Kinetics of the Reaction of the "Masked" and "Free" Sulfhydryl Groups of Human Hemoglobin with p-Mercuribenzoate

Abstract

The rates of reaction of the "free" (&3) and "masked" (alo and &) sulfhydryl groups of liganded human hemoglobin with p-mercuribenzoate (PMB) have been studied under a variety of conditions.The presence of PMB on /393 promotes the dissociation of hemoglobin into dimers and the subsequent reaction of the masked sulfhydryls promotes further dissociation into single chains.Decreasing pH and increasing NaCl concentration have opposite effects on the reaction of the masked and free groups.When PMB is in excess, the apparent first order rate constant for the reaction of the masked -SH groups is proportional to the PMB concentration, as would be expected for a simple bimolecular reaction; on the other hand, at sufficiently low values of the hemoglobin concentration, it increases with a decrease of hemoglobin concentration.This suggests that the rate of reaction of the masked -SH groups is controlled by a dissociation process beyond the dimer stage.Human hemoglobin (cu&) contains six sulfhydryl groups of which four, namely those at alo4 and &z, are unreactive (or "masked") in relation to a variety of specific reagents, such as p-mercuribenzoate, and two, namely those at ,&, are reactive (or "free").The reaction of the masked -SH groups with PMBl has acquired special importance as it promotes extensive dissociation of the hemoglobin molecule into its constituent OL and p chains and can be used for their isolation (1) ; hence, a study of the mechanism of the PMB reaction should provide a better understanding of chain to chain interactions in the molecule.The effect of several variables on the dissociation into chains produced by PMB has already been investigated by Bucci et al.(2) and by Rosemeyer and Huehns (3).The latter authors,