尖吻蝮蛇蛇毒出血毒素DaHT-3的纯化与性质  

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[2] 俞帆[2] 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：《中国生物化学与分子生物学报》1999年第5期762-767,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：云南省应用基础研究基金资助项目!( 95 B0 93 M)

摘　　要：被尖吻蝮蛇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的筛选.通过SephadexG-75,DEAE-SephadexA-50,SephadexG-200和两次PBE聚焦层析,从尖吻蝮蛇(Dienagkistrodonacutus)蛇毒中纯化到一个分子量为56000的出血毒素(DaHT-3),经氨基酸组成测定计算,由487个氨基酸残基组成.此成分在SDS-PAGE上显示出一条均一的蛋白染色带,用等电聚焦电泳测定,其pI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,但没有精氨酸酯酶和磷脂酶A2活力.用红外光谱仪研究DaHT-3在溶液中酰胺I带的吸收谱,该毒素含有31.8%的α螺旋、56.1%的β折叠和12.1%的转角;当加入EDTA螯合剂去除金属离子后,它们的α螺旋、β折叠、转角和无规卷曲分别变为11%、26.4%、46.2%和16.5%,而出血活力和蛋白水解酶活力均丧失。A new hemorrhagic toxin,named DaHT 3,was purified from the venom of Dienagkistrodon acutus using five steps of Sephadex G 75 filtration, DEAE Sephadex A 50 chromatography,Sephadex G 200 filtration and two Poly buffer exchanger chromatofocusings. The purified sample was homogeneous as judged by SDS polyacrylamide gel electrophoresis. DaHT 3 had molecular weight of 56 000 estimated by SDS PAGE,while it was 54 573 using determination of amino acid autoanalyzer with 487 amino acid residues. When the purified sample was judged on the gel of isoelectric focusing electrophoresis, it had isoelctric point (pl) of 5.50. The minimum hemorrhagic dose was measured to be 2 6μg, when DaTH 3 was injected into mice. 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, which exhibited an active unit of 3 68. No arginine esterase and phospholipase A 2 activity were observed with TAME, BAEE and lecithin as substrates, respectively. When absorbent bands of amide I of DaHT 3 was estimated by Fourier transform infrared (FT IR) spectrometer, its contents of secondary structure in aqueous solution were α helix of 31 8%, β pleat sheet of 56 1%, and turn of 12 1%, respectively There was no unordered structure in the sample.After the addition of EDTA, which moved metal ions, the α helix, β pleat sheet, turn, and unordered structure were 11%, 26 4%,46 2%,and 16 5%,respectively.Their hemorrhagic and proteolytic activities at the same time were lost.It could be considered that metal ions, particularly Zn ion, played an important role in maintaining secondary structure of the hemorrhagic toxins purified from snake venoms.It was very clear that the purified sample was a metalloproteinase. The yield of DaTH 3 was 0.5% only.

关 键 词：尖吻蝮蛇毒 出血毒素 理化性质 纯化 金属蛋白酶 

分 类 号：Q556.3[生物学—生物化学] R996.3[医药卫生—毒理学]