Reactivities of the sulphydryl groups of dog hemoglobin.

Dog hemoglobin has four sulphydryl groups at positions olfl (G18) and p93 (F9), all of which are titratable with mercurials. Only two of these, however, react with non-mercurial sulphydryl reagents. Kinetic results indicate that the reacting site might be the p”” (F9). An examination of the environment of the alI1 (G18) shows that this sulphydryl must be unreactive towards non-mercurials because of the presence near it of several interacting groups. These are the carboxyl group of Glu 27u, which is only 4.5 A away; the carbonyl of Val 1070; and the hydroxyl of Tyr 24a. There is also a strong interaction with the carboxyl of Glu 116~ which, though 12 A away, is separated from the (Y”’ (G18) not by water but by protein, a low dielectric constant medium. All these interactions would considerably raise the pK of the Cys llla thiol. Therefore reaction with non-mercurial sulphydryl reagents via nucleophilic attack by the thiol anion becomes impossible. The effect of inositol hexaphosphate on the kinetics of the sulphydryl group reaction was investigated. Inositol hexaphosphate slows down the reaction by a factor of three for a 10 M excess of inositol hexaphosphate per hemoglobin tetramer and makes about 25% of the sulphydryl contents of the 0’” and CX”’ sites unavailable for reaction by any sulphydryl reagent.