The kinetics of the formation of the complex between bovine P-trypsin and the bovine basic pancreatic trypsin inhibitor (BPTI) was investigated using three different signals: the displacement of proflavine, the optical density changes in the UV region, and the loss of the enzymatic activity. For the three different signals, with inhibitor in excess over bovine /3-trypsin ([BPTI] > 5 x [bovine P-trypsin]), the time course of the reaction corresponds to a pseudo-first-order process. The concentration dependence of the rate is second order at low BPTI concentrations and tends to first order a t high inhibitor concentrations. This hehavior may he explained by relatively rapid preequilihria followed by limiting first-order processes according to E + IF+ (EI),i + (EI),?
K , k +I
The values of K,, k+,, and k(,,,, ( = k+,/K,) have been determined for the different reactions at three pH values: 6.80,4.80, and 3.50. The kinetic parameters differ widely for the processes reflected by the various signals; the difference increases upon lowering pH. The results indicate that the formation of the bovine P-trypsin-BPTI complex is not an all-or-nothing process, but involves several intermediates corresponding to discrete reaction steps, which are differently affected by ionization processes.