降纤酶的分离纯化及其性质

Defibrase purification and properties

蛇毒蛋白对抑制血栓形成和溶解血栓具有较好的疗效 ,但由于得不到性质稳定、组分单一的酶蛋白 ,影响了其临床效果。该研究通过亲和层析方法 ,从长白山白眉蝮蛇乌苏里种蛇毒中分离提纯到 SDS- PAGE图谱为单一组分糖蛋白——降纤酶 ,相对分子质量约 3.6× 10 4。该酶具有体外凝血、体内抗凝作用。等电点约为 4 .2 5。HPL C分析结果表明其纯度为 99%。最适温度为 70℃ ,最适 p H =8.0。动力学研究表明该酶为一 Michaelis- Menten酶 ,Michaelis常数为0 .32 0 mm ol/ L,最大反应速度为 0 .117μm ol/ m in。EDTA,Mg2 + ,Cu2 + 对其有抑制作用。测定该酶 N端 2 0个氨基酸的序列是 Val Ile Glu Glu Asp Glu Cys Asn Ile Asn Glu His Arg Phe L eu,与其他的蛇毒类凝血酶有高度同源性。

Snake venom enzyme can inhibit and dissolve blood coagulum but its clinical function is weakened if it is not be complete purified. Defibrase purified from Baimei Snake Venom by affinity chromatography showed one band on SDS PAGE with HPLC purity of 99%. Defibrase improves blood clotting in vitro and dissolves blood coagulum in vivo. The relative molecular mass of the enzyme is 3.6×10 4 and it has optimal pH=4.25. Its optimal pH is 8.0 and its optimal temperature is 70 ℃. A dynamic study demonstrated that defibrase was a typical Michaclis Menten enzyme with Michaelis constant of 0.320 mmol/L and maximum reaction velocity of 0.117μmol/min. EDTA, Mg 2+ and Cu 2+ were found to inhibit defibrase. The sturefure of the enzyme molecule from the N terminal was found to be ValIleGluGluAspGluCysAsn IleAsnGluHisArgPheLeu, which has high homology with the thrombin of other snake venom.