Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, Cidade Universitária Zeferino Vaz, 13083-887 Campinas, SP, Brazil.
Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, Avenida Brazil 1500, São Paulo, SP, Brazil.
Comp Biochem Physiol C Toxicol Pharmacol. 2018 Nov;213:27-38. doi: 10.1016/j.cbpc.2018.06.003. Epub 2018 Jun 30.
In this work, we examined the proteolytic and phospholipase A (PLA) activities of venom from the opisthoglyphous colubrid Pseudoboa neuwiedii. Proteolytic activity (3 and 10 μg of venom) was comparable to that of Bothrops neuwiedii venom but less than Bothrops atrox. This activity was inhibited by EDTA and 1,10-phenanthroline but only slightly affected (≤30% inhibition) by PMSF and AEBSF, indicating it was mediated by snake venom metalloproteinases (SVMPs). The pH and temperature optima for proteolytic activity were 8.0 and 37 °C, respectively. The venom had no esterase activity, whereas PLA activity was similar to B. atrox, greater than B. neuwiedii but less than B. jararacussu. SDS-PAGE revealed venom proteins >100 kDa, 45-70 kDa, 21-24 kDa and ~15 kDa, and mass spectrometry of protein bands revealed SVMPs, cysteine-rich secretory proteins (CRISPs) and PLA, but no serine proteinases. In gelatin zymography, the most active bands occurred at 65-68 kDa (seen with 0.05-0.25 μg of venom). Caseinolytic activity occurred at 50-66 kDa and was generally weaker than gelatinolytic activity. RP-HPLC of venom yielded 15 peaks, five of which showed gelatinolytic activity; peak 7 was the most active and apparently contained a P-III class SVMP. The venom showed α-fibrinogenase activity, without affecting the β and γ chains; this activity was inhibited by EDTA and 1,10-phenanthroline. The venom did not clot rat citrated plasma but reduced the rate and extent of coagulation after plasma recalcification. In conclusion, P. neuwiedii venom is highly proteolytic and could potentially affect coagulation in vivo by degrading fibrinogen via SVMPs.
在这项工作中，我们研究了后沟鳞蛇科剧毒蛇 Pseudoboa neuwiedii 的毒液的蛋白水解和磷脂酶 A（PLA）活性。蛋白水解活性（3 和 10 μg 毒液）与 Bothrops neuwiedii 毒液相当，但低于 Bothrops atrox。这种活性被 EDTA 和 1,10-菲啰啉抑制，但仅被 PMSF 和 AEBSF 轻微影响（≤30%抑制），表明它是由蛇毒金属蛋白酶（SVMPs）介导的。蛋白水解活性的最佳 pH 和温度分别为 8.0 和 37°C。毒液没有酯酶活性，而 PLA 活性与 B. atrox 相似，大于 B. neuwiedii，但小于 B. jararacussu。SDS-PAGE 显示毒液蛋白大于 100 kDa、45-70 kDa、21-24 kDa 和~15 kDa，对蛋白带进行质谱分析显示 SVMPs、富含半胱氨酸的分泌蛋白（CRISPs）和 PLA，但没有丝氨酸蛋白酶。在明胶酶谱中，最活跃的带出现在 65-68 kDa（用 0.05-0.25 μg 毒液观察到）。溶菌酶活性发生在 50-66 kDa，通常比明胶酶活性弱。毒液的 RP-HPLC 产生 15 个峰，其中 5 个显示明胶酶活性；峰 7 是最活跃的，显然含有 P-III 类 SVMP。毒液具有α-纤维蛋白原酶活性，不影响β和γ链；这种活性被 EDTA 和 1,10-菲啰啉抑制。毒液不会凝固大鼠枸橼酸盐血浆，但会在血浆重新钙化后降低凝固的速度和程度。总之，P. neuwiedii 毒液具有高度的蛋白水解活性，通过 SVMPs 降解纤维蛋白原，可能会在体内影响凝血。
