment, common to animals, yeast, and higher plants, Proc. Roy. Soc., Series B. 98: 312, 1925. DISCUSSION Dr. Felix Horowitz: There are two points on which I would like to comment. One of these is the crevice problem and the other the problem of imidazole residues as points of attachment of the heme groups. I think that we can state one thing definitely, namely that the hemes must form some bridges between the globin moieties. This is clear from the follow- ing simple experiment. The initial interfacial tension between p-xylene and an aqueous solution free of hemoglobin is 35 dynes/cm. As increasing quantities of hemoglobin are added this tension decreases to a minimum of about 24 dynes/cm., corresponding to a drop of 11-12 dynes/cm. Globin, in contrast to all other proteins, lowers the interfacial tension to about 12 dynes/cm, at pH 9. Evidently, the drop in interfacial tension is twice as high as that caused by hemoglobin.) On adding further amounts of globin, the interfacial tension remains unchanged at 12 dynes/cm. How- ever, if one equivalent hemin is added to this solution, the interfacial tension increases again to 24 dynes/cm. There are two interpretations which come to our mind. One of these is that globin has only one half of the molecular weight of hemoglobin and that, therefore, twice as many particles are present in the interface, resulting in high interfacial pressure and low interfacial tension. Another interpretation is that heme is bound to a lipophilic group of the globin molecule so that the affinity of globin to the organic solvent, p-xylene, is higher than that of hemo- globin. Both interpretations may be right. When hemin is added to the globin film, it seems to form bridges between globin molecules, thus causing the formation of the larger hemoglobin molecules; consequently both the number of molecules in the interfacial film and the interfacial pressure decrease. I conclude from these observations that the hemes form bridges between the globin moieties. However, I do not know whether the hemes are buried in crevices. They may be bound in such a fashion that there is ample space for the iron atoms to combine with other ligands. Ir1 a hemoglobin article written a few years ago~ I discussed the problem of the hemaffinic (heme-linked) groups and stated that imidazole was not very probable as a ligand. I am glad that Dr. George has also some doubts in this

DISCUSSION 49 respect. The principal reason for the general belief that imidazole groups are involved, is the high histidine content of mammalian globins. However, the Robins of invertebrates are very poor in histidine. My belief that imidazole groups are not involved in binding heme is based on the great stability of many hemoglobins over the whole range from pH 4 to pH 12. The imidazole groups undergo a change from the cationic to the uncharged form in this plI range. The great lability of all hemoglobin at pH values of less than 3.5 suggests rather an acidic hemaffinic group which, at lout pH values, gains a proton and, thereby, loses its negative charge. REFEREN CES 1. Haurowitz, F., Boucher, P., Dicks, M., and Therriault, D.: Interfacial pressure of proteins, Arch. Biochem.~ Biophys. 59: 52, 1955. 2. Haurowitz, F`., and Hardin, R. L.: Respiratory proteins, in Neurath-Bailey, Pro- teins, Vol. II, A, 279, 1954. Dr. Jacinto Steinharclt: This is a comment on Dr. Hauro~vitz's remarks on the surface effects of globin and their reversal on the addition of heme. His point would be more convincing if globin had a quarter of a molecular v~eight of hemoglobin instead of half, because the molecular weight of hemo- globin on acid denaturation under the mildest conditions (pH 2.8 at 0°) appears to be reduced to a quarter. Since the globin itself has a molecular weight of half in the region of its stability, there is something else holding the pieces together besides heme. This is a very minor point and it does not exclude the possibility that there are heme bridges in the higher structure. Incidentally, the evidence for the molecular weight being reduced to a quarter its normal value is not absolutely conclusive. The evidence is merely that the kinetics of the regeneration of denatured hemoglobin to native hemo- globin is very accurately second order, i.e., the native molecule must have a higher molecular weight than the denatured molecule. However, at the pH ot regeneration native ferrihemoglobin has half the molecular weight it shows at pH 7. Of course, a second-order rate process does not unambiguously refer tc an association reaction, but it usually does for reactions in solution. Dr. Edsall: May I ask if there are any direct measurements by ultracen- trifuge or other determinations about the molecular weight of the acid material by which one could show directly that it was a quarter ? Dr. Steinhardt: I have not at present the facilities to make such measure- ments. They s~rould not be easy to make or to interpret. The interpretation of sedimentation measurements made in such acid solutions with a molecule as small as this would be a very troublesome one. The dissociation into half molecules at pH 5 has been observed at Upsala bsT Gralen.

