原矛头蝮蛇毒中一个新颖双链纤溶酶的分离纯化与性质研究

Purification and characterization of a novel double-stranded serine protease from Protobothrops mucrosquamatus venom

目的从原矛头蝮蛇毒中寻找新型纤溶酶,对其理化性质与生物学活性进行研究,以了解其在蛇伤中的作用与毒性机制,并评估其潜在的应用价值。方法采用蛋白层析技术分离纯化获得目标蛋白,检测其分子量、等电点、肽指纹图谱、多种蛋白水解活性、抗补体活性、出血活性、水肿活性及对流血时间的影响。结果通过阴离子交换层析、凝胶过滤层析、亲和层析从原矛头蝮蛇毒中纯化出一个酸性纤溶酶PMSP-A,它是由两条等电点分别为5.7与6.1的非均等肽链共价结合而成。SDS-PAGE和凝胶过滤测定其分子量分别为26.1 ku与25.3 ku。肽指纹图谱分析表明PMSP-A与黄绿烙铁头蛇毒中的血液凝固结合因子有部分序列吻合。PMSP-A能够依次降解纤维蛋白原的Bβ、Aα链,该活性能被PMSF、1,10-phenanthroline抑制,EDTA、EGTA、SBTI不能抑制其活性。PMSP-A具有纤维蛋白、精氨酸酯水解活性,没有偶氮酪蛋白水解活性。它还具有抗补体活性。动物实验表明,PMSP-A明显延长小鼠尾静脉流血时间,能诱导小鼠足趾轻度水肿,无皮下出血活性。结论 PMSP-A是一个新颖的原矛头蝮蛇毒双链丝氨酸蛋白酶,具有多种影响机体的生物学活性。

Aim To discover the new or novel fibrinolysin from Protobothrops mucrosquamatus venom.The physico-chemical property and biological activity of the interest enzyme were investigated for better understanding of its role in snakebite and evaluating its clinical potential.Methods The object protein was isolated by chromotography techniques.Assays were performed to measure molecular mass,isoelectric point,peptide mass fingerprinting analysis,variety of proteolytic activity,anticomplementary activity,edema-inducing activity,hemorrhagic activity,and mice tail bleeding time.Results By anion exchange chromatography,gel filtration chromatography,and affinity chromatography,an acidic fibrinolysin PMSP-A was purified from Protobothrops mucrosquamatus venom.It consists of two non-equal polypeptides connected by covalent bond.The isoelectric points of the two subunits were 5.7 and 6.1,respectively.The molecular weight of PMSP-A was determined to be 26.1 ku and 25.3 ku by SDS-PAGE and gel filtration,respectively.Peptide mass fingerprinting analysis revealed that PMSP-A shared sequence similarity with the blood clotting factor from Trimeresurus flavoviridis.PMSP-A degraded Bβ-chain and Aα-chain of fibrinogen in order.This fibrinogenolytic activity was inhibited by PMSF and 1,10-phenanthroline,but not by EDTA,EGTA,and SBTI.PMSP-A hydrolyzed fibrin and arginine ethyl ester,but no hydrolytic activity against azocasein.PMSP-A induced slight edema of mice paw.No hemorrhagic activity was observed.Interestingly,PMSP-A prolonged the bleeding time of mice tail vein significantly.Conclusion PMSP-A is a novel double chains serine protease from Protobothrops mucrosquamatus,and it possesses diversified biological activity.