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Human a,-antichymotrypsin reacts with bovine chymotrypsin to form an equimolar complex and this reaction is accompanied by the formation of a free, modified form of the inhibitor. Time-course studies, performed on mixtures containing an excess of native inhibitor and kept at 0°C or at 25 "C, show that the equimolar complex dissociates spontaneously ; this dissociation results in the release of inactive modified a,-antichymotrypsin and of some active enzyme, which is able to recycle with active inhibitor in excess. When all the native inhibitor is used up, the released active enzyme degrades the remaining intact complex into intermediate forms. At the endpoint of the reaction only inactive modified inhibitor and some active chymotrypsin remain. Immunochemical data indicate that, in the complex, a steric hindrance of the antigenic determinants of the inhibitor prevents the formation of the precipitate with specific antiserum. Inactive modified inhibitor, which has dissociated from the complex, has retained antigenic determinants of the native a,-antichymotrypsin. a,-Antichymotrypsin is probably one of the most specific human serum antiproteases since it is known specifically to control the activity of chymotrypsin-like proteases from phagocytic cells (neutrophils, basophils, tissue mast cells) [l]. a,-Antichymotrypsin forms an equimolar complex with human chymotrypsin-like enzymes such as leukocyte cathepsin G or pancreatic chymotrypsin [2,3].More recently [4] interactions of El-antichymotrypsin with leukocyte cathepsin G and bovine chymotrypsin were compared using circular dichroism spectroscopy and analytical electrophoresis. It was shown that, in both cases, formation of an equimolar complex occurred and that, moreover, these complexes dissociated within few days even if they were kept at 0°C.The reaction of human serum a,-antichymotrypsin with bovine chymotrypsin was studied as a model for the reaction between this inhibitor and chymotrypsin-like enzymes. After preliminary studies on mixtures containing different inhibitorto-enzyme molar ratios, this report is focused on the timecourse of formation and dissociation of the complex when the inhibitor is in excess over the enzyme. First we started from previous experiments in which the mixture was kept at 0 "C [4], then we performed the reaction at 25 "C to avoid a temperature difference between the incubation step and the analyses. Taking into account the fact that formation of such a complex was primarily an interaction between two proteins, we followed each of the two components during the reaction using some of their own characteristics : esterase activity visualization and spectrophotometric measurements for the enzyme and immunochemical analyses with specific antiserum for the inhibitor. Moreover, methods such as sodium dodecyl sulfate (SDS) and alkaline polyacrylamide gel electrophoreses were carried out to obtain information about all the components present in the reaction mixture.Abbreviatians. PMSF, phenylmethylsulfonyl fluoride; Suc-AlaAh-Pro-Phe-NA, s...

Incubation of human serum a,-antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. a , -Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor:enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when a,-antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1).Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equiinolar complex ( M , near 77000) and the release of a fragment migrating as a peptide of M , near 5000. Moreover a free proteolytically modified form of a,-antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when a,-antichymotrypsin reacts with porcine elaslase than with human elastase. As a consequence of our findings, the specificity of a,-antichymotrypsin, so far limited to the inhibition of chymotrypsin-like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work.

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