Characterization of hemorrhagic principles from Trimeresurus gramineus snake venom

Abstract

In addition to α-fibrinogenase (hemorrhagin I, HR 1), a potent hemorrhagic principle (hemorrhagin II, HR 2) was purified from Trimeresurus gramineus venom. It was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. HR 2 was a single peptide chain containing 10% carbohydrate with a molecular weight of 81,500. It possessed 669 amino acid residues per molecule, while HR 1 contained only 203 amino acid residues per molecule with a molecular weight of 23,500. Both hemorrhagins possessed proteolytic activities toward fibrinogen, casein and azocoll. However, the proteolytic activities of HR 1 were much more potent than those of HR 2. They were devoid of TAME-esterase and phospholipase A 2 activities which were found in crude venom. β-Mercaptoethanol and antivenin completely inhibited the hemorrhagic activities of HR 1 and HR 2, while ε-aminocaproic acid, trasylol, p-bromophenacyl bromide, phenylmethanesulfonylfluoride and soybean trypsin inhibitor did not. EDTA completely inhibited the hemorrhagic, fibrinogenolytic and caseinolytic activities of HR 1. EDTA also completely inhibited the caseinolytic and fibrinogenolytic activities of HR 2, but only partially inhibited its hemorrhagic activity. Subsequent addition of Zn 2+ (5 mM) reversed the EDTA-induced inhibitory effect on the hemorrhagic activity of HR 1. However, Zn 2+ did not reverse the EDTA-induced inhibitory effect on the HR 2-induced hemorrhagic activity. These hemorrhagins were found to be Zn 2+-containing metalloproteinases. Therefore, the hemorrhagic activity of HR 1 seems to be related to its proteolytic activity while that of HR 2 seems to be unrelated to its proteolytic activity.