降纤酶的分离纯化及其性质  

Defibrase purification and properties

作　　者：蒋宇扬[1] 张歧山[2] 张玉魁[2] 万荣[1] 赵玉芬[1] 

机构地区：[1]清华大学化学系,生命工程研究院,生命有机磷教育部重点实验室,北京100084 [2]中国医科大学生物工程研究中心,沈阳110001

出　　处：《清华大学学报（自然科学版）》2001年第12期62-64,共3页Journal of Tsinghua University(Science and Technology)

基　　金：清华大学"九八五"骨干人才研究经费 ;清华大学;北京中医药大学与香港浸会大学中医药合伙研究基金资助

摘　　要：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.

关 键 词：白眉蝮蛇 降纤酶 分离纯化 蛇毒蛋白 蛋白酶 酶活力 体外凝血作用 

分 类 号：Q556.9[生物学—生物化学]