黄黑小斑蝥纤溶活性蛋白分离纯化及性质研究

Purification and Biochemical Character of A Fibrinolytic Protein from Mylabris

目的以药用斑蝥(Mylabris)为原料,从其中分离纯化得到一种具有纤溶性的蛋白MFP(Fibrinolyric Protein of Myla-bris)。方法采用硫酸铵分段盐分、透析和DEAE-32纤维素离子交换层析等纯化方法进行分离纯化,SDS-PAGE电泳测定其分子量,纤维蛋白平板法测定其纤溶活性。结果该目的蛋白相对分子质量约为95.5 kD,其水解纤维蛋白的相对比活力为14.7 u/mg;该成分在35℃下最稳定,65℃及以上活性丧失,金属离子K+,Na+对其活力无影响,Mn2+,Ca2+对其有明显抑制作用,最适pH为6.0。结论研究分离出的斑蝥活性蛋白确为一种具有纤溶性的蛋白组分,并证明其纤溶作用机理为纤溶酶原激活;体外溶栓实验表明,其对血栓的溶解具有一定作用;溶血实验结果表明,该纤溶活性蛋白溶液无溶血反应。

Objective To separate and purify a fibrinolytic protein from the tissue of the Mylabris. Methods Separate and purify the protein by ammonium sulfate fractionation,dialysis and DEAE-cellulose chromatography,determine molecular weight by ion exchange sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyze,determine its fibrinolytic activity by fibrin plate. Results The protein showed an apparent molecular weight of 95.5 kD.Its specific activity to hydrolyze fibrin was 14.7u/mg,and the mechanism of fibrinolysis was plasminogen activator.The protein was stable under 35℃,and would lost activity above 65℃.Metal ions K＋,Na＋ had no effect on its activity,but was significantly inhibited by Mn2＋,Ca2＋.Its optimum pH was 6.0. Conclusion The fibrinolyric protein of mylabris was proved as a protein of fibrinolytic activity.Thrombolysis in vitro experimetns showed that it has a certain effect on dissolution of thrombus,and has no hemolytic reaction.