14 September 1954

Dear Peter:

I shall pay the bill for the book when it arrives.

I am afraid that I do not have any very good suggestion to make about getting a heavy metal atom onto myoglobin or hemoglobin. It seems to me that it would be best if the atom were directly attached to the iron atom, and not dangling at the end of a chain somewhere. Our experience is that with hemoglobin a large molecule has difficulty in combining with the iron atom -- you remember that tertiary butyl isocyanide was found to combine with hemoglobin 200 times less strongly than ethyl isocyanide.

I was interested in making some alkyl isocyanides with an ionic group, such as a carboxyl group, attached to the side chain. Much to my surprise, the synthesis did not work out. I have a feeling that the ordinary isocyanide synthesis is sensitive to electronic interactions along the chain. I do not remember that anyone has ever made an isocyanide in which there is a heavy atom attached to the side chain. It might be possible to make an ethyl isocyanide with an iodine atom on the beta-carbon atom. On the other hand, I think that probably the treatment with alkali in the manufacture of the isocyanide would hydrolyze the carbon-iodine bond.

I have one suggestion to make. It seems to me possible that iodide ion would combine with ferrimyoglobin. You remember that hemoglobin combines preferentially with neutral molecules - oxygen, carbon monoxide, imidazole, the alkyl isocyanides. When the iron atom is oxidized to the tripositive state, hemoglobin combines with negative ions -- cyanide ion, fluoride ion, azide ion. Accordingly your best bet is to try to attach a negative ion to myoglobin. I think it is unlikely that iodide ion combines strongly, or I would remember that the compound has been made. Nevertheless, you might take some myoglobin, oxidize it to the ferric state (this can be done with ferricyanide ion), observe its spectrum, using a Hartridge reversion spectroscope, then add some potassium iodide solution, and see if the spectrum changes in the way that it does when you add cyanide ion. Roughton probably could lend you a Hartridge reversion spectroscope -- it is a little instrument in which there are two spectra, running in opposite directions. The wavelength of an absorption band or line is measured by moving the two spectra relative to one another until the line coincides in the two spectra, and then measuring the wavelength on a scale. Perhaps you have one in the unit.

I may say that it is still more likely that astatide ion would combine with ferrimyoglobin, but I do not know whether astatide ion can be obtained or not. Probably not.

I have just had another idea, although I hesitate somewhat to mention it to you. Ferrihemoglobin will combine with the hydrosulfide ion, HS^-. It is likely that it will combine with the hydroselenide ion, and also with the hydrotelluride ion. Accordingly, you might prepare some hydrogen telluride, H₂Te, by action of acid on a metal telluride, and run this gas into a solution of ferrihemoglobin -- or, better still, see if you can get some sodium telluride, Na₂Te, and add a small quantity of it to a solution of ferrihemoglobin. You must be very careful in working with hydrogen telluride or sodium telluride. Several chemists have been killed by it, because it is very poisonous. If I remember correctly, there was one such case in the chemistry department at Cambridge, about 50 years ago.

If this procedure works, and I think it likely that it will, you would have a tellurium atom attached directly to the iron atom of the heme group. The tellurium atom has one electron less than an iodine atom. It is accordingly far from being as strong a scatterer of x-rays as a mercury atom is, but it might be enough to do the job. Moreover, you can predict its distance from the iron atom, and you know that it will be out from the plane of the porphyrin ring. You might be able to orient the porphyrin ring by the optical properties -- except for azimuthal orientation around the iron-tellurium axis, which would have to be evaluated separately. If you had the coordinates of the tellurium atom and all of the atoms in the heme group, you would have a good start on the determination of phases.

I may say that I am not absolutely sure that the hydrogen telluride ion will combine with ferrimyoglobin. There is one argument against it, the fact that the electronegativity of tellurium is less than that of sulfur, and that the tellurium atom has accordingly a small tendency to form a bond with an iron atom. Nevertheless, I think feel pretty sure about making this prediction. You might want to try out hydrogen selenide first, perhaps determining the equilibrium constant of the HSe^- ion with ferrimyoglobin. You must be careful in working with selenium, also. It is not so poisonous as tellurium, although it is poisonous. On the other hand, most people who do any work with the element soon get a strong garlic breath, which causes their friends to be concerned.

