Purification And Characterization Of PLA₂from Gloydius Ussuriensis Venom

Phospholipase A2(phospholipase A2, PLA2) exists in almost all snake venom, inaddition to the enzyme activity, the venom PLA2also has many kinds of biologicalactivity, such as muscle toxicity, neurotoxicity and membrane toxicity, and also hashemolysis, anticoagulation, inhibition of platelet aggregation and inducing theformation of edema, in addition PLA2also has inhibit bacterial growth, induce cellapoptosis, to prevent the HIV virus into the host cell. Because of the different kinds ofsnakes containing PLA2, so even the same specie of poisonous snakes, also changingthe activity and structure of venom in the PLA2strongly by the different environment.PLA2has the complexity of the physiological function and unique chemical properties,not only in the study of biological membrane phospholipids structure, lipidmetabolism, blood coagulation and hemolysis mechanism, also play an important roleas a drug in the treatment of cardiovascular diseases, tumor and etc, which hasimportant application prospect.About the separation and purification of PLA2in snake venom, and hasquite a few literature reported both at home and abroad, but because of the differenceof each experimental materials and purification methods, experimental results are alsodifferent.Objective:In this study, First, using alcohol precipitation methods to remove most of theprotein impurities. Then through the two Sephadex G-50gel-filtrationchromatography to separate and purify PLA2related protein from Gloydiusussuriensis venom. By SDS-PAGE and phospholipase activity, fibrinogen activity ofhydrolysis, bleeding cytotoxic activity and many other biological assay, we detecteand assess the protein’s structural features and biological functions.Content:Utilization of Sephadex G-50gel-filtration chromatography to separate and purify phospholipase PLA2from Gloydius ussuriensis venom. By SDS-alkalinediscontinuous vertical polyacrylamide gel electrophoresis and phospholipidhydrolysis activity determination, Bleeding cytotoxic activity detection and otherbiological activity assay method for detection and evaluation of their structuralcharacteristics and biological function, according to the experimental results, itsattributable to the phospholipase PLA2。In the process of separation and purification, this experiment pioneered usingSephadex G-50of gel filtration chromatography method for separation andpurification twice. Testing PLA2protein component from Gloydius ussuriensis venomunder reducing and non-reducing conditions by SDS-polyacrylamide gel alkalinecontinuous vertical electrophoresis detection, electrophoretic pattern showed a singleband. Under reducing conditions calculated molecular weight of approximately16.2kD.Coomassie brilliant blue G-250staining by UV-visible spectrophotometerdetermination of the concentration of the protein solution after separation andpurification the PLA2from Gloydius ussuriensis venom. The concentration of thePLA2solution is0.945mg/ml.In the experiment of determination of phospholipid hydrolysis activity of PLA2its activity was measured using two methods proved the hydrolytic activity of PLA2with phospholipase.In the determination of PLA2fibrinogenolytic activity. After testing thepurification PLA2does not have hydrolytic activity of fibrinogen,in this study, whichnot hydrolyze fibrinogen to form a jelly-like fibrous protein aggregates. TheSDS-PAGE electrophoresis detection of α, β, γ chain don’t have degradationphenomenon.In the determination of the optimum pH of the activity of PLA2phospholipidhydrolysis. Adjust the pH value of each group of substrates, plus50μl of enzymesample incubated in37℃for30min, then measured the pH changes of each set ofsamples. Increases with the pH of the substrate, the changing difference for eachgroup of samples is also increased, pH=10the change in maximum. The optimum pH value at about10.In the PLA2activity determination of induced edema, statistical analysis of theexperimental data, The draw t value=0.398, t＜t0.052,14，P＞0.05. According to theα=0.05inspection level, that the experimental group and the control group, thedifference were not significant, the Gloydius ussuriensis venom of PLA2nopro-edema activity.In the PLA2bleeding cytotoxicity assays, mouse subcutaneous surface of theexperimental and control groups were not found bleeding point. The Gloydiusussuriensis venom of PLA2having no bleeding cytotoxic activity.Conclusion:In this experiment, using Gloydius ussuriensis Venom as material, the alcoholsolvent precipitation to remove most of the contaminating proteins in the venom,through two gel filtration chromatograph（ySephadex G-50stationary phase selection）to purify crude protein，solated a new protein monomer component from the Gloydiusussuriensis venom. By SDS-polyacrylamide gel alkaline continuous verticalelectrophoresis detection, electrophoretic pattern showed a single band. Underreducing conditions calculated molecular weight of approximately16.2kD.Determination of enzyme activity（Hydrolysis of egg yolk lecithin，Hydrolysis offibrinogen, The optimum pH）and pharmacological activity（Edema activity, Bleedingcytotoxic activity）of the purified protein. The results show that the protein has strongphospholipase A2hydrolysis activity, suggesting this enzyme as a venomphospholipase A2, which optimum pH at about10. This protein does not cause edemaactivity, hydrolytic activity of fibrinogen and bleeding cytotoxic activity. So namedthis phospholipase A2as Gloydius-PLA2.