The covalent nature of the human antithrombin III–thrombin bond

Abstract

Cleavage of the human antithrombin II[ATIII]-thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labeled ATIII. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulfate reveals 2 ATIII 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 MW that is .apprx. 4000 less than the MW of native ATIII. Treatment of the cleavage products of the complex with carboxypeptidas B yields 1 .mu.mol of arginine, a new C-terminal amino acid per .mu.mol of thrombin dissociated. During formation of the ATII-thrombin complex, the inhibitor is apparently cleaved at an arginine-X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulfide bridge(s).