Interaction of chymotrypsinogens with .alpha.1-protease inhibitor

Abstract

It has been demonstrated that human proelastase 2 and .alpha.1-protease inhibitor react slowly to form a complex that is stable to denaturation with sodium dodecyl sulfate [SDS] and .beta.-mercaptoethanol and that the zymogen can be recovered from the isolated complex following dissociation by hydroxylamine. Bovine chymotrypsinogen A was reacted with human .alpha.1-protease inhibitor in a very similar manner. The rate of complex formation was measured by 2 methods. In the 1st, the reaction was followed by determining the loss of the inhibitory activity of .alpha.1-protease inhibitor as a function of time. A 2nd-order rate constant for complex formation (pH 7.6, 37.degree. C) of 12.9 .+-. 2.4 M-1 s-1 was obtained. In the 2nd procedure the reaction of fluorescein isothiocyanate labeled chymotrypsinogen A with .alpha.1-protease inhibitor was measured by fluorescence polarization. A 2nd-order rate constant (pH 7.6, 37.degree. C) of 13.9 .+-. 2.1 M-1 s-1 was obtained. The rate of complex formation is .apprx. 10-5 of that measured for the reaction of bovine chymotrypsin with .alpha.1-protease inhibitor. Dissociation of the complex was not observed after dilution or the addition of excess bovine .alpha.-chymotrypsin. As judged by SDS-polyacrylamide gel electrophoresis experiments, human chymotrypsinogens I and II react with .alpha.1-protease inhibitor at rates that are approximately equivalent to that determined for bovine chymotrypsinogen A. Bovine trypsinogen reacts very slowly with .alpha.1-protease inhibitor, at a rate that is at most 10-2 of that of bovine chymotrypsinogen A. Zymogens react with .alpha.1-protease inhibitor by virtue of partially formed active sites, and the potential active-site specificity of the zymogen partly determines the rate of complex formation.