X-RAY ANALYSIS OF HAEMOGLOB1N ANN F. CULLIS, H. M. DINTZIS AND M. F. PERUTZ* 1. PHYSICAL PRINCIPLES We are trying to determine the structure of haemoglobin by direct x-ray analysis, using isomorphous substitution with heavy metals. So far this has given us a picture of the electron density distribution in the haemoglobi~ ' molecule in projection on a plane, at a resolution of about 3 A. This picture can be proved to be correct, but on ac- count of the great thickness of the molecule and the con- sequent overlapping of its components in proj ection we have been unable to recognize in it any polypeptide chains or haem groups. The only definite information obtained so far concerns the shape of the molecule, and the positions of the sulfhydryl groups. Before describing the results we should like to outline some of the physical principles involved in the C-ray anal- ysis. These are best illustrated by analogy with optical diffraction, using a device invented by Braggi and per- fected by Hanson, Lipson and Taylor.' As figure 1 shows, this consists essentially of a light source, a pinhole, two plane-convex lenses and a microscope. A picture of a simple molecule, e.g. hexamethylbenzene, is stamped out on a mask of black paper, each atom being represented by a circular hole. The mask is placed between the two plane-convex lenses and its Fraunhofer diffraction pattern observed through the microscope. The pattern, illustrated in figure 2a, consists of bright patches of positive or neg- at~ve amplitude separated by nodes, where the amplitude passes through zero on changing sign. We must now consider the relation between the dif- \\ o---l l A —B ., D 1 it, FIG. 1. Optical device used for ob- serving diffraction patterns of simple molecules (Repro- duced from Han- son, Lipson and Taylor, Proc. Roy. Soc., Series A, 21S: 371, 1953 ) . fraction by a single molecule and that of a crystal in which many molecules are arranged in a regular array. As a first step we may examine the diffraction by two mole- cules of hexamethylbenzene placed side by side (fig. 3a). Interference between them causes the diffraction pattern of figure 2a to be divided by a series of vertical dark fringes whose spacing is inversely proportional to the distance between the molecules. Note that the d ffraction pattern of the pair of molecules exhibits bright patches only at positions where the pattern of the single molecules also shows a bright patch. * This paper was presented by Dr. Perutz. 50

X-RAY ANALYSIS CULLIS, DINTZIS AND PERUTZ 51 · - ~ ~ ;W, ·. ,,,.,,- ., ,,. , i,, ,¢..;, ,., ..~ . '"." '. ~'.'""'""""'' _ ' ~ ~..... _............................................ ,............................................ - . ~ I.... - ....'.. _ "~ ~ _ '"''""''""'""~ ~' '"~, ~. ............................................................. (~6 - ~: ~ ; ~,~ e : : ~ FIG. 2. Diffraction from single molecule of hexamethylbenzene without and w ith an extra atom added at the centre. The diffraction patterns shown in this figure, and in figures 3 and 4, were kindly provided by Professor H. Lipson at the University of Manchester. ...,.~................ ii~ .,,.,. ... i it_ ........................... ........................ .:.::::::: :.:::::::::: :: :::::::::: :::::: ::::: ::: :::: ::: .... :: : : . . :.:: .: By... ~ _L ........ - . ~_Z - : _ _]k - . ~ . ..: ~ ::.. }:: :: ~ i: :: ::.... ~ .::~ id: it: ~ :::: ~ ~ ~ :::: :: ~ ~ ~ ~ ~ : ~ ~ :~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ i: - - - : ~ ~ ~ :~ : ~:~ ~ ~ ~ ~ ::::: :: ~ ~ ~ ~ :::::: ~ ~ :~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Hi::: : :::::::: ::.: . :::: :: :: :: ;:;; ;;;;;;;;;;;;;;;;;. ;; ;;; ~ ::::::::::::::::::::~:::::: ~:: ~ ::::^ ... 2.'.'.2"""""'""""'"""''"""'."'."'.'.""".""""""""" "' '' ''"'I''''"'"' :' ' ~.'.'.~.'. ~t, f . ~.~.~.~.. ~ ~. ~.~.~. ~... - i:: ::: ::::::: ~ I ~ - L:: Id: :.: ::: ..... Em. a ..... - :: :: FIG. 3. Diffraction by two molecules of hexamethylbenzene. In other words the effect of the additional molecule is merely to modify the diffraction pattern by cutting it into strips. If four molecules are placed at the corners of a parallelogram the diffrac- tion pattern is divided by a second set of dark fringes cutting across the first one (fig. 4a). As a result the pattern is now divided into spots lying at the corners of a parallelogram outlined by the molecules. Note again that bright spots appear only in positions corresponding to bright patches in the diffraction pattern of the single molecule. If the four molecules are replaced by a large number in a regular t~vo-dimensional array, as in the pattern of phthalocyanin illustrated in figure 5, the spots become sharper and form a completely regular