I know that polonium can be obtained in at least microgram quantities, so that you might try the hydrogen polonide ion. Probably the difficulty of getting polonium is so great, however, as to make this not worth doing. Moreover, you would have trouble because of its fogging the photographic plate. I think that the tellurium idea is a good one, however.

I am interested to learn about the shrinkage states in the crystals of Finbak whale myoglobin. It is nice to have an orthorhombic crystal, but on the other hand four molecules are rather too much for the unit of structure -- I think that a monoclinic crystal with two molecules in the unit is the thing to look for, or even a triclinic crystal with one molecule in the unit. Dr. Corey has, I think, just about decided to give up on lysozyme as a crystal for an attempted complete structure determination, because of the four molecules in its unit (tetragonal).

I shall look forward to learning about any new results you obtain on the 1.5-A reflection.

I passed your comment about the rotating anode tube on to Holmes. He said that our rotating anode tube is pretty well along, and I went downstairs to look at it. Sheldon is installing the auxiliary apparatus for it now, and I hope that it will be operating in a few months.

The new laboratory is coming along in good shape. Concrete is being poured on the sub-basement walls and basement walls now, and on the columns along the center line of the building.

Give Millie Fowler my regards when you see him -- also Linda, when she gets back from France.

I am afraid that I cannot do anything about providing you with an index for your copy of Lucas. Professor Lucas left a couple of weeks ago -- he is going to teach organic chemistry full time in Columbus, Ohio, this year. It is unlikely that he would have an extra index, even though he might have saved page proofs of the book.

I am glad that you are taking a course on operation of the electronic computer. We have been having some trouble here with the electronic computer that was being used by our x-ray men - the Consolidated computer. The company now has enough business, I judge, to have caused them to break off the arrangement that had been made with us, before we were able to make the calculations that had been agreed upon. It seems possible, however, that we can arrange to use the Navy computer at UCLA. Moreover, it is not so much trouble to get to UCLA, now as it used to be. The freeway goes through Hollywood, and up into the San Fernando Valley. McCullough told me that he can get from the Institute to the point where he leaves the freeway -- probably Sunset Boulevard in Hollywood -- in the same length of time that it takes to get from there to UCLA -- altogether about 45 minutes from the Institute to UCLA, I think. I have not made the trip.

I have just been to Kalamazoo and Evanston, Illinois. I spent one day in Kalamazoo, visiting the Upjohn Research Laboratories. The Upjohn Company is an Industrial Associate of the Institute, and staff members are supposed to visit the Industrial Associates to give them some return for their $10,000 per year. I gave a talk on sickle cell anemia. At Evanston I attended a conference held by the Society for Engineering Education, and gave a talk on new concepts in chemistry. John Slater was there, and several other friends of mine. I was gone three days. Perhaps four days and three nights is more accurate.

I am still working hard on the revision of COLLEGE CHEMISTRY. I don't know just where I stand, because I have done some work on almost every chapter. Several people are reading the manuscript, and I have the job of going over it again, with their comments in mind.

Did I tell you that we have made some more dural models, in which the bond angles and bond distances are exactly right for an ordinary amide group? We have also made some in which the angles have the right values for a proline residue. I decided the other day that these models are with us to stay, and that we should order 10,000 Allen set screws, for $227, in order to get the reduction in price. This is enough for about 100 models, each with about 25 amino-acid residues in it.

If you do anything with tellurium, you probably should talk with Herman Emeleus first, and get advice from him. It is likely that he has some sodium telluride at hand. If so, there is no need to prepare the gas, hydrogen telluride -- all that you need to do is to add a milligram or two of the sodium telluride to your solution.

