Purification of haemorrhagic proteinase from the venom of Agkistrodon caliginosus (Kankoku-Mamushi).

A haemorrhagic proteinase was purified from A. caliginosus venom by ion-exchange chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50, S-Sepharose Fast Flow and Q-Sepharose Fast Flow, and gel-filtration on a Sephadex G-100 column. By this procedure, about 17.6 mg of the enzyme were obtained from 4 g of the venom. The enzyme showed a single band on polyacrylamide gel electrophoresis at pH 8.3 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had weak caseinolytic and potent haemorrhagic activities, and the minimum haemorrhagic dose of the enzyme was about 0.04 microg. The haemorrhagic activity of the enzyme was only suppressed by treatment with ethylenediamine tetraacetic acid-disodium or o-phenanthroline. The enzyme is composed of a single polypeptide chain with a molecular weight of about 70 kDa and is a glycoprotein. These results indicate that the enzyme is a metalloproteinase as are haemorrhagic proteinases from other snake venoms.