Mediation Fu (gloydius Intermedius) Snake Venom Phospholipase A < Sub > 2 < / Sub > Neurotoxin Purification And Identification Of Biological Activities

Gloydius intermedius is the most toxic species among six pit-vipers in China, which subordinates to the Genus Gloydius, Crotalinae of Viperidae, and distributes in northwest and north China. Snake venom phospholipase (SVPLA2) is the most toxic components of snake venom, and also possess a variety of toxicological and pharmacological activities besides enzymatic activity, which may have potentials in application. However, PLA2toxicological activity, structure and function relationship, molecular mechanism of envenomation remain to be elucidated, while the obtaining of pure neurotoxin PLA2is a premise for the addressing of these issues.Methods and Results:1. PLA2PurificationAt first, PLA2s were separated by using size exclusion chromatography (Sephacryl-200HR) from Gloydius intermedius crude venom. After elution with50mmol/L ammonium bicarbonate buffer (pH8.0), five fractions were obtained (P1-5). Each peak component was measured by the phospholipase A2activity and acute toxicity experiments, the purity was analyzed by SDS-PAGE. The results showed that P2and P3contained the neurotoxin phospholipase A2, which were not a single band as revealed by SDS-PAGE analysis. Therefore further separation was needed.Secondly, P2and P3fractions were separated with anion exchange chramatography on a HiTrap DEAE column. After loading each sample, proteins were eluted with a linear gradient from start buffer A (50mmol/L ammonium acetate, pH7.3) to elution buffer (50mmol/L ammonium acetate,0.5mol/L NaCl, pH7.3), unbound proteins and bound proteins were collected. Each peak component was measured by the phospholipase A2activity and acute toxicity experiments, purity was analyzed by SDS-PAGE. The results showed that, P2-5, P3-2and P3-3were neurotoxins PLA2, which were not a single band as revealed by SDS-PAGE.Then, P2-5, P3-2and P3-3were further separated by cation exchange chromatography on a HiTrap CM column. After loading each sample, proteins were eluted with a linear gradient from start buffer A (50mmol/L ammonium acetate, pH5.3) to elution buffer (50mmol/L ammonium acetate,0.5mol/L NaCl, pH5.3), unbound proteins and bound proteins were collected. Each peak component was measured by the phospholipase A2activity and acute toxicity experiments, purity was analyzed by SDS-PAGE. The results showed that P2-5-1, P3-2-1, P3-3-2were neurotoxins PLA2. SDS-PAGE analysis showed that the purity of the P2-5-2, P3-2-1, P3-3-1was very high, close to a single band, with a relative molecular weight of14kDa.2. Characterization of the Biological Activities.Firstly, the PLA2activity of P3-2-1was examined to be352nmol/minmg, by using the synthetic substrate4-nitro-3-benzoic acid (NOB). The Vmax was estimated to be7.21nmol/min. Maximum enzyme activity occurred at35℃, and the optimum pH was8.0, which required Ca2+for full activity.Secondly, with1%Triton X-100and Tris-HCl as positive and negative controls, hemolytic activity was tested by red blood cell hemoglobin release method, which reveals the indirect PLA? activity. The results showed that, P3-2-1component has a certain hemolytic activity, the hemolysis rate was21%.Thirdly, mice were injected with dose of25μg/kg of P3-2-1, and then their serum creatine kinase were assayed at Oh,2h,4h,6h or8h respectively, by using CK kit. The result showed that the mice serum creatine kinase rose markedly and reached the highest value in2h, which indicating that P3-2-1has myotoxic activity.Fourthly, with crude venom and sterile deionized water as positive and negative controls, the antibacterial activity of P3-2-1component was evaluated on Escherichia coli and Staphylococcus aureus. The result showed that P3-2-1caused obvious transparent circle, indicating that P3-2-1has antibacterial activity.Conclusion:Neurotoxic PLA2has been successfully separated from Gloydius intermedius venom by using size exclusion chromatography and ion exchange chromatography, whose molecular weight was approximately14kDa. The optimum temperature was35℃and the optimum pH was8.0. P3-2-1possessed potent indirect hemolytic activity, myotoxic activity and antibacterial activity. This work may therefore establish the theoretical foundation for the further study of molecular evolution and molecular mechanism of envenomation.