November 23, 1942

Dr. Hugo Theorell

Medicinska Nobelinstitutet

Biokemiska Avdelningen

Stockholm, Sweden

Dear Dr. Theorell:

Last week I presented to our chemistry seminar the new results contained in the papers on horse radish peroxidase which you sent to me. The chemists here were very much interested in your work.

While I was reading carefully through the papers preparatory to giving the seminar, I made some calculations about the possible explanation of the variation in the equilibrium constant for peroxidase fluoride as given in Figure 2 of your paper. The sequence of points in this figure suggested that the fluoride ion was replacing a hydroxide ion in peroxidase hydroxide. It is indeed found that a theoretical curve which passes through the points very nicely results from this assumption. The equilibrium constant K' for the postulated hydroxide complex is such as to give pK' a value about 5.2 or 5.3. The addition of a hydroxide to peroxidase in this region is apparently not accompanied by any observable change in either magnetic properties or absorption spectrum, since you did not observe a change. The calculation of the competition of fluoride and hydroxide for the peroxidase is very easily made, of course. It is found that the apparent equilibrium constant for the fluoride as given in your Figure 2 varies according to the equation

K_apparent = K 1 + (OH-)

K'

Here K' is the hydroxide equilibrium constant mentioned above, and K is the true equilibrium constant for the reaction peroxidase ion + fluoride ion to form peroxidase fluoride. The value of K is about antilog (-3.45).

It is mentioned in a paper by Dr. Coryell and me in the Journal of Biological Chemistry, 132, 769 (1940) that both hemoglobin and ferrihemoglobin have a heme-linked acid group with pK about 5.25. That for ferrohemoglobin is not effective magnetically, whereas that for ferrihemoglobin is, being responsible for the small change in the magnetic susceptibility in this region. Our evidence, however, did not indicate that this hydroxide competes with fluoride in ferri-hemoglobin; it is, on the other hand, the acid group with pK about 8.1 which competes with fluoride in ferrihemoglobin.

I do not have a clear picture of the structure of peroxidase and its hydroxide and fluoride complexes. If we assume that the 5.2 hydroxide occupies the sixth coordination position about the iron atom, and must be displaced in order for the fluoride to be formed, then the question arises as to the nature of the acid-base transition with pK 11 which is shown by your magnetic and spectroscopic measurements. Possibly this represents the replacement of a protein side chain group in the fifth coordination position about iron by hydroxide.

With best wishes for the continued success of your very interesting experiments, I am

Sincerely yours,