I may say that it might be well worth while, as a small extra investigation, to study the reaction of hydrogen selenide and hydrogen telluride (that is, of the hydrogen selenide ion and hydrogen telluride ion) with ferrihemoglobin and ferrimyoglobin, as a separate biochemical problem. These acids, H₂Se and H₂Te, are weak acids -- the first ionization constant of H₂Se is given in COLLEGE CHEMISTRY as 1.7 x 10^-4, and that of H₂Te as 2.3 x 10^-3. The second ionization constants are 1 x 10^-1 and 1 x 10^-5, respectively. These values mean that in the neutral range the solution that you would obtain by adding sodium selenide to the ferrihemoglobin solution would contain practically all of the selenium as HSe-, that is, in the form to combine with the ferrimyoglobin. The tellurium, however, would, if the value of the second ionization constant is correct, be largely in the form of Te^--. In order to convert it into HTe^- it would be necessary to acidify the solution somewhat, to about pH 5. I may say that it is quite possible that the ion Te^-- would also combine with ferrimyoglobin, so that you might have a compound at all pH values. You can follow the compound formation spectroscopically. It might be worth while for you to look through the literature, to see whether anybody has investigated possible compounds of ferrihemoglobin with the hydroselenide ion and the hydrotelluride ion, but no matter what you find, you should not be prevented from making your own investigation. See PPS

Do you know Joan Keilin? She published some work on compounds of isocyanides with heme, a few years ago, if I remember correctly. She might be willing to collaborate with you, if you wanted to get somebody to help you, in the investigation.

Crellin came back from Honolulu yesterday. He seems to have learned a good bit, in his work as an organic chemist. He leaves for Portland tomorrow. He is taking the little trunk, with a jog in it, which I bought before we went to Europe -- you remember we thought that it might have been a trunk on the back of an automobile. By the way, do you have two trunks with you in Cambridge? I tried to remember, and decided that you had taken the rawhide trunk for your books, and the big greenish trunk for your clothes. Is this right?

If you go to London sometime, you might be interested to hunt up Hermann Lehmann, pathologist in St. Bartholomew's Hospital. He spends his spare time traveling around in Africa, India, Arabia, the Andaman Islands, etc. testing blood samples for sickling. He visited us here last spring. Dr. Anthony Allison, of Christ Church and the Radcliffe Infirmary in Oxford, has just left, on his way home. He is another sickle-cell-anemia investigator -- the one who inoculated Africans with malaria parasites, and found that the sickle-cell-anemia hemoglobin provides protection against malaria. His wife is an American girl, interested in Egyptology, I believe.

I have not been able to do anything on collagen during the last week, but I hope to get some results before long.

Remember to be careful in working with tellurium. You should not have any trouble, because you need only a very small amount to combine with your heme groups.

Do you have any opportunity to speak in public -- that is, to talk before a group of people? You probably are going to follow an academic career, and it is important for you to be as good a speaker as possible. Practice counts for a lot. Are you a member of the Space Group, or does it not exist any more? I remember that I spoke before the Space Group in Cambridge in 1930 -- Bernal had organised it. The new group, in the Cavendish, probably is an outgrowth of the old one. If you could join some other group which has been formed for the purpose of holding seminars, I think that it would be a fine idea for you to do so.

Much love from

[Linus Pauling]

P.S. Have I told you that this summer I have been doing my work almost entirely in the new study, just west of the front porch. It is much cooler here than in my old study. In fact, I usually work in my old study for a little while in the morning, unless the morning is especially warm, and then I work in the new study in the afternoon. I have only been spending a couple of hours every day in the lab.

P.P.S. I am afraid that I have found a fatal flaw in the above suggestion about H₂Te. Hydrogen telluride and the hydrogen telluride ion are very strong reducing agents, their oxidation-reduction potentials being in the neighborhood of 1 volt. I do not remember the oxidation-reduction potential of the ferrihemoglobin-ferrohemoglobin couple, but I think it is a little above the ferricyanide-ferrocyanide value, about -0.3 volt. The difference of 1.3 volts is so great that there is, I think, no possibility whatever of adding hydrogen selenide to a ferrimyoglobin solution without getting reduction of the ferrimyoglobin to ferromyoglobin, and formation of metallic tellurium.

We might ask whether there is any chance that the hydrogen telluride ion or hydrogen telluride itself would combine with ferromyoglobin. I do not think that the ion would combine with ferromyoglobin -- the only negative ion that is known to combine with ferrohemoglobin is cyanide ion. Fred Stitt, when he was working here, discovered that ferrohemoglobin will combine with cyanide ion if the concentration is great, half saturation occurring at about 0.7 molal concentration of cyanide ion. Hence we are left with hydrogen telluride itself -- will it combine with ferromyoglobin? It might possibly be worth while to carry out the experiment, simply by reducing all free oxygen, say by the use of sodium dithionide in the usual way (atmospheric oxygen oxidizes hydrogen telluride), in the myoglobin solution, and then adding a bit of sodium telluride, if it is available. If there is a change in the spectrum, it would be worth while to try to crystallize the myoglobin-hydrogen telluride compound.

