A New Procedure for Purification of Crotalase From Crotalus Adamanteus Venom

Abstract

A new procedure has been developed for large scale, rapid purification of crotalase, the thrombin-1ike enzyme from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). The three step procedure involves: (1) molecular sieve chromatography on Sephadex G-100 in 0.04 M Tris buffer containing 0.10 M sodium chloride, pH 7.1; (2) gradient elution from DEAE-cellulose with sodium acetate buffer, pH 7.0; and (3) affinity chromatography on p-aminobenzamidine Sepharose using a spacer of 6-aminohexanoic acid. Crotalase was eluted from the affinity resin by 0.05 M Tris buffer containing 0.10 M sodium chloride and 0.15 M benzamidine-hydrochloride, pH 9.0, after first washing with the Tris buffer containing 0.40 M sodium chloride. From the crude venom, pure enzyme was obtained with an overall recovery of 40-60% of clotting activity and a 90-100 fold increase in specific activity. Crotalase was shown to be pure by Polyacrylamide disk gel electrophoresis which gave one band. The molecular weight was estimated to be approximately 31,000 by gel filtration on a calibrated Sephadex G-100 column. Amino acid analysis was performed and the composition was shown to be very similar to that reported earlier (F.S. Markland and P.S. Damus, J. Biol. Chem. 246: 6460, 1971). Clotting activity of the enzyme was not inhibited by heparin, either with or without plasma, whereas, thrombin was rapidly inactivated by heparin in the presence of plasma. In conclusion, we have developed a rapid and reproducible procedure for isolation in high yield of large quantities of the thrombin-like enzyme from the venom of the eastern diamondback rattlesnake. Studies are continuing on the primary structure and possible clinical applications of this enzyme.