| Phospholipase A2(svPLA2) is the most toxic component snake venom,svPLA2 possess several toxicological activities besides enzymatic activity, which may have potentials in application. Snake venom from one species may contain several kinds of PLA2, their primary structures are similar, but the corresponding function may be markedly diverse, therefore PLA2s have served as a good model for the study of protein structure-function relations. It also become a hotspot in snake venom research.Gloydius intermedius subordinates to the Genus Gloydius, Crotalinae of Viperidae, which mainly distributes in northwest and north China, is the most toxic species among six pit-vipers in China. Little is still known about its structure and molecular mechanism of envenomation, while the obtaining of pure PLA2 is a precondition for the addressing of these issues.Methods and Results:1. PLA2 Purification.At first, PLA2s were separated by using Size exclusion chromatography (Sephacryl-200HR) from Gloydius intermedius crude venom. After elution with 50 mmol/L ammonium acetate buffer (pH7.3), eight fractions were obtained (designated Peakl-8). Acute toxicity was assessed by injection intraperitoneal with 1 mL of each peak fraction in mice, purity was analyzed by SDS-PAGE. The results showed that, Peakl and Peak8 were not lethal for mice and the protein concentration were very low. Peak2, Peak6 and Peak7 were lethal and hemorrhagic, but didn't cause paralysis, so were speculated as hemorrhagic toxins. Fractions Peak3, Peak4 and Peak5 were both lethal and paralytic neurotoxins, but were still mixed, as revealed by SDS-PAGE analysis.Secondly, fractions Peak3, peak4 and Peak5 were separated with anion exchange chramatography on a HiTrap Q column. After loading each sample, Protein was eluted with a linear gradient from start buffer A(50 mmol/L ammonium acetate, pH7.8) to elution buffer (50 mmol/L ammonium acetate,0.5 mol/L NaCl, pH7.8), unbound proteins and bound proteins were collected. Acute toxicity was assessed by injection intraperitoneal with 1 mL of each peak fraction in mice, purity was analyzed by SDS-PAGE. The results showed that, Peak3-1, Peak4-1 and Peak5-2 were hemorrhagic and lethal. Peak3-2, Peak4-2 and Peak5-1 were lethal and paralytic neurotoxins, but were still mixed as revealed by SDS-PAGE.Then, three neurotoxic fractions(Peak 3-2, Peak 4-2 and Peak 5-1) were further separated by cation exchange chromatography on a HiTrap SP column. After sample loading of each fraction, Protein was eluted with a linear gradient from start buffer A(50 mmol/L ammonium acetate, pH5.5) to elution buffer (50 mmol/L ammonium acetate,0.5 mol/L NaCl,pH5.5), unbound proteins and bound proteins were collected. Acute toxicity was assessed by injection intraperitoneal with 1 mL of each peak fraction in mice, purity was analyzed by SDS-PAGE, enzyme activities were determined by lecithin hydrolysis on agarose plates. The results showed that, Peak3-2-2, Peak4-2-1, Peak4-2-2 and Peak5-1-2 were not lethal. Peak3-2-1 and Peak5-1-1 were lethal and the enzyme activity of the former was higher than that of the latter. Peak3-2-1, with a relative molecular weight of 14kDa was almost pure and Peak5-1-1 contained multi-bands.2. Characterization of the Biological Activity.Firstly, with crude venom as positive control, PLA2 enzyme activities were compared by method of lecithin hydrolysis on agarose plates. The result showed that fraction Peak3-2-1 caused larger transparent circle than crude venom on agarose plate.Secondly, with crude venom and PBS as positive and negative controls, the antibacterial activity were evaluated on E. coli.24 hour later, crude venom caused obvious transparent circle, while Peak3-2-1 only showed subtle antibacterial activity.Next, hemolysis activity, which reveals the indirect PLA2 activity, were tested with RBC hemoglobin release method. On human RBCs, Peak3-2-1 showed lower hemolytic activity than the positive control.Finally, with phrenic nerve-diaphragm muscle preparation of SD rat as a model, the blocking activity of fraction Peak3-2-1 were measured. Muscle contraction inhibitory effect was observed 20min after addition of Peak3-2-1(50μg/mL) into the MOPS medium, diaphragm contraction declined to only about 10% of the normal magnitude. Peak3-2-1 only blocked the indirect stimulated diaphragm contraction, but didn't influence direct stimulated muscle contraction, indicating that the blocking effect was on nerve-muscle junction(synapse), but not diaphragm itself. So protein in Peak3-2-1 is a neurotoxin.Conclusion:by combined use of size exclusion chromatography and ion exchange chromatography, a neurotoxin with PLA2 activity and about 14kDa has been successfully separated from Gloydius intermedius venom. It possesses potent hemolytic and weak antibacterial activity. This work may therefore establish the basis for the investigation of function, strcture and molecular mechanism of envenomation in the future. |
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