The Oxygen Equilibrium of Cystine-treated Human Hemoglobin without Free Sulfhydryl Groups.

In recent years there has been a good deal of speculation as to the possible role of sulfhydryl groups in determining the oxygen equilibrium of hemoglobin, especially the Bohr effect and the sigmoid character of the equilibrium curve. It is now known that human hemoglobin A contains six sulfhydryl groups (l-3), one in each of the LY chains and two in each of the /3 chains (3), but that only two of these are reactive (2, 4). The reactive -SH groups have been located in the /3 chains (5) belonging to residues 93 (6). Recent x-ray studies have shown that the reactive -SH group of each p chain stands next to the histidine residue which is coordinated with one side of the iron atom of the heme (7, 8). These -SH groups are thus in a favorable position to exert a strong influence on the oxygen equilibrium generally. Because the Bohr effect is considerably reduced when the protons of the reactive -SH groups are displaced by treatment with N-ethylmaleimide (5), it seems clear that SH groups can influence this property. Indeed, it has actually been suggested (9) that these -SH groups are themselves the very groups that are indirectly involved in the Bohr effect. But there are a number of compelling arguments against this view, some of which have recently been presented by Benesch and Benesch (10). These authors have ably discussed the Bohr effect and have offered their own suggestion as to its mechanism, which does not of itself involve sulfhydryl groups at all. When the free -SH groups of hemoglobin are titrated with p-mercuribenzoate or some other -SH reagents, there is a striking reduction in the coefficient n of the empirical Hill equation (11). This observation has been interpreted to mean that the reactive -SH groups are intimately involved in the hemeheme interactions which are invoked to account for the sigmoid character of the oxygen equilibrium curve (11). The present study deals with the effect on the oxygen equilibrium of treating hemoglobin with cystine so as to eliminate the free -SH groups. Definite evidence regarding the nature of the resulting compound is incomplete, but it seems probable that each of the reactive -SH groups has been converted into a