Formation and Dissociation of the Covalent Complexes Between Trypsin and Two Homologous Inhibitors, α₁‐Antitrypsin and Antithrombin III

The mechanisms by which alpha 1-antitrypsin and antithrombin III inhibit trypsin were investigated by chemical stability studies and amino acid sequence analyses of the enzyme-inhibitor complexes. One-to-one covalently linked complexes were formed between each of the inhibitors and trypsin. The complexes were stable over the course of the experiments at pH 8 or lower, and benzamidine, hydroxylamine, thiols, guanidine, or dodecyl sulfate had no apparent effect on the stability of the complexes. The complexes dissociated slowly at pH 9 or greater. Neither of the inhibitors was active after dissociation from its complex with trypsin. Sequence analysis indicated that no new amino terminus was generated when alpha 1-antitrypsin formed its complex with trypsin, but that two new amino termini were formed in approximately one-to-one ratio when the complex dissociated. This may indicate that alpha 1-antitrypsin contains two inhibitory sites for trypsin in close spatial proximity on the molecule.