Purification and Properties of Prothrombin Activator from the Venom of Echis carinatus1

Abstract

A prothrombin activator from Echis carnatus venom was highly purified by gel-filtration on Sephadex G-150 followed by chromatogrphies on DEAE-Sephadex A-25 and DEAE-cellulose, gel-filtration on Sepharose 6B, and rechromatography on DEAE-cellulose. Through these procedures, about 2 mg of the purified material was obtained from one gram of the lyophilized venom and about 57-fold purification was acheived. The purified preparation was found to give a single band on polyacrylamide-gel disc electrophoresis in the presence and absence of sodium dodecyl sulfate (SDS). Moreover, no additional protein band was observed on the reduced SDS-gel, suggesting that the protein consists of a single polypeptide chain. The molecular weight of prothrombin activator was estimated to be approximately 56,000 by SDS-gel electrophoresis and by molecular sieving on Sepharose 6B in the presence of 6 M guanidine. The activatory appeared to be a glycoprotein, as judged by a positive periodic-acid-Schiff reaction against the protein band on SDS-gel from the amino acid analysis. The isoelectric point was determined to be 4.5 by isoelectric focusing. The purified activator showed strict specificity towards prothrombin and its derivative; it did not activate factor X, factor IX, prekallikrein,plasminogen, or trypinogen from a bovine source. The K_m values towards bovine prothrombin and prethrombin 1 were estimated to be 1.03×10⁻⁶ M and 1.05×10⁻⁶ M, respectively. Further, the activator did not hydrolyze any of the synthetic ester and anilide substrates widely used for α-thrombin, factor Xa, and kallikrein; neither caseinolytic nor fibronogenolytic activities were detected in it. Chelating agents including EDTA, o-phenanthroline, and cysteine strongly inhibited the enzyme activity, while dusopropyl phosphorofluoridate, anitithrombin III, and naturally occurring proteinase inhibitors, which inhibit serine proteinases, had no effect. These results indicate that the venom prothrombin activator is not a serine proteinase like coagulation factors found in mammalian plasma, but is one of the metal proteinases. Thus, the enzymatic properties differ remarkably from those of factor Xa.