The First and Second Dissociation Constants of Subtilisin BPN-Plasminostreptin Complex Determined by a Polarographic Method, and the Effects of pH on Them

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A polarographic method based on the Brdicka current (the polarographic catalytic hydrogen evolution current produced by proteins in the presence of cobalt salts) was applied to direct titration of subtilisin BP1ST (S.BPINT) with plasminostreptin (PS) at a concentration level of 10-8 m.The first and second dissociation constants of the S.BPN -PS complex were determined by fitting theoretical curves to the titration data, in which the multiple equilibria involving microscopically distinct forms of S.BPN -PS complex were taken into account. The intrinsic free energy change in the first binding of S.BPN to dimeric PS was larger than that in the second binding. The dependence of the microscopic dissociation constants of S.BPN-PS complex on pH indicates the participation of ionizable groups of pKa 8.0 and 9.4 in the complex formation. The repulsive effect between negatively charged molecules of S.BPN and PS in the complex formation at elevated pH is suggested.
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