REACTIVITIES OF THE SULFHYDRYL-GROUPS OF DOG HEMOGLOBIN

Dog hemoglobin has four sulphydryl groups at positions α111 (G18) and β93 (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 β93 (F9). An examination of the environment of the α111 (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 27α, which is only 4.5 Ǎ away; the carbonyl of Val 107α; and the hydroxyl of Tyr 24α. There is also a strong interaction with the carboxyl of Glu 116α which, though 12 Å away, is separated from the α111 (G18) not by water but by protein, a low dielectric constant medium. All these interactions would considerably raise the pK of the Cys 111α 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 β93 and α111 sites unavailable for reaction by any sulphydryl reagent. © 1979.