部分还原和分步序列测定法定位虎纹捕鸟蛛毒素-Ⅳ二硫键  

Assignment of the Disulfide Bonds of Huwentoxin-Ⅳ by Partial Reduction and Sequence Analysis

作　　者：刘中华[1] 梁宋平[1] 

机构地区：[1]湖南师范大学生命科学学院,长沙410081

出　　处：《高等学校化学学报》2003年第10期1815-1819,共5页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金 (批准号 :3 93 70 15 2 )资助

摘　　要：结合部分还原和分步序列测定法确定了虎纹捕鸟蛛毒素 - 的二硫键配对方式 .在 p H=3和 40℃的条件下与还原剂三羧甲基磷酸 ( TCEP)反应 1 0 min,利用 RP-HPLC分离并分别收集含有一对和两对二硫键被还原的中间体 ,分别与 0 .5 mol/L碘乙酰胺溶液 ( p H=8.3 )反应 1 min,使游离巯基烷基化后 ,测定各中间体的氨基酸序列 ,从而确定虎纹捕鸟蛛毒素 - 的 3对二硫键分别为 Cys2 -Cys1 7,Cys9-Cys2 4和Cys-Cys 3 1 ( 1 -4,2 -5和 3 -6) .Huwentoxin-Ⅳ, a neurotoxic peptide from the spider Ornithoctonus huwena, contains three disulfide bonds in a chain of 35 residues. Fully the assignment of disulfide bonds was made by partial reduction and sequence analysis. Huwentoxin-Ⅳ was partially reduced with tris(2-carboxyethyl)-phosphine at 40 ℃ for 10 min at pH=3 and the intermediates were isolated by reversed-phase HPLC. Appropriate isomers containing free thiols were alkylated by adding 100 μL of 0.5 mol/L iodoa- cetamide, respectively, following by sequencer analysis, which enabled all three bridges Huwentox- in-Ⅳ to be identified as Cys 2-Cys 17, Cys 9-Cys 24 and Cys-Cys 31.

关 键 词：部分还原 分步序列 测定法 定位 虎纹捕鸟蛛毒素-Ⅳ 二硫键 三羧甲基磷酸 毒素多肽 

分 类 号：Q516[生物学—生物化学] Q503