52 PART I. STRUCTURE OF HEMOGLOBIN FIG. 4.- Diffraction by four molecules of hexamethylbenzene as arranged in the crystal. pattern. The distance between the spots is inversely proportional to the dis- tc~nce between the molecules, but their brightness depends only on the bright- r~ess of the corresponding region in the diffraction pattern of the single phthal- ocvanin molecule. J FIG. 5. Left: pattern representing the b projection of the phthalocyanin structure; right: optical diffraction given by it. (Reproduced from C. W. Bunn, Chemical Crys- tallography, Oxford University Press, 1945).

X-RAY ANALYSIS—CULLIS, DINI'ZIS \ND PERUTZ 53 In x-ray analysis one is faced with the task of reconstituting the unknown picture of a molecule from the x-ray diffraction pattern of a crystal. This can be done because diffraction is reversible. For instance, if a mask were prepared corresponding to the diffraction pattern of the hexamethylbenzene molecule (figure 2a), the patches of positive amplitude being left open while those of negative amplitude are covered with a phase plate giving half a wavelength retardation, and if this mask were now placed between the plane- conve~x lenses of the diffraction machine, then the superposition of the dif- fracted waves would produce an image of the hexamethylbenzene molecule at the focus of the microscope. Similarly the array of phthalocyanin mole- cules can be reconstituted by punching a mask with a regular array of holes of varying sizes, corresponding to the spots of varying brightness in figure 5, and leaving open the holes of positive amplitude, while covering those of negative amplitude with a phase plate. Thus the image of an object can be reconstituted from its diffraction pat- tern provided the sign of each diffracted spot is known. In an x-ray diffraction picture, however, such as that of haemoglobin show-e later in figure 8, only the intensities of the spots can be measured, while their signs are unknown. The difficulty of x-ray analysis consists in the determination of the unknown signs or phases. One well-known method of sign determination depends on the use of two kinds of crystal, identical in all respects, except that in one crystal each molecule contains a heavy atom in a position occupied by a light atom in the other. The effect of such an ison~orphous substitution upon the diffraction pattern may again be explained with the help of an optical analogue. Suppose for the sake of argument that an additional atom could be put at the centre of the hexamethylbenzene molecule, as in figure 2b. Due to its central position, this atom makes a positive contribution over the whole area of the diffraction pattern, thus increasing the amplitude of the positive patches and diminishing that of the negative ones, as is evident from a com- parison of figures 2a and 2b. By changing their brightness the additional atom thus enables us to recognize the signs of the different patches, and to gather the information needed for the reconstitution of the image from the diffraction pattern. The effect of the additional atoms on the diffraction pat- terns by two and by four molecules is shown in figures 3b and 4b. In each case it enables us to~ find the signs of the different patches or spots, simply by observing whether their brightness goes up or down. It will also be noted that the positions of spots of positive or negative amplitude in the diffraction pattern from two or more parallel molecules coincide with those of the corresponding positive or negative patches in the diffraction pattern of the single molecule. This is the physical basis underlying the x-ray analysis of organic compounds by isomorphous substitution. In actual x-ray analysis the reconstitution of the image from its diffraction pattern is done by calculation rather than optically, but the principle is the

