Comparison of two methods for proteolytic enzyme detection in snake venom

An acrylamide gel system containing fibrinogen was used to detect proteolytic enzymes in snake venoms. Proteolytic activity was observed as a clear area on a blue background after electrophoresis and overnight incubation in Tris buffer, prior to staining with Coomassie blue. Venoms from eastern and western diamondback and west coast Mexican rattlesnakes, Crotalus adamanteus, C. atrox and C. basiliscus basiliscus, respectively, and southern copperhead, Agkistrodon contortrix contortrix, were analyzed at the level of 1 mg of venom. The effect of the serine proteinase inhibitor diisopropylfluorophosphate (DFP) and the metalloproteinase inhibitors tetraethylenepentamine (TEP) or EDTA on fibrinogen and normal gel profiles were evaluated. Normal gels (without fibrinogen) were Coomassie stained to visualize migration of 250 micrograms of venom proteins on the gels. Several proteolytic enzymes detected in C. atrox and C. b. basiliscus venoms were inhibited by TEP, whereas DFP had no effect on activity. The fibrinogen gels detected no protease activity in C. adamanteus venom, although it is known from other studies that there are several proteolytic enzymes in this venom. Several proteases were detected in A. c. contortrix venom, one of which was inhibited by TEP. By comparison, proteolytic activity in 5-10 micrograms of all venoms was readily detected using the mammalian plasma kallikrein specific chromogenic substrate, S2302 (H-D-Pro-Phe-Arg-p-nitroanilide). The fibrinogen gel method does not appear to have the specificity nor the sensitivity of the recently developed chromogenic substrates for detection of proteolytic enzymes in snake venom.