Multiple intermediates in the reaction of bovine β‐trypsin with bovine pancreatic trypsin inhibitor (kunitz)

The kinetics of the formation of the complex between bovine β‐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 β‐trypsin ([BPTI] ≥ 5 × [bovine β‐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 at high inhibitor concentrations. This behavior may be explained by relatively rapid preequilibria followed by limiting first‐order processes according to\documentclass{article}\pagestyle{empty}\begin{document}$$\hbox{E + I} \> \mathop{\rightleftharpoons}_{K_i} \> (\hbox{E\,I})_{i1} \> \mathop{\rightarrow}_{k_{+i}} \> (\hbox{E\,I})_{i2}$$\end{document} The values of K i , k + i , and k (on) i ( = k + i / K i ) 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 β‐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.