54 PART I. STRUCTURE OF HEMOGLOBIN 1 1 1 1 1 1 1 1 o o 5 A FIG. 6.—Fourier projection of sodium penicillin (Reproduced from Crowfoot e' al., The Chemistry of Penicillin, pp. 310-367, Princeton University Press, 1949). same. The result of the image reconstruction is expressed in the form of a contour map showing the molecule in projection on a plane. The contours are drawn at levels of equal electron density. The maps are called Fourier projections, because Fourier series are used in their computation. As an example figure 6 shows a Fourier projection of penicillin.3 Some atoms are clearly resolved and appear as individual peaks, awhile others overlap in pro- jection, especially in the p-lactam ring. A projection of a structure on a plane, such as figure 6, is calculated from only part of the diffraction pattern of a crystal, namely the x-ray reflexions from lattice planes lying normal to the plane of projection. Overlapping of atoms can be avoided and complete resolution achieved by extending the x-ray analysis to the entire diffraction pattern from the crystal, which may be visualized as a three-dimensional network of spots of varying brightness. At this stage, however, the analysis of a complex enantiomorphous structure becomes exceedingly difficult, because the amplitudes of the general spots are no longer restricted to plus or minus. Instead there is associated with each

X-RAY ANALYSIS CULLIS, DINTZIS AND PERUTZ 55 FIG. 7. Electron density distribution along a series of parallel sections through the penicillin molecule (Reproduced from Crowfoot et al., The Chemistry of Penicillin, pp. 310-367, Princeton University- Press, 1949 ) . spot a phase angle which may have any value between 0° and 360°, and reconstitution of the image is possible only by determining the precise values of all the phase angles. This is the situation at present confronting us in the >;-ray analysis of proteins. There is a way of determining these phase angles by direct x-ray analysis, but it is a difficult one, especially because of the exacting demands it makes upon the ingenuity of the chemists assisting the C-ray crystallographer. In- stead of a single isomorphous pair, it is necessary to have a series of at least three, and if possible more, isomorphous compounds, each having a heavy atom attached to a different site on the protein molecule. By measuring the amount by which the intensity of each diffraction spot changes in each of these isomorphous substitutions, the value of the phase angle can be deter- mined, and the electron density of the molecule can then be calculated in three dimensions. As an example of the advantages to be gained by three-dimensional crystal structure analysis, as compared to two-dimensional prod ections, figure 7 shows the results of such an analysis in penicillin. The electron density has been calculated along a series of parallel sections through the penicillin mole-

56 PART I. SlRUCTURE OF HEMOGLOBIN `:ule, rather like the sections the histologist cuts through the organ of an animal. For purposes of illustrations the sections are all superimposed and drawn together on one plane. They show the atoms of penicillin lying at different levels above the plane of projection clearly resolved, thus allowing the investigator to build a three-dimensional model of the molecule. The building of such a model is the ultimate aim of the protein crystallog- rapher. In principle it is possible to attain it, provided a series of isomorphous heavy atom compounds can be made. In practice it will be difficult, at any rate with a protein as big as haemoglobin, to obtain a resolution comparable with that of figure 7. The best that can be hoped for is a picture allowing one to recognize the configuration and lay-out of the polypeptide chains, together with the positions of the haem groups and of the more prominent side-chains If the amino-acid sequence of haemoglobin w ere to become known from chemical analysis, it should then be possible to build a fairly accurate model of the haemoglobin molecule. 2. APPLICATION TO HORSE METH.\EMOGLOBIN Our work has been done on horse, rather than human, haemoglobin, be- cause it leas the more favourable crystal form, all the molecules in the crystal being arranged in parallel. We have used methaemoglobin on account of its stability. Crystals of horse methaemoglobin are monoclinic, with two mole- cules in ~ face-centred unit cell, each lying on an axis of dyed symmetry. Ire such a structure there is only one projection, along the dyed axis, in which the phase angles of the diffracted rays are restricted to 0 or ~ (plus or minus). The first attempt at solving this projection of haemoglobin by iso- morphous substitution with heavy atoms was made by Green, Ingram and Perutz.4 They prepared compounds in which two of the sulphydryl groups of haemoglobin were combined with parachloromercuribenzoate or with silver ions. By comparing the intensities of the diffracted rays from the substituted and the unsubstituted haemoglobin, they w ere able to determine the signs of 87 out of 94 reflexions from lattice planes of spacings greater than 6.5 A (fig. 8~. From this and other, supplementary, information Bragg and Perutz' it. ~~ ..: I, I,. . .~.~...~. ~ A... ~~ ~.~,,,~,,,,' ~ .. , ,. ~'~.x'.: I.... ~ . ..... .~ . - . . I FIG. 8.—X-ray photograph of hOI re- flections of horse methaemoglobin extend- ing to a spacing of 6.5 A.

