Kinetics of the Reaction of p-Chloromercuribenzoate with the Sulfhydryl Groups of Glutathione, 2-Mercaptoethanol, and Phosphorylase b

The reaction of p-chloromercuribenzoate (PCMB) with the sulfhydryl groups of phosphorylase b and the model compounds glutathione and 2-mercaptoethanol was followed spectrophotometrically on a stopped flow apparatus. Model sulfhydryl compounds, glutathione and 2-mercaptoethanol, under identical conditions of pH (6.8), buffer, and temperature (30°) reacted at a rate only slightly higher (6 × 106 M−1 s−1) than the fastest-reacting sulfhydryl groups of phosphorylase b (3 × 106 M−1 s−1). The sulfhydryl groups which are concerned with structure–activity relationships react at rates which are four to five orders of magnitude less than these.An analysis of the pH dependence of the rate of reaction of PCMB with the fastest-reacting groups of phosphorylase b and with 2-mercaptoethanol indicated that the ionized sulfhydryl group is reacting at a much faster rate than is the unionized group. The reaction of PCMB with the ionized sulfhydryl group is so fast (~ 106M−1 s−1) that it is presumably diffusion controlled, while the reaction with the unionized group, although three orders of magnitude slower, is still much greater than with those sulfhydryl groups concerned with structure and activity. Thus the reactivity of sulfhydryl groups of protein molecules is determined both by their pKa values and their accessibility to PCMB.