A thrombin-like enzyme from timber rattlesnake venom

Abstract

The procoagulant component has been purified from timber rattlesnake ( Crotalus horridus horridus) venom by DEAE-cellulose ion-exchange chromatography followed by affinity chromatography on immobilized p-aminobenzamidine and a final DEAE-Sepharose chromatography. As obtained, the procoagulant gave a single band of M r 29 500 ± 2000 on SDS-polyacrylamide gel electrophoresis whether or not the sample was reduced prior to electrophoresis. Schiff's stain indicated the presence of some carbohydrate. The procoagulant showed one predominant and four minor bands on discontinuous gel electrophoresis. All caused fibrinogen solutions to clot. Treatment of the preparation with neuraminidase did not cause the minor bands to comigrate with the major band. Amino acid analysis revealed the presence of eight half-cystines, all of which were present as cystines in the intact molecule. The procoagulant has 13 tryptophans per molecule and an extinction coefficient for a 1% solution at 280 nm of 26.3. This venom procoagulant was found to induce clotting by catalyzing the hydrolysis of only the A fibrinopeptide from the Aα-chain of fibrinogen. It was not inhibited by protein trypsin inhibitors, N-ethylmaleimide or dithiothreitol, but it was inactivated by phenylmethylsulfonyl fluoride, indicating an active-center serine. The procoagulant catalyzed the release of negligible acid-soluble peptides from bovine serum albumin, casein and hemoglobin.