X-RAY ANALYSIS CULLIS, DINTZIS AND PERUTZ 57 were then able to calculate the first electron density projections of haemo- glol~in. Two kinds of projection were obtained. One was calculated from the intensity changes produced in the diffraction pattern when salt solution re- placed water as the suspension medium of the crystals and shows a contour map of the volume of hydrated protein into which salt cannot penetrate (fig. 9~. The contour levels represent thickness of the hydrated protein in projection. This picture has a very low resolution, because only the lowest order reflexions are affected by salt, but it gives a fairly accurate picture of the external shape of the molecule. This is seen to be an ellipsoid of dimen- sions 55 x 55 .x 70 9~. There is a depression at the centre corresponding to a dimple or a pair of dimples at the surface of the molecule. Act;. 9. (left) Volume of hydrated protein molecule into which salt cannot penetrate, . . as seen In projection along the dyed axis. The dyed is marked at the centre of the molecule. The broken part of the contours is extrapolated. (Reproduced from Bragg and Perutz, Proc. Roy. Soc., Series A, 225: 315, 1954). FIG. 10. (right) Electron density distribution in a single haemoglobin molecule sus- pended in salt-free water, seen in projection along the dyed axis. The contours near the right and left hand edges of the picture may be distorted due to overlapping of neighbouring molecules in the crystal. Note the difference in scale between this figure and figure 6. The other picture shows the electron density distribution within ~ haemo- globin molecule suspended in saltfree water (fig. 10~. It contains a flat area at the centre, corresponding to the central depression of figure 9, and a com- plex system of peaks and depressions which have so far defied any attempt at interpretation. The enigmatic appearance of the projection is due to two causes. First, the overlapping of a large thickness of matter in projection, and secondly, insufficient resolution. At the next stage of the analysis we have therefore attempted both to improve the resolution and to prepare some more heavy atom complexes in preparation for a three-dimensional analysis of the structure. Native horse haemoglobin has four available -SH groups arranged in two pairs.6 These combine with four silver ions or with two moles of parachloro- mercuribenzoate or other mercurials. If the -SH groups of haemoglobin are

58 PART I. STRUCTURE OF HEMOGLOBIN blocked by iodoacetamide, another mercury-birding site is uncovered form- irlg a complex with mercuriacetate. So far three complexes have proved useful for x-rav analysis: J I. Methaemoglobin + 2 moles of paramercuribenzoate 2. NIethaemoglobin + 2 moles of Hg~CHCOO- (dimercuriacetate) 3. Blocked methaemoglobin + 2 moles of Hg(CH3COO-~2 (mer- curidiacetate ~ ~ The first and most crucial task in the actual analysis consists in finding the positions of the heavy atoms, for on their correct location depends the subsequent calculation of the signs or phase angles of the x-ray reflexions. In our work on haemoglobin, the heavy atoms were found by a method which is largely automatic and free from assumptions about the nature of the pro- tein molecule or the number of heavy atoms attached to it. For each x-ray re- flexion the intensity charge produced by the heavy atom is measured. All the changes together are then put through a mathematical process of image reconstitution in which all the signs are taken as positive, and the result is plotted ire the form of a contour map (fig. 11~. This map shows a high peak at the origin together with a system of smaller peaks distributed around it. If the substitution with the heavy metal is truly isomorphous, and if it has attached itself to one unique kind of site, then the map shows either one peak or a small number of well-defined peaks which are high compared to the general fluctuations of the background. The line connecting the peaks to the origin correspond to the vector joining the different heavy atoms on the protein molecule. In figure 10 there is just one such peak, corresponding to the vector between the two mercury atoms in PCMB-haemoglobin. Fig- ure 12 shows the positions of the mercury atoms in the three different deriv- atives as determined by the method j ust outlined. One group of mercury , . / .. ~ . . , . . ~ c , · · . , ., ~ . · , / · . . / ., , , , . , FIG. 11. Difference Patterson pro- jection of me rcu ribenzo ate ha emoglob- in, showing the vector between the two mercury atoms attached to the haemo- globin molecule.

