AIR MAIL

Balliol College

Oxford, England

Feb. 18, 1948

Dr. Robert B. Corey

Crellin Laboratory

California Institute of Technology

Pasadena 4, California

Dear Bob:

I am glad to have your letter, and to learn how you are getting along.

Has there been anything done about cooperation with Palmer? I am beginning to feel a bit uncomfortable about the English competition. They have a gift for driving straight at the heart of a problem, and getting its solution by hook or crook.

I enclose a copy of a letter to Carl Niemann, about an address that Chibnall gave here last night. They have not succeeded in crystallizing the quarter-molecules, with molecular weight 2,500 or 3,500, but they may succeed, and if they so they will [sic] the crystals to Mrs. Hodgkin. I think that the whole problem is so important that it would be worthwhile to tackle it independently in Pasadena, and see what progress we could make. Would you talk with Carl, and consider the question of how much effort should be put into an immediate attack along pretty much the same lines as those used by Chibnall. You know that the matter of crystallization of a new compound depends so much on chance that, despite the head start that Chibnall has, we might well succeed in obtaining the first crystals. Moreover, the structure determination of peptides with 26 or thereabout amino residues is such a difficult job that I think we could consider it the next step on the journey toward the complete solution of the crystal structure of a protein, a step that sooner or later we shall have to take.

-2-

Dr. Corey Feb. 18, 1948

My proposal is that Carl have a man or two begin work at once on the degradation of insulin, and that as fast as reasonably pure materials are made they be turned over to you, for crystallization and x-ray investigation.

The progress that has been made seems to me to be truly astounding. I judge that paper chromatography has been very largely responsible for it.

I am continuing to get along very well—perhaps being kept a little too busy, with so many extra lectures to deliver. However, I feel that when there is so much interest in what I have to say it is proper that I make the effort to say it.

Cordially yours,

Linus Pauling:par

enclosure

I have written to Corey that I thought it important to make an immediate and vigorous attack on the insulin problem, even though it involves duplication of work being done by Chibnall and his collaborators. Would you go to see him, and talk over the problem of what might be done at once. Chibnall spoke here before the Alembic Society last night. I judge that a good bit of what he said has been published already. The amino acid analysis of insulin indicates very clearly that there is a molecule of sub-molecule with molecular weight 12,000. The free amino groups have been determined by a method (due, I believe, to Saenger) involving treatment at about pH 7 with dinitrofluorobenzene, which couples dinitrobenzene groups onto the free amino groups, without changing the molecule otherwise. Then the molecule is hydrolyzed and analyzed by paper chromatography, the bright color of the dinitrobenzene group permitting easy identification of the substituted amino acid. It is found that the two lysine residues present in the molecule have their epsilon amino groups free, because these are coupled with the reagent. Also there are two glycine molecules that turn up as coupled derivates, and also two phenylalanine derivatives. It is accordingly concluded that there are four polypeptide chains in the molecule of molecular weight 12,000 (which contains 106 amino residues), two of these chains having glycine residues at their free amino ends, and the other two having phenylalanine residues at the free amino end.

There are six cystine residues in the molecule, and the four polypeptide chains are presumably held together by these sulfur-sulfur bonds of the cystine residues. These can be reduced, but the more effective method of destroying the bonds

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is oxidation with performic acid, also carried out by Saenger. The oxidation produces sulfonic acid groups in place of the sulfide bonds, the residues then being cysteic acid. An ultracentrifuge study has been made by Gutfreund and Ogsten here at Oxford, which is reported to show that the boundary just barely moves, indicating molecular weight slightly less than 5,000. The molecular weight of four equal residues from the 12,000 molecule would be 3,000, and later evidence indicates that the two chains containing glycine at the end have molecular weight 2500 and the other two have molecular weight 3,500.

Saenger has separated the two kinds of chains. He has obtained a 30% yield - that is, 30% of the original protein, which would be about 75% yield - of the glycine-end polypeptide. He has carried out an amino-acid analysis, and has found that most of the simple amino acids are in this molecule, whereas the complicated ones are in the other polypeptide chains, with molecular weight 3,500. Each of the polypeptides seems to contain about 26 residues, the difference in molecular weight resulting from the difference in complexity. He has begun the analysis of the glycine-ended polypeptide by coupling it with the dinitrofluorobenzene reagent and then hydrolyzing, and then separating the various peptides by paper chromatography. He can identify the peptides that come from the end of the group where the glycine residue was by their color. He finds that the dipeptide is the substituted glycine attached to isoleucine. The tripeptide is the substituted glycine attached to isoleucine and valine, and the tetrapeptide is similar but with glutamic acid also. A pentapeptide has also been investigated. Thus it is found that in this peptide containing 26 amino acid residues the first four are, in this order, glycine, isoleucine, valine, glutamic acid, and the fifth one is believed to be serine.

It is clear that there is already considerable progress made on the job of a complete structure determination of insulin. However, there

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is still a very great deal of work that remains to be done, and I do not think that it is assured that the British school will finish the job. I believe that this is the problem that we should begin to work on, with as much vigor as possible, under our insulin project. I would accordingly suggest, and I ask you to talk the matter over with Corey, that the thing to do is to get insulin, and to begin its degradation by essentially the same methods as those used by Chibnall, with such variations as seem reasonable to us. In particular, I think that the preparation of the 26-peptides should be carried out in quantity, and that the two (presumably two) kinds of molecules should be isolated in as pure form as possible. I suggest that the men working under you on the insulin project do this, and that the material obtained be turned over to Corey for crystallization. Chibnall has not succeeded in crystallizing either of the 26-peptides. It might be a very big job to do this crystallization, and I think that a specialist should have the job, namely Dr. Schroeder or some similar man. The effort should be made to grow crystals of many different possible kinds, in the hope that one derivative of the 26-peptide would crystallize, and in a suitable form for x-ray examination. It might not be necessary to determine by chemical methods the complete order of residues in the chain, because the x-ray structure determination should provide this information.

I would think that you might want to try to split the 26-peptides into half-size molecules by enzymatic methods. If these were made and crystallized their x-ray investigation would be, of course, much easier than that of the 26-peptides.

With best regards, I am

Sincerely yours,

Linus Pauling:par

cc: R. B. Corey