After thinking further about the matter, I have dug up another idea. It is seen that in general the singly-charged ions of weak acids combine with ferrihemoglobin. HF has acid constant 6.7 x 10^-4, H₂S has the constant 1.1 x 10^-7, HCN 4 x 10^-10, HN₃ 1.8 x 10^-5. In addition, we might include H₂0, with acid constant about 10^-16, because ferrihemoglobin in alkaline solution forms ferrihemoglobin hydroxide. I may mention that it seems likely that there is a water molecule attached to the iron atom in the ferrihemoglobin ion itself, and probably any ligand that attaches itself to the iron atom has to displace this water molecule.

It is true that not all weak acids combine with ferrihemoglobin. For example, acetic acid does not do so, nor do the other carboxylic acids. Their acid constants are about 10^-4. It may be that the strength of the bond that they can form with the iron atom is just not quite strong enough to do the job, but the example of ferrihemoglobin hydroxide [unreadable] an oxygen acid with acid constant small enough, approaching that of water, should work.

There is one obvious acid that you should try - this is telluric acid, H₆TeO₆, or Te(OH)₆. Its first acid constant is 1.6 x 10^-9. You might be able to get some telluric acid from Emeleus, but you could make it easily. I remember making telluric acid just thirty years ago, in order to study its crystal structure. If I remember correctly, it is made simply by dissolving tellurium in nitric acid. In checking on its combination with ferrimyoglobin you should work at moderately high pH, perhaps pH 8, or possibly even pH 9 -- I have forgotten when ferrihemoglobin is converted to ferrihemoglobin hydroxide, but I might as well check up on it. I find on referring to the paper on ferrihemoglobin that Stitt, Coryell, and I published in the J.A.C.S. in 1937 that the equilibrium constant for the formation of ferrihemoglobin hydroxide has p_k = 8.15. At pH 8.15 half of the ferrihemoglobin is in the form of the hydroxide. At this pH about 30 percent of the telluric acid is in the form of the single charged anion. You probably should work at about this pH, then, to see whether a ferrimyoglobin-tellurate is formed.

The experience with the alkyl isocyanides and hemoglobin suggests that the size of the tellurate ion might cut down its combination constant. At present there is no information as to whether this argument applies to myoglobin. I have a man here, Professor Lein from Northwestern University Medical School, who is, I think, working on this problem this year. At any rate, I have suggested that he do so. That is, he is going to investigate the combination of myoglobin with various alkyl isocyanides, in order to see whether or not the argument that St. George and I applied to hemoglobin applies to myoglobin also.

If we ask what other possibilities there are, we are left, I think, with only the acids of antimony as the answer. Antimonic acid, HSb(OH)₆, is a strong acid, and so is to be ruled out. Antimonous acid, H₃Sb0₃, is reported to have acid constant 10^-11; this is only a rough value. By reference to Latimer's book on oxidation potentials I find that the antimonite-antimonate potential is about -0.6 volt, and that accordingly antimonite ion could exist together with ferrimyoglobin. If tellurate ion does not combine with ferrimyoglobin, you probably should try antimonite ion.

Although lead and bismuth may form oxygen anions at high pH, it does not seem worth while to test them. The anions are, I think, not stable at pH below 10, and above pH 10 there would be strong competition with hydroxide ion, to say nothing about the denaturation of the protein.

To make Te(OH)₆

8 g Te dissolved in 50 ml HNO₃(conc) + 75 ml water. 5 g CrO₃ added. Evaporated on water bath to 15 ml. Residue washed 6 times with conc HNO₃. 100 ml of 50% HNO₃* added to dissolve TeO₃, and Te(OH)₆ crystallized out. Little octahedra. (A hydrate, monoclinic, may form.)

*Perhaps hot - my notes don't say