X-RAY ANALYSIS CULLIS, DINTZIS \ND PERUTZ 59 / \/ , c / ~~ / ~ / A / ~ /C O I I t I I 19 1 1 1~ 1 1 1~ 1 1 14 1 ~ A 7 ~ H9(OOCCH;2 \/ .-HclCHCOO- / \ °-~sCbH.coo / FIG. 12.—Positions of mercury atoms / in different haemoglobin derivatives. atoms clusters around the two pairs of sulphydryl groups, while the others. representing the mercuriacetate on blocked haemoglobin, are combined with a site whose chemical nature is still unknown. The positions of the mercury atoms bound to the sulphydryl groups show that these groups are spaced 30 A apart in the haemoglobin molecule. The positions of the heavy atoms having been found, the way was now opened for the calculation of signs. In the first round Green, Ingram and Perutz had confined themselves to 94 reflexions, extending to a spacing of 6.5 A. In the second round eve have tried to find the signs of the 470 re- flexions in figure 13, extending to a spacing of 2.8A. We have so far suc- ceeded in determining the signs of 422 of these. Our new electron density projection is thus calculated from 4.5 times as many reflexions as the original one of Bragg and Perutz and shows corres- pondingly greater detail (fig. 14~. For the sake of clarity the contours of the high density regions or peaks, and those of the low density regions or de- pressions have been drawn on separate diagrams. They show the density distribution within the area of the unit cell covered by one haemoglobin molecule suspended in ~ M ammonium sulphate solution. The contours neat the periphery of the molecule are not exact, being partly modified by the overlapping of neighbouring; molecules in the crystal lattice. The map con- ...... ~ ......... ,i .... ~0. · t ~ ; ~ ... .... ~ ~ ~ . my. ~ : 2' ,' ' :':': A: :~ : : : : : : :~: k :~ : c::: : : ,,,,,, ,,,.,,,,,,,.,, ,. , . , . . ~ . . .,,. ,. ,. ,, ~ . ,, I, . FIG. 13. X-ray photograph of hOI reflexions of horse methaemoglobin ex- tending to a spacing of 2.8 A.

60 PART I. STRUCTURE OF HEMOGLOBIN (a) <~—1 in. 9, I i, 9,,,, I,,,, I,,, I I ~ A FIG. 14. Elects on density distribution in the haemoglobin molecule seen in pro jec- tion along the dyed axis at a resolution of 2.8 A. The black dots in (a) represent the >;ulphydryl groups. (a) shows the contours of high electron density or peaks, (b) shows the contours of low density or depressions. Contours are drawn at intervals of 2.5 electrons/^', the base line being the density of 2 M ammonium sulphate solution, marked B. The lowest contour in figure 14a corresponds to 5 electrons/ above the level of 2 M ammonium sulphate solution. firms the existence of Ha flat region at the centre of the molecule where the density is hardly higher than in the surrounding salt solution. It shows, in addition, ~ striking system of peaks and depressions which appear as enig- matic as ever. Presumably the peaks represent projected positions of chains crossing over, or portions of chains running normal to the plane of projection, but clearly the projection contains too little information as yet to give any clue to the structure of the molecule. This was disappointing, but not un- expected, in view of the great thickness of matter projected on one plane. It is evident that the riddle cannot be solved without a solution of the crystal structure in three dimensions, giving us the density distribution in the molecule along a series of sections. This will have to be the next step. Acknowledgment: The authors wish to thank Professor H. Lipson of the University of Manchester for his kindness in preparing the optical diffraction patterns shown in figures 2, 3 and 4. REFEREN CES 1. Bragg, W. L.: A new type of x-ray microscope, Nature, 143: 678, 1939. 2. Hanson, A. W., Lipson, H., and Taylor, C. A.: The application of the principles of physical optics to crystal-structure determinations, Proc. Roy. Soc. (London), Series A, 218: 371, 1953. 3. Crowfoot, D., Bunn, C. W., Rogers-Low, B. W., and Turner-Jones, A., in "The Chemistry of Penicillin" pp. 310-367, Princeton University Press, 1949. 4. Green, D. W., Ingram, V. M., and Perutz, M. F.: The structure of haemoglobin IV. Sign determination by the isomorphous replacement method, Proc. Roy. Soc., (London), Series A, 225: 287, 1954. 5. Bragrg, Sir Lawrence and Perutz, M. F.: The structure of haemoglobin VI. Fourier projections on the 010 Plane, Proc. Roy. Soc. (London), Series A, 225: 325, 1954. 6. Ingram, V. M.: Sulphydryl groups in haemoglobin, Biochem. J. 59: 653, 1955.

