| Snake Venom Serine proteinases (SVSPs) is an important kind of proteolytic enzymes. SVSPs affect, for example, platelet aggregation, blood coagulation, flbrinolysis, the complement system and blood pressure, with a lot of clinical application value. Although the primary structure share great homology, but different SVSPs is working on different substrates, and often a SVSP can hydrolyze many kinds of protein or small molecular substrate. So they are good models of researching the relationship between structure and function of proteins.Gloydius intermedius subordinates to the Genus Gloydius, Crotalinae of Viperidae, is the most toxic species among six pit-vipers in China, which widely distributed in northwest and north of China. It is close to people’s life. Therefore, research of the SVSPs has important implications, however separation of SVSPs from Gloydius intermedius is a premise.Methods and Results::1. SVSPs PurificationAt first, SVSPs were separated by using Size exclusion chromatography (Sephacryl-200HR) from Gloydius intermedius crude venom, with50mmol/L ammonium bicarbonate buffer(pH8.0, and contains0.15mol/LNaCl and0.2g/L Sodium azide) as elution buffer. Five fractions were obtained (designated Peakl to5). Using BApNA as the substrate to determine the serine proteinase activities of the different fractions, and using the SDS-PAGE to analyze protein purity. The results showed that, Peak2can hydrolyze BApNA, but were still mixture, as revealed by SDS-PAGE analysis.Secondly, fraction Peak2was separated with anion exchange chramatography on a HiTrap DEAE-FF column. After loading sample, protein was eluted with a linear gradient from start buffer A(50mmol/L ammonium acetate, pH8.0) to buffer B(buffer A plus0.5mol/L NaCl), unbound proteins and bound proteins were collected. Using BApNA as the substrate to determine the serine proteinase activities of the five fractions, and using the SDS-PAGE to analyze protein purity. The results showed that Peak2-3, Peak2-4has the hydrolysis activity of BApNA. The SDS-PAGE analysis showed that Peak2-3and Peak2-4were still multi-banded, still needed further separation.Then, fractions Peak2-3was further separated by cation exchange chromatography on a HiTrap CM-FF column. After sample loading, protein was eluted with a linear gradient from start buffer A(50mmol/L sodium acetate, pH6.0) to elution buffer (buffer A plus0.5mol/L NaCl, pH6.0). Unbound proteins and bound proteins were collected. Using BApNA as the substrate to determine the serine proteinase activities, and using the SDS-PAGE to analyze protein purity. The results show that Peak2-3-1can hydrolyze BApNA, SDS-PAGE analysis showed that Peak2-3-1was a single band.2. Characterization of the Biological Activities.(1) Protein purity identification:SDS-PAGE of fraction Peak2-3-1were carried out under reducing conditions and non-reducing conditions, the results showed that both were single band.(2) Separated Peak2-3-1was detected by SDS-PAGE on reducing condition. The subject band in the gel was cut down and identified by tandem mass spectrometry.(3) The enzyme activity of Peak2-3-1was8.4×103nmol/min/mg with BApNA as the substrate.(4) The inhibitory effect with specific inhibitors:The effects of different proteinase inhibitors on purified enzyme were examined with its activity on BApNA. Purified enzyme, Fraction Peak2-3-1was co-incubated with different inhibitors:PMSF, EDTA, β-Mercaptoethanol for20min at37℃. The results show that:PMSF can inhibit the hydrolysis activity of Peak2-3-1to BApNA significantly, while EDTA can not. It can account for that fraction Peak2-3-1is SVSPs, not snake venom metalloproteinases.(5) Fibrinogen hydrolysis activity:Peak2-3-1and fibrinogen were mixed and incubated at37℃. At various time points (0,15and30min,1,2,4,8,12h), end the reaction by adding of6μl loading buffer and aliquots were taken to examine the fibrinogen degradation products at each time point. Samples were analyzed by SDS-PAGE under reducing conditions using a12%polyacrylamide gel. The result showed that fraction Peak2-3-Γ is able to cleave Bβ-chain rapidly and cleave Aa-chain slowly with time going on.(6) Fibrinolytic activity assay:The fibrinolytic activity was assayed on the fibrin plates. Peak2-3-1showed fibrinolytic activity when applied to fibrin plates(7) Thrombin-like activity:with thrombin as positive control, Peak2-3-1and fibrinogen were co-incubated at37℃for10min, then observed the white precipitation produced. The results showed that Peak2-3-1can’t make fibrinogen coagulation, and doesn’t possess the thrombin-like activity. (8) Bleeding activity:Balb/c mice were divided into six groups and injected with a series dosage of fraction Peak2-3-1subcutaneously on the back of mice. Mice were sacrifice after24h of injection, and peel off the skin. The results showed that fraction Peak2-3-1has not bleeding activity, even with the dose up to50μg.(9) The bleeding time of mice tail:Balb/C mice were divided into two groups and injected fraction Peak2-3-Γ with a dose of0.5g/kg, with saline as control. The results showed that, compared to the saline control (599±165s), Peak2-3-1significantly prolonged the tail vein bleeding time of the subject mice (984±182s).Conclusion:By combined use of size exclusion chromatography and ion exchange chromatography, a SVSP with fibrinogenase activity and with molecular weight about28.8kDa has been successfully separated from Gloydiits intermedius venom. Peak2-3-1can cleave Bβ chain of fibrinogen rapidly and Aa chain of fibrinogen slowly. The enzyme activity can be significantly inhibited by PMSF, and EDTA do not affect the activity. Peak2-3-1can not induce the coagulation of bovine fibrinogen and bleeding. It can significantly extend the time of mice vein bleeding. 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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