The Purification of Thrombin and Isolation of a Peptide Containing the Active Center Histidine

Abstract

A chromatographic purification of bovine thrombin from commercial starting material is described which yields preparations judged to be essentially pure, that is, with a clotting activity of 2100 to 2500 NIH units per mg. In addition to the two-chain form of thrombin, the purified enzyme contains a three-chain form arising from cleavage of the B chain at arginine-73 (or 76). This form must be fully active since the specific clotting activity of various preparations does not vary with the content of the three-chain form, amounting at times to 70%. Inactivation of thrombin with Nα-tosyl lysyl chloromethyl ketone and Nα-(p-nitrobenzyloxycarbonyl)arginyl chloromethyl ketone results in alkylation of nitrogen-3 of the active center histidine with loss of both clotting and esterase activities. A radioactive peptide has been isolated from thrombin inactivated with tritiated Nα-tosyl lysyl chloromethyl ketone and shown to contain histidine-43.