Markland F S, Perdon A
Toxicon. 1986;24(4):385-93. doi: 10.1016/0041-0101(86)90198-4.
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.
使用含有纤维蛋白原的丙烯酰胺凝胶系统来检测蛇毒中的蛋白水解酶。在 Tris 缓冲液中进行电泳和过夜孵育后，用考马斯亮蓝染色之前，蛋白水解活性表现为蓝色背景上的清晰区域。分别对东部菱斑响尾蛇、西部菱斑响尾蛇和墨西哥西海岸响尾蛇（即 Crotalus adamanteus、C. atrox 和 C. basiliscus basiliscus）以及南部铜头蝮（Agkistrodon contortrix contortrix）的蛇毒进行了 1 mg 水平的分析。评估了丝氨酸蛋白酶抑制剂二异丙基氟磷酸酯（DFP）和金属蛋白酶抑制剂四乙烯五胺（TEP）或乙二胺四乙酸（EDTA）对纤维蛋白原和正常凝胶图谱的影响。对正常凝胶（不含纤维蛋白原）进行考马斯染色，以观察 250 微克蛇毒蛋白在凝胶上的迁移情况。TEP 抑制了在 C. atrox 和 C. b. basiliscus 蛇毒中检测到的几种蛋白水解酶，而 DFP 对活性没有影响。尽管从其他研究中已知该蛇毒中有几种蛋白水解酶，但纤维蛋白原凝胶未检测到 C. adamanteus 蛇毒中的蛋白酶活性。在 A. c. contortrix 蛇毒中检测到了几种蛋白酶，其中一种被 TEP 抑制。相比之下，使用哺乳动物血浆激肽释放酶特异性显色底物 S2302（H-D-脯氨酸-苯丙氨酸-精氨酸-对硝基苯胺）可以很容易地检测到 5 - 10 微克所有蛇毒中的蛋白水解活性。纤维蛋白原凝胶法似乎既没有最近开发的用于检测蛇毒中蛋白水解酶的显色底物的特异性，也没有其灵敏度。
