竹叶青(Trimeresurus stejnegeri)蛇毒五种磷脂酶A_2的分离纯化和氨基酸序列分析

Isolation and sequencing of five variants of phospholipases A_2 from venom of snake Trimeresurus stejnegeri

用超细SephadexG 75凝胶色谱和C4反相高效液相色谱从竹叶青(Trimeresurusstejnegeri)蛇毒中分离纯化5种磷脂酶A2,并分别命名为PLA2 Ⅰ(SWISS PROT,P82892)、Ⅱ(SWISS PROT,P82893)、Ⅲ(SWISS PROT,P82894)、Ⅳ(SWISS PROT,P82895)、Ⅴ(SWISS PROT,P82896).SDS PAGE测定它们的分子量分别为14.0、15.8、15.0、14.0和14.0kDa.等电聚焦电泳测得PLA2 Ⅰ、Ⅱ、Ⅲ呈碱性,等电点大于8.8;PLA2 Ⅳ和Ⅴ呈酸性,等电点分别为5.2和4.7.PLA2 Ⅳ和Ⅴ有水解卵磷脂活性.用自动Edman降解法测定了PLA2 Ⅴ的全部氨基酸序列和PLA2 Ⅰ、Ⅱ、Ⅲ、Ⅳ的N 端部分氨基酸序列.PLA2 Ⅴ由122个氨基酸残基组成,有14个Cys,并与其它来源的PLA2的氨基酸序列进行了比较.

Five variants of phospholipases A2, PLA2Ⅰ(SWISS PROT, P82892), PLA2Ⅱ(SWISS PROT, P82893), PLA2Ⅲ (SWISS PROT, P82894), PLA2Ⅳ (SWISS PROT, P82895) and PLA2Ⅴ(SWISS PROT, P82896) were isolated from venom of the snake Trimeresurus stejnegeri (Chinese green tree viper) by means of superfine Sephadex G75 gel permission chromatography and C4 reserve phase high performance liquid chromatography. Their molecular weights were calculated to be 14.0, 15.8, 15.0, 14.0 and 14.0 kDa by SDSPAGE. The PLA2Ⅰ, Ⅱ and Ⅲ were basic protein. The PLA2Ⅳ and Ⅴ were acidic protein with isoelectric points of 5.2 and 4.7 respectively. PLA2Ⅳ and PLA2Ⅴ showed phospholipase A2 activity towards eggyolk phosphatidylcholine. Nterminal sequences of PLA2Ⅰ, Ⅱ, Ⅲ, Ⅳ and full sequence of PLA2Ⅴ were determined by automatic Edman degradation analysis. PLA2Ⅴ had 122 amino acid residues with 14 cystines.