DISCUSSION DISCUSSION 61 Dr. Reinhold Benesch: I would like to draw attention to some work which we have done which had as its aim the de nova introduction of an -SH group into proteins through a peptide bond. Partly we had in mind the application to x-ray work since the -SH groups introduced de novo in this way could be transformed into mercury derivatives and the protein examined in this form. The compounds which we selected for this purpose are homocysteine thio- lactones, which would react with protein amino groups according to the fol- lowing scheme ~ Benesch, R. and Benesch, R. E.: I. Am. Chem. Soc. 78: 1597, 1956): S CH., ~ H., CH:3-CO-NH-CH-CO + PrNH~ ~ SH CH CH., ClI3-CO-NH-CH-CO-NH-Pr Dr. David B. Smith: Regarding the number of subunits in hemoglobin, I would like to bring to your attention some results from our laboratoryi~~~3 on horse globin. Horse globin at pH 2 and ionic strength 0.05 separates into a material whose weight-average molecular weight is 21-22,000 and whose number-average molecular weight is 17,000. These results are interpreted a. indicating four subunits with partial aggregation to give the higher weight- average molecular weight. Molecular weights were measured by osmometry, light scattering and sedimentation using Archibald's method. Incidentally, the effect of pH 2 and ionic strength 0.05 on sedimentation was checked in two ways. The sedimentation rate of ribonuclease in this medium was the same as at neutrality. The molecular weight of lysozyme by Archibald's method was about 14,000 in agreement with results obtained at neutrality and higher ionic strength. Under conditions where the molecular weight of globin has its minimum value, that is pH 2 and ionic strength 0.05, the electrophoretic pattern is at its simplest and shows two components. Extrapolation to allow for the dis- torting effect of the extreme conditions on the relative areas of the peaks indicates that the two components are present in equal amounts. We obtained small amounts of each component from the ends of the electrophoresis appa- ratus and investigated their properties separately. The faster-moving component at pH 2 and ionic strength 0.05 had a weight- average molecular weight of about 2S,000 and a number-average value of about 17,000. Any increase in pH or ionic strength resulted in association. The slower-moving component had a ~veight-average molecular weight of about 17,000 and alterations in the medium had no effect on this value. The

62 PART I. STRUCTURE OF HEMOGLOBIN behavior of unfractionated globin is in some respects intermediate between that of these two components. We have made some investigations by Edman's methods on the amino acid sequence at the N-terminal ends of the separated components. Both compo- nents, of course, have N-terminal valine. The second amino acid residue of the faster-moving component is glutamic acid. The slower component has principally leucine in the second position; slight contamination with glutamic acid is ascribed to the difficulty of obtaining the slow component free from the faster in the descending limb of the electrophoresis cell. In conclusion, it appears that horse globin can be readily split into four subunits, all of similar molecular weight and divided equally between two types. REFEREN CES 1. Reichmann, M. E.. and Colvin, J. R.: The number of subunits in the molecule of horse hemoglobin, Can. J. Chem. 34: 411, 1956. 2. Haug, A., and Smith, D. B.: Separation, molecular weight and interactions of horse globin components, Can. l. Chem., 35: 945, 1957. 3. Smith, D. B., Haug, A., and Wilson, S.: Physical and chemical studies on horse globin components, Federation Proceedings 16: 766, 1957. Dr. V. M. Ingram: Have you any information on human globin? Dr. D. B. Smith: No. Dr. b~al~er Hughes: I would like to report an observation which may be important relative to heme-heme interaction. In searching for gentle methods or removing heme from hemoglobin, I observed that approximately half of the heme may be extracted from precipitated carbonmonoxy hemoglobin by acetone containing small amounts of pyridine and water. The resulting product appears very "native." It shows two peaks in the ultracentrifuge suggesting partial dissociation into 34,000 M.W. units. I have not been able to remove the remaining heme except by more rigorous conditions with concomitant de- naturation. Myoglobin under these conditions releases no heme. If all of the hemes are equivalent in hemoglobin, this finding must also be interpreted through heme interaction, here of a negative (repulsive) nature. Lewist has published a similar finding in the acid denaturation of carbonmonoxy hemo- globin. However, he found the removal of only the first heme to be easier than the rest. REFEREE CE 1. Lewis, U. J.: The acid cleavage of hemoglobin, J. Biol. Chem. 206: 109, 1954. Dr. M. T. Perutz: May I make a short point? I should like to remind you of a result of Kendrew and Parrishi which has some bearing on the crevice theory of iron attached in myoglobin. They prepared the 1-methyl and 4- methyl imidazole derivatives of myoglobin, which thus have large groups attached to the iron atom. They crystallized those compounds and took x-ray

