Purification of two thrombin-like enzymes from the venom of Agkistrodon caliginosus (kankoku-mamushi).

Two thrombin-like enzymes were purified from the venom of Agkistrodon caliginosus. Both were homogeneous on polyacrylamide gel electrophoresis, hydrolyzed N-alpha-tosyl-L-arginine methylester and did not show any caseinolytic activity. the molecular weights of the enzymes were estimated to be about 34,000 - 37,000 and 42,000 - 43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel-filtration on Sephadex G-100 column. One enzymes was purified from the venom by gel-filtration on Sephadex G-100 column, CM-Sephadex C-50 and DEAE-Sephadex A-50 chromatographies, affinity chromatography on p-aminobenzamidine-epsilon-aminocaproic acid-Sepharose 4B and gel-filtration on Sephadex G-100 column. The second enzyme was separated from the first enzyme by the DEAE-Sephadex A-50 chromatography mentioned in the above procedure and was isolated by affinity chromatography on arginine-Sepharose 4B and gel-filtration on Sephadex G-100 column. From 4 g of the venom, 0.88 mg of the first enzyme and 1.18 mg of the second enzyme were obtained, with specific activities of 118 and 139 NIH units per mg of protein, respectively.