竹叶青(Trimeresurus stejnegeri)蛇毒凝血酶样酶的分离纯化及其理化性质的研究

PURIFICATION AND SOME PROPERTIES OF THE THROMBIN- LIKE ENZYMES FROM TRIMERESURUS STEJNEGERI VENOM

用CM—Sephadex C—25分离竹叶青粗毒,再经DEAE—Sephadex A—50和CM—Sephadex C—25纯化得到凝血酶样酶组分Ⅰ。用DEAE—Sephadex A—50分离竹叶青粗毒,再经DEAE—Sepharose CL—6B纯化得到凝血酶样酶组分Ⅱ。用聚丙烯酰胺凝胶电泳和SDS—聚丙烯酰胺凝胶电泳鉴定,组分Ⅰ在两种电泳中均为一条带; 组分Ⅱ在两朴电泳中均为二条带,经切割后鉴定证明组分Ⅱ的二条带都是凝血酶样酶。组分Ⅰ的分子量为54,500,由261个氨基酸残基组成,其中Asp和Glu含量较高,含14％中性己糖,13.1％己糖胺和14.7％唾液酸,等电点为3.5,在280nm处的消光系数为E_(1cm)^(0.1％)=0.855。组分Ⅱ的分子量分别为54,000和47,000。经凝胶电泳分离后用过碘酸—Schiff's试剂染色,证明组分Ⅰ和组分Ⅱ都是糖蛋白。

Two thrombin-like enzymes (principle Ⅰand principle Ⅱ ) had been purified from the venom of Trimeresurus stejnegeri by CM-Scphadex C-25, DEAE-Sephadex A-50 and DEAE-Sepharose CL-6B column chrornatography. Principle I showed one band and principle I two bands on polyacrylamide gel clcctrophoresis and SDS-polyacrylamide gel elcctrophoresis.We had identified the two bands of principleⅡboth were thrombin-like enzymes by the method of cutting polyacrylamide gel clectrophorcsis. Principle \ has a molecular weight of 54,500, contains approximately 261 amino acid residues and has a relatively high content of aspartic acid and glutamic acid, contains 14% neutral hexose, 13.1% hexosamine and 14.7% sialic acid, the isoelectric point is 3.5, the extinction coefficient at 280nm for a 0.1% solution is 0.855. Principle I has molecular weights of 54,000 and 47, 000 respectively as determined by SDS-polyacrylamidc gel clcctrophoresis, principleⅡis also a glycoprotein as showed by Schiff's stain.