Intermedius (Gloydius Intermedius) Venom Serine Protease GI-SP ₂ Purification And Biological Activity

Gloydius intermedius subordinates to the Genus Gloydius, Crotalinae of Viperidae, which distributes extensively in the Northwest and North China. The Venom is rich in a variety of proteins and enzymes. The investigation of the venom will be of importance for the treatment of the envenomation and exploitation of drugs for clinical use.Snake venom Serine proteases (SVSPs) is an important class of proteolytic enzymes in the snake venom, which include Plasminogen activator, Snake fibrin(ogen)olytic enzyme, snake venom thrombin-like enzyme(SVTLE), protein C activator, coagulant V activator, coagulant X activator and platelet aggregation activator. They can cause diverse effects in the blood clotting and thrombolytic system and affect platelet aggregation, blood coagulation and fibrinolysis. Although the primary structure of SVSPs has great homology, they exhibit specific biological activities based on substrate specificity. It has become a good source to develop related drugs for the treatment of cardiovascular disease, but the obtaining of SVSP by purification is a premise.In the present study, we purified SVSPs(GI-SP2) by combined use of Gel filtration chromatography and Ion exchange chromatography, and characterized the biological activities.The experimental details and results are as follows:1. SVSPs Purification(1) Gel filtration chromatography:crude venom of Gloydius intermedius was separated by using Size exclusion chromatography(Sephacryl-200HR), with50mmol/L ammonium bicarbonate solution(contains0.15mol/L NaCl and0.2g/L Sodium azide, pH8.0) as elution buffer. Five fractions were obtained, designated as P1-P5. The protease activity of the five fractions were determined with BApNa(N-benzoyl-D,L-arginine-p-nitroanilide) as substrate and protein purity was detected by SDS-PAGE. The result showed that three fractions(P2, P3and P5) could act on BApNa and P2was the best, but were still a mixture as revealed by SDS-PAGE. So further purification steps were needed.(2)Anion exchange chromatography:P2was then separated with anion exchange chramatography on a HiTrap DEAE-FF column. with buffer A (50mmol/L ammonium bicarbonate) and B (50mmol/L ammonium bicarbonate and0.5mol/L NaCl, pH8.0) as buffer at a flow rate of0.2mL/min. First, the unbound proteins were firstly eluted with start buffer A. Later, the bound proteins were eluted by gradient elution from start buffer A to buffer B. Different principles were colleted and the serine protease activities were determined by using BApNAaas the substrate. The resuts showed that P2-3, P2-4and P2-5showed enzyme activity and P2-4was the best. But SDS-PAGE analysis revealed that P2-4still was a mixture. Further isolation is necessary.(3)Cation exchange chromatographic separation:Fraction P2-4was further separated with anion exchange chramatography on a HiTrap DEAE-FF column, with solution A (50mmol/L sodium acetate) and B (50mmol/L sodium acetate and0.5mol/L NaCl, pH4.5) as buffer, at a flow rate of0.2mL/min. At first, the unbound proteins were eluted with start buffer A. The bound proteins were eluted by gradient elution from start buffer A to buffer B. Two fractions (P2-4-1and P2-4-2) were colleted. Again, BApNa was used as the substrate to evaluate the serine protease activities.The results showed that the protease activity of P2-4-1was optimal, and SDS-PAGE analysis revealed that P2-4-1was a single band and pure protein, here designated as GI-SP2.2. Characterization and the Biological Activity of GI-SP2(1) BApNAhydrolyzing activity of GI-SP2.(2) Detect the effects with different inhibitors:the effects of β-mercaptoethanol, PMSF, EDTA were tested. These Inhibitors were used respectively, with a goup without inhibitors as positive control. The results showed that the hydrolysis activity of GI-SP2on BApNAa could be inhibited by PMSF, while EDTA coulden’t. So GI-SP2is a kind of SVSP, instead of snake venom metalloproteinases.(3) Fibrinogen hydrolysis activity assay:GI-SP2was mixed with fibrinogen and incubated at37℃. Samples at different time points were subjected to SDS-PAGE(12%) to analyze the hydrolysis of α-chain, β-chain and y-chain cleavage. The results showed that GI-SP2was able to cleave P-chain of fibrinogen after30minutes.(4) Mouse tail bleeding time:mice were divide into different groups, one were injected with GI-SP2at a dose of0.3μg/g while mice of another group were injected the same dose of saline as control. The results showed that GI-SP2could prolong the mice tail bleeding time.(5) Hemorrhagic activity in mice:mice were injected subcutaneously with GI-SP2at different doses and sacrificed24hours. Then the skins were peeled off and the hemorrhagic sites were observed. The results showed that, even at dose up to50μg, there was no haemorrhagic activity in mice.(6) Thrombin-like activity assay:with thrombin as positive control, GI-SP2and fibrinogen were co-incubated at37℃for10min, then observed the white precipitation formation.The results showed that GI-SP2couldn’t cause fibrinogen coagulation, and diden’t possess the thrombin-like activity.(7) Fibrinolytic activity assay:The fibrinolytic activity was detected on the fibrin plates. GI-SP2showed fibrinolytic activity when applied to fibrin plates.