January 6, 1939

Dear Linus,

I would like to know your views on certain questions concerning protein denaturation.

(1) When a protein spreads to form a film it unfolds. At the same time it becomes insoluble and its SH groups become available. We would say that it is denatured. And yet it has certain important properties of the native protein. Gorter has studied the enzymatic activity of trypsin after spreading. The interpretation of his observations is, however, ambiguous. Langmuir's exp[eriments] on urease seem pretty clear. While spread out as a film, this protein acts as an enzyme. In this sense it is not denatured. We regard unfolding as taking place in denaturation and yet here is a case of unfolding without loss of a certain configuration. I feel the explanation of the apparent contradiction is as follows: when a protein spreads unfolding takes place in a very orderly manner. Langmuir came over here on day and demonstrated some of his exp[eriments]. As a protein spreads it forms certain visible patterns. The same protein always forms the same pattern; each protein forms a different and characteristic pattern. The whole film forms a continuum, bonds holding everything in one piece. When a protein unfolds at a surface it seems possible for it to do so without passing into a multitude of random configurations. The configuration of the protein in the film may in certain respects be very similar to that of the original native protein. It may therefore still act as an enzyme. At the same time it is insoluble and has its SH groups available. It has some of the properties of native protein and some properties of denatured. The controversy about whether or not it is denatured is in a certain sense surely verbal. What do you think of this?

(2) Another point about configuration and specific properties. When the layers of a protein fall apart from each other to assume random relations to each other, certain specific properties of proteins are lost - as mentioned in our paper. Other properties distinguishing one protein from another will remain. The amino acid contents of proteins vary and the sequence of amino acids in the chains vary from one protein to another. Even after unfolding these differences will persist and manifest themselves in certain properties. There is good evidence that antibodies are proteins. These proteins lose the power to act as antibodies on denaturation in the same way that Hb and the enzymes lose their properties on denaturation. Unfolding (with subsequent random arrangements) is enough to denature antibodies, Hb and enzymes. An antigen is a foreign protein which when injected into an animal gives rise to antibodies. There is some evidence (by no means conclusive) that when an antigenic protein is denatured in the same way as Hb, enzymes etc. are it loses some of its specific properties, but not all of them. It certainly is possible on our views that certain properties of a specific nature would remain after denaturation and there is experimental evidence that this is so. In a certain sense, denaturation will be complete only when the protein is finally resolved into its constituent amino acids. There do, however, appear to be certain definite steps in the loss of specific configurations and our paper was concerned with one of the first and apparently most definite steps.

(3) Have you seen the paper by LaMer in Science of Dec. 31st? Please let me know what you think of this. It seems to me that LaMer does not consider the view that denaturation takes place only after many bonds are broken - after a considerable number of steps (and one after the other) have been taken, so to speak. What do you think of Eyring's views? What about the meaning of S, the steric factor, in the velocity equation? What about the role of collisions in denaturation?

(4) I have made some progress in understanding the coagulation of myosin in muscle. You may remember I told you about comparing the effect of heat on the contraction of muscle (in which myosin coagulates) and on the coagulation of isolated myosin. Frog muscle is caused to contract by raising the temperature to 37 degrees and isolated frog myosin coagulates at 37 degrees. But the rate of this latter process is very slow. I now find that if myosin is treated by NH₄Cl or LiCl (and the excess or free salt carefully washed away) that the myosin has become sensitized. It is now exceedingly unstable. At 37 degrees it is coagulated with very great speed. Heretofore I have prepared myosin by using KCl. After treating myosin with LiCl or NH₄Cl the ppt. of myosin can be washed for at least 10 hours with constantly renewed dilute KCl and still this myosin appears sensitized when compared with myosin that has never been treated with anything but KCl. How much LiCl or NH₄Cl remains attached to the myosin I do not yet know. The instability of the myosin shows itself in other ways too. If ordinary (KCl) myosin is dried it also loses its solubility - it cannot be redissolved. If a surfusion of this myosin is frozen at -10 C it slowly in the course of weeks loses its solubility. It is presumably being dried by freezing away its water. Myosin that has been treated with NH₄Cl loses its solubility after it has been frozen for several hours. The possible bearings of all this on muscle - In the muscle fibre K is the common cation and it is located in the same place in the fibre as is myosin. During contraction K leaves the fibre, passing into the blood, and returning during rest. At the same time (approx) myosin becomes insoluble. Also at the same time the fibre becomes opaque. If gels (the myosin in both are still soluble) of myosin of the same concentration are compared (one of the gels having previously been treated by NH₄Cl) it is striking how much more opaque the NH₄Cl treated myosin is than ordinary KCl myosin. It looks as if K stabilizes myosin in the muscle fibre and that in activity NH₄ or something like it comes in contact with myosin and sensitizes it. All this is quite new and incomplete, but the observations I have just described to you are, I believe, probably sound. Perhaps this has been too incompletely described to be comprehensible.

If you still are in the East, it would be the greatest pleasure for me to see you again. I would be quite willing to travel a bit for a talk with you.

In the meantime, yours as ever,

Alfred Mirsky

O, by the way, I was talking with Gasser about you the other day. He referred to you as a "wizard." I hope you don't find being one a burden.