尖吻蝮蛇蛇毒出血毒素的纯化与部分性质  

Purification and Partial Characterization of Hemorrhagic Toxin from the Venom of Dienagkistrodon acutus

作　　者：叶文娟[1] 冉永禄[1] 赖仞[1] 龚光 林俞帆 曾昭权[2] 

机构地区：[1]中国科学院昆明动物研究所,昆明650223 [2]云南大学实验中心,昆明650091

出　　处：《蛇志》1999年第3期21-24,共4页Journal of Snake

基　　金：云南省应用基础研究基金

摘　　要：目的　被尖吻蝮蛇 （ Ｄｉｅｎａｇｋｉｓｔｒｏｄｏｎ ａｃｕｔｕｓ） 咬伤会引起严重的出血， 对蛇毒出血毒素的研究有利于治疗蛇伤出血药物筛选。 方法　采用 Ｓｅｐｈａｄｅｘ Ｇ７５， Ｄ Ｅ Ａ Ｅ Ｓｅｐｈａｄｅｘ Ａ５０， Ｓｅｐｈａｄｅｘ Ｇ２００ 和两次 Ｐ Ｂ Ｅ 聚焦层析纯化。 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 电泳和等电聚焦电泳测定纯化样品的纯度和等电点。氨基酸组成用自动氨基酸分析仪测定。以小鼠背部皮下注射部位出血斑的面积来确定最小出血剂量和常规的方法测定酶活性。结果　从尖吻蝮蛇毒中纯化到一个相对分子量为５６ ０００ 的出血毒素 （ Ｄａ Ｈ Ｔ３）， 经氨基酸组成测定计算，它由 ４８７ 个氨基酸残基组成。此成分在 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ上显示出一条均一的蛋白染色带， 其ｐ Ｉ为５５０。该出血成分的最小出血剂量是 ２６μｇ， 具有蛋白水解酶活力， 其活力为 ３６８， 但没有精氨酯酶和磷脂酶 Ａ２ 活力。当加入 Ｅ Ｄ Ｔ Ａ 螯合剂去除金属离子后， 它们的出血活力和蛋白水解酶活力均丧失。 结论　这是从大陆尖吻蝮蛇毒中获得的一个新的出血金属蛋白酶 （ Ｄａ Ｈ Ｔ３）。Objective Studies on hemorrhagic toxins of snake venoms are beneficial for medical screening Methods Five step separations of Sephadex G 75 filtration, DEAE Sephadex A 50 chromatography, Sephadex G 200 filtration, and two Polybuffer exchanger chromatofocusing were employed The purified sample and molecular weight were estimated on SDS polyacrylamide gel electrophoresis pI was detected by the gel of isoelectric focusing electrophoresis Amino acid composition was determinated with amino acid autoanalyzer The minimum hemorrhagic dose(MHD) was defined as the lowest dose which caused an area of hemorrhage of 5mm in diameter 6 hours after subcutaneous injection The purified sample was assayed by using casein as substrate Arginine esterase activity and phospholipase A 2 activity were observed with TAME, BAEE and lecithin as substrates Results A new hemorrhagic toxin(DaHT 3) was purified from the venom of Dienagkistrodon acutus DaHT 3 had molecular weight of 56 000 with 487 amino acid residues and isoelctric point(pI) was 5 50 The minimum hemorrhagic dose was measured to be 2 6μg It exhibited proteolytic active unit of 3 68 and there were no arginine esterase activity and phospholipase A 2 activity When the hemorrhagic toxin treated with EDTA was again assayed for hemorrhagic and proteolytic activity, the both activities disappeared The yield of DaHT 3 was only 0 5% Conclusion This hemorrhagic toxin was a metalloproteinase

关 键 词：蛇毒 尖吻蝮蛇 出血毒素 纯化 物理化学性质 

分 类 号：Q959.620.6[生物学—动物学]