DISCUSSION 63 pictures. In taco species of myoglobin the imidazole group produced no change in the unit cell dimensions of the crystal. If there were the kind of crevice where the molecule is forced apart, as it were, through the insertion of a group like propyl isocyanide, then this ought to have the effect of making the molecule somewhat bigger and enlarging the unit cell. This, as I say, was not observed. In a third species crystallization in the usual crystal form was inhibited and replaced by another. Kendrew and Parrish conclude that the heme is most likely to be on the surface of the myoglobin molecule, the imi- dazole group finding space in the interstices between neighboring molecules in the crystal lattice. REFEREN CE 1. Kendrew, J. C. and Parrish, R. G.: Imidazole complexes of myoglobin and the position of the haem group, Nature (Lond.) 175: 206, 1955. Dr. Davidson: Dr. Perutz, would you not expect something like PC~B to change the size of a hemoglobin molecule? Dr. Edsall: You mean whether there is or is not a crevice so that merely tacking on a group as large as that to a hemoglobin will alter its dimension? Dr. Perufs: In the picture vou saw here the distance between the -SH groups was 30 Angstroms. This picture does not tell you whether the -SlI groups are on the surface or within the molecule. However, further results have now been obtained by Dr. David Green at the Royal Institution in London, which seem to show that the -SH group is about 7 Angstroms in- side the external boundary of the molecule. In other words, there must be a sort of crevice or canal where the -SH group is located, so that the PCMB does not make the molecule any bigger. I should like to mention a paper published by Drs. David Ingram. Gibson and myselt,l concerning the orientation of the neme groups. We measured the paramagnetic resonance or electron spin resonance, as it is sometimes called, of the iron atoms in single crystals of horse me/hemoglobin. We got a very beautiful and sharp anisotropic effect with the help of which it was possible to determine the angular orientation of the heme groups with a very high accuracy indeed. The accompanying figure ~ shows the hemoglobin molecule, egg shaped, 55 Angstroms wide, 55 Angstroms thick and 70 Angstroms long, lying on an axis of dyed symmetry (the lo-axis). The heme groups are arranged in two pairs, related by the dyed axis. One pair of heme normals lie in the a,b-plane of the crystal, while the other pair is tilted by 13° above and below that plane. I should like to stress that this result tells nothing about the position of the heme groups. As figure 2 shows, they can be anywhere you like, except that they must be related in pairs by the two-fold axis of symmetry. In order to find their position, we made the para-iodo-nitroso-benzene com- plex of hemoglobin, hoping that the iodines would label the iron atoms and

64 PART I. STRUCTURE OF HEMOGLOBIN /c g ~~ ~' ~ / ~,'~ dead _ ~emoglo~ /~N ~ ·Fe ~ F[G. 2. Three of the many possible arrangements of the four heme groups in the hemoglobin molecule, repre- sented diagrammatically as a spheroid. In each pair of drawings the left- hand spheroid shows the molecule in projection normal to the dyed axis, and the right^hand one shows the same arrangement seen along the dyed axis. The small black points in ( c) represent the positions of the -SH groups deduced from the x-ray anal- ysis. (From Nature 178: 908, 1956.) FIG. 1.—Perspective drawing of the orientation of the heme groups with respect to the crystal axes and the hemoglobin molecule. ( a ) and ( b ) show the two pairs of heme groups related to the dyed axis; (c) shows the external shape of the molecule, as determined by }3ragg and Perutz,0 drawn on a much smaller scale than the heme groups. (From Nature 178: 907, 1956.) a) \ a_ IT' Ah /~ ': ~ a a a ' 1 ~ /'' (C) in\—i~ ~ THAI

DISCUSSION help us to find their positions in an electron density map. Unfortunately, the results obtained so far have been inconclusive. REFERENCE 65 1. Ingram, D. J. E., Gibson, J. F., and Perutz, M. F.: Electron spin resonance in myoglobin and haemoglobin. Orientation of the four haem groups in haemo- globin, Nature (Lond.) 178: 905, 1956.

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