Differential Pulse Polarographic Determination of the First and Second Dissociation Constants of Dimeric Subtilisin BPN′-Streptomyces Subtilisin Inhibitor Complex

Abstract

A differential pulse polarographic method based on the Brdi?ka current (polarographic catalytic hydrogen evolution current produced by proteins in the presence of the cobalt ion) was applied to the direct titration of subtilisin BPN′ (S.BPN′) with Streptomyces subtilisin inhibitor (SSI) at a concentration level of 10^-9m. The first and second dissociation constants of the dimeric S.BPN′-SSI complex were determined by fitting theoretical curves to the titration data, in which the multiple equilibrium involving microscopically distinct forms of the S.BPN′-SSI complex was taken into account. The intrinsic free energy change in the first binding of S.BPN′ to dimeric SSI was larger than that of the second binding.