Abstract
1. Cleavage of the human antithrombin III--thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulphate reveals two antithrombin III bands that do not resolve during electrophoresis without reduction. The heavy band has the electrophoretic mobility of the native protein. The light band has an apparent mol.wt. that is approx. 4000 less than the molecular weight of native antithrombin III. 3. Treatment of the cleavage products of the complex with carboxypeptidase B yields 1 mumol of arginine, a new C-terminal amino acid, per mumol of thrombin dissociated. The results indicate that during formation of the antithrombin III--thrombin complex, the inhibitor is cleaved at an arginine--X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulphide bridge(s).