Comparative Study of Biological Activities of Venom from Colubrid Snakes Rhabdophis tigrinus (Yamakagashi) and Rhabdophis lateralis

Abstract

Rhabdophis lateralis, a colubrid snake distributed throughout the continent of Asia, has recently undergone taxonomic revisions. Previously, Rhabdophis lateralis was classified as a subspecies of R. tigrinus (Yamakagashi) until 2012, when several genetic differences were discovered which classified this snake as its own species. To elucidate the toxicity of venom from this poorly studied colubrid, various biological activities were compared between the venom from the two snake species. The components of their venom were compared by the elution profiles of reversed-phase HPLC and SDS-PAGE, and gel filtrated fractions were tested for effects on blood coagulation. Proteolytic activities of these fractions were also assayed by using synthetic substrates, fibrinogen, and matrix proteins. Similar to the R. tigrinus venom, the higher molecular weight fraction of R. lateralis venom contained a prothrombin activator. Both prothrombin time (PT) and activated partial thromboplastin time (APTT) of human plasma were shortened by the addition of R. lateralis and R. tigrinus venom. The thrombin formation was estimated by the uses of SDS-PAGE and chromogenic substrates. These venom fractions also possessed very specific proteinase activity on human fibrinogen, but the substrates for matrix metalloproteinase, such as collagen and laminin, were not hydrolyzed. However, there were some notable differences in reactivity to synthetic substrates for matrix metalloproteinase, and R. tigrinus venom possessed relatively higher activity. Our chemical investigation indicates that the components included in both venoms resemble each other closely. However, the ratio of components and proteolytic activity of some ingredients are slightly different, indicating differences between two closely-related snakes.

Keywords: Rhabdophis lateralis; Rhabdophis tigrinus; blood coagulation; prothrombin activator; snake venom.

Figure 1

Comparison of crude venoms of R. tigrinus and R. lateralis. (A) SDS-PAGE of crude venoms; (B) Elution profiles from gel filtration column. Two milligrams of crude venoms were applied to the column and eluted with 0.01 M Tris-HCl buffer (pH 7.2) at a flow rate of 1.0 mL/min; (C) Elution profiles from a reversed-phase-HPLC column were obtained with a linear gradient from 20% to 90% acetonitrile over 25 min, with at a flow rate of 1.0 mL/min.

Figure 2

Time course of prothrombin degradation by R. tigrinus and R. lateralis venom. A mixture of human prothrombin (0.5 mg) and crude venom (5 μg) in a total volume of 110 μL 0.05 M Tris-HCl buffer, pH 7.5, containing 0.1 M NaCl was incubated at 37 °C. At the time intervals indicated, aliquots of the mixture were subjected to SDS-PAGE under unreduced conditions.

Figure 3

Time course of fibrinogen degradation by R. tigrinus and R. lateralis venom. A mixture of human fibrinogen (0.1 mg) and venom fraction 1 (5 μg) in a total volume of 150 μL 0.01 M Tris-HCl buffer, pH 7.5, containing 0.1 M NaCl, was incubated at 37 °C. At the time intervals indicated, aliquots of the mixture were subjected to SDS-PAGE under reduced conditions.