高等学校化学学报

高等学校化学学报 ›› 2003, Vol. 24 ›› Issue (10): 1815.

• 论文 • 上一篇    下一篇

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

刘中华, 梁宋平   

  1. 湖南师范大学生命科学学院, 长沙 410081
  • 收稿日期:2002-09-17 出版日期:2003-10-24 发布日期:2003-10-24
  • 通讯作者: 梁宋平(1946年出生),男,博士,教授,博士生导师,从事多肽化学研究.E-mail:liangsp@public.cs.hn.cn E-mail:liangsp@public.cs.hn.cn
  • 基金资助:

    国家自然科学基金(批准号:39370152)资助

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

LIU Zhong-Hua, LIANG Song-Ping   

  1. College of Life Sciences, Hunan NormaLUniversity, Changsha 410081, China
  • Received:2002-09-17 Online:2003-10-24 Published:2003-10-24

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被引次数

7

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pH=3和40℃的条件下与还原剂三羧甲基磷酸(TCEP)反应10min,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0.5mol/L碘乙酰胺溶液(pH=8.3)反应1min,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-Cys17,Cys9-Cys24和Cys-Cys31(1-4,2-5和3-6).

关键词: 虎纹捕鸟蛛毒素-Ⅳ, 二硫键定位, 三羧甲基磷酸, 部分还原, 序列测定

Abstract: 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 10min at pH=3 and the intermediates were isolated by reversed-phase HPLC.Appropriate isomers containing free thiols were alkylated by adding 100μL of 0.5 mol/Liodoa-cetamide, respectively, following by sequencer analysis, which enabled all three bridges Huwentox-in-Ⅳ to be identified as Cys2-Cys17, Cys9-Cys24 and Cys-Cys31.

Key words: Huwentoxin-Ⅳ, Assignment of disulfide bonds, TCEP, Partial reduction, Sequence determination

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