Release of a two-chain form of antithrombin from the antithrombin-thrombin complex.

Abstract

The stable, inactive complex between antithrombin and thrombin was prepared and subsequently purified free of reactants by a procedure which minimized secondary thrombin proteolysis. Dissociation of the complex was effected by hydroxylamine or ammonia in the presence or absence of sodium dodecyl sulphate, or by prolonged treatment at 100°C in the presence of dodecyl sulphate. All these procedures released from the complex a proteolytically modified, two‐chain form of antithrombin and not the intact protein. Dissociation in the absence of denaturant also released active thrombin. Control experiments indicated that the inhibitor was indeed released from the complex as the modified form of antithrombin rather than as intact antithrombin which was subsequently modified by the dissociation agents or by thrombin. The modified form of antithrombin dissociated from the complex could not be differentiated electrophoretically from a proteolytically modified, inactive form of antithrombin which has been shown previously to be produced free in solution during the reaction between antithrombin and thrombin. These observations suggest that limited proteolysis of antithrombin by thrombin may be involved in the formation of the inactive complex. The inhibitor may thus exist in the complex as a proteolytically modified, two‐chain species. However, a state in which the scissilc peptide bond of the inhibitor only forms a tetrahedral adduct with the active site serine of the enzyme, as shown by X‐ray crystallography in other protease‐protease inhibitor complexes, cannot be excluded.