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.
Highlights
Inactivation of thrombin with Na-tosyl lysyl chloromethyl ketone and Na-(p-nitrobenzyloxycarbonyl)arginyl chloromethyl ketone results in alkylation of nitrogen-3 of the active center histidine with loss of both clotting and esterase activities
B Chain 3H-TLCK Thrombin-The peptide (1.28 pmoles), ob- same amino acid composition as thrombin eluted subsequently tained as described in Fig. 6, was digested with 1.5 mg of pepsin with high specific activity (Table I) it was devoid of clotting at 25”in 3.5 ml of 50/, formic acid (24) for 22 hours
Titration of the active center of esterase thrombin action mixture was evaporated in a vacuum and the residue was with p-nitrophenyl p-guanidinobenzoate (14) gave from 40 to gel filtered on Sephades G-50 (Fig. 6)
Summary
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 tq 2500 NIH units per mg. Change resin was introduced by Rasmussen (6) This method has yielded highly active preparations of thrombin when scmpurified prothrombin has been used (7, 8). Chromatography on carboxylate resins since this had been the Inhibition of I’hrombin with 3H-TLCK--The JH-TLCK used method of choice in earlier work (6-8) It was eventually found had a specific activity of 8.75 x lo[5] dpm per pmole. B Chain 3H-TLCK Thrombin-The peptide (1.28 pmoles), ob- same amino acid composition as thrombin eluted subsequently tained as described, was digested with 1.5 mg of pepsin with high specific activity (Table I) it was devoid of clotting at 25”in 3.5 ml of 50/, formic acid (24) for 22 hours. Titration of the active center of esterase thrombin action mixture was evaporated in a vacuum and the residue was with p-nitrophenyl p-guanidinobenzoate (14) gave from 40 to gel filtered on Sephades G-50 (Fig. 6)
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