25 September 1953

Dear Peter:

I am writing to answer your question about the heme-heme interactions in hemoglobin, and also to say something about the work on globular proteins, now that Bragg, Perutz, and Kendrew have presented their work.

The work about heme-heme interactions in hemoglobin that I mentioned to you was done by Dr. Robert C. C. St. George, at my suggestion, and is published in Science, 114, 629 (1951). I am sending a reprint to you under separate cover. It has been reprinted together with our paper on sickle cell anemia - we decided to reproduce these two reprints, when they became exhausted.

The work on hemoglobin consisted in determining, by spectrophotometric techniques, the equilibrium constant between hemoglobin and three different alkyl isocyanides, ethyl isocyanide, isopropyl isocyanide, and tertiary butyl isocyanide. These molecules have the composition RNC. Their nature is such that the carbon atom has a strong tendency to form an additional bond, with use of its unshared pair of electrons, and in particular it can form a bond with the iron atom of a heme group, either a free ferroheme group or a heme group in hemoglobin. We found that heme alone combines with the isocyanides to form a compound with one isocyanide molecule and also a compound with two isocyanides molecules - the iron atom, which forms four bonds to four nitrogen atoms of the four pyrrole rings of the porphyrin in heme itself, these four bonds lying in the plane of the porphyrin molecule, can form two additional bonds, extending to either side of the plane, at right angles to it. If it forms one additional bond with isocyanide the compound of the first sort is obtained, and if it forms two bonds the compound of the second sort is obtained. We found that the combining powers of the three alkyl isocyanides with a free heme group were essentially the same, and concluded that the ability of the carbon atom of the isocyanide to combine with an iron atom was essentially independent of the nature of the attached alkyl group.

When, however, the combining powers of hemoglobin with the three isocyanides were determined it was found that tertiary butyl isocyanide combined only 1/200th as strongly as ethyl isocyanide, with isopropyl in between. We suggested as an explanation of this fact that steric hindrance was involved. It is known that the heme groups in the hemoglobin molecule are fastened on one side to the nitrogen atom of an imidazole ring of a histidine residue of the globin, and that the oxygen molecule or carbon monoxide molecule or, in this case, alkyl isocyanide molecule hooks onto the iron atom on the other side. Our conclusion that steric hindrance is operating in the combination of the alkyl isocyanides led us to conclude

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then that there is also protein on this side of the heme group, and accordingly that the four heme groups are buried within the hemoglobin molecule. In our paper a number of other arguments supporting this hypothesis are presented. Also, Jeffries Wyman made the same suggestion on the basis of some results that he had obtained on the heats of combination of hemoglobin with different substances.

Now I want to say something to you about the Cambridge work on the structure of crystalline globular proteins. It seems to me that this work is going along very well. I now understand what Perutz has been doing with his mercury atoms - he is not using the mercury atoms to determine the sines of his reflections in general, but rather only to answer the questions about sines that had remained nodes in the transform for the crystal by consideration of the dependence of observed intensities on position in the reciprocal lattice, and with use of data from a number of crystals containing different amounts of water. I think that the use of the two mercury atoms may well have added enough power to their efforts to determine sines to have permitted them to find nearly all of the sines, out to the spacing that they have gone to, for the h0l zone. Although there seem to be a few doubtful points still, probably the Fourier that they have obtained - not a very sharp one as yet - is pretty good, and it seems to me to be reasonably interesting. I think, however, that they will have a very great difficulty in determining signs farther out in the reciprocal lattice, although they may succeed. In any case, they will obtain only a projection - the determination of the phases for the layer-line reelections will be very much more difficult, essentially impossible. The problem of the structure of hemoglobin is going to have to be solved by use of other methods as well.

I am very pleased with the new results that Kendrew has obtained on the holomyoglobin. I think that his Pattersons provide very interesting information. He seems to be able to determine the approximate orientations of rods (presumably helixes) in several of the modifications that he is working with. This field of work seems to me to be well worthy while, and, of course, an effort should be made to make it still more productive.

I feel that with the situation as it is you might well be justified in concentrating on holomyoglobin - taking over some part of the whole problem, involving perhaps some new point of view - in working for your doctorate. I am not sure that this work would be as profitable as work on simple substances, but you have the advantage of a year's experience, and would accordingly be better off to continue for two years in this field than to shift to the field of simple crystals.

I feel, however, that you should not continue with myoglobin or some similar globular protein unless you plan to work very hard. It is evident that Kendrew must have been working extremely hard during the last year, to have obtained the results on which he reported.

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This letter, then, is being sent to you in order that I may convey to you the impression that I have formed about the globular protein work at Cambridge, as a result of the conference here. I feel not that the globular protein field might well be a satisfactory one for you, for your doctoral work, whereas during the summer I think that I was pessimistic about it.

Our conference has been going along very well. Our main difficulty is that nearly everyone had been hoping to talk about twice as long as he has been able to talk, because we are meeting only during five days, and there are many questions to be argued about.

With love,

[Linus Pauling]