Reaction of Chloroacetamide with the Sulfhydryl Group of Papain

The kinetics of the reaction of the essential sulfhydryl group of papain with chloroacetamide was studied over a wide range of conditions. The variation of the second order rate constant (k2) with pH, at temperatures from 15.0–39.5°, is described by simple S-shaped curves at 0.50 ionic strength, characteristic for alkylation of a normal sulfhydryl anion. The k2-pH profiles are S-shaped at 0.07 ionic strength also (4.5–30.5°), although the values of k2 at low pH values are higher than those expected for simple ionization curves; this increase is eliminated by raising the ionic strength from 0.07 to 0.50. The pH dependence for the rates of inhibition are described by formulations relating the apparent rate constants with the states of ionization of the sulfhydryl group and another pH-dependent event. On the basis of these formulations, pK' values and absolute rate constants were obtained which appear to be intrinsic parameters characteristic for the sulfhydryl group. Thermodynamic and activation constants described by these sulfhydryl-specific parameters were also calculated.