虎纹捕鸟蛛毒素Ⅴ的二硫键定位分析  

Assignment of The Disulfide Bonds of Huwentoxin-Ⅴ From The Venom of Chinese Bird Spider Selenocosmia huwena

作　　者：张鹏飞[1] 肖顺勇[2] 梁宋平[2] 

机构地区：[1]中南大学湘雅医院卫生部肿瘤蛋白质组学重点实验室,长沙410008 [2]湖南师范大学生命科学学院

出　　处：《生物化学与生物物理进展》2004年第6期550-555,共6页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金重大项目资助 (3 9990 60 0 )~~

摘　　要：虎纹捕鸟蛛毒素Ⅴ是从虎纹捕鸟蛛毒液中分离得到的一种昆虫毒素 .它含有 35个氨基酸残基 ,其中 6个半胱氨酸形成三对二硫键 .首先采用多酶将天然的肽链裂解后 ,通过MALDI TOF质谱分析酶解肽段 ,推断出 1对二硫键位于Cys9 Cys2 1,然后利用改进的部分还原分步测序法 ,确定虎纹捕鸟蛛毒素Ⅴ的另外 2对二硫键的配对方式为Cys2 Cys16和Cys15 Cys2 8.因此 ,虎纹捕鸟蛛毒素Ⅴ的 3对二硫键分别以Cys2 Cys16 ,Cys9 Cys2 1,Cys15 Cys2 8(即 1 4、 2 5和 3 6 )的方式配对 .Huwentoxin-V (HWTX-V) is an insecticidal peptide purified from the venom of spider SeInocosmia huwena. HWTX-V contains 35 amino acid residues, including six cystein residues that form three pairs of disulfide bond. The positions of the disulfide bonds of HWTX-V have been determined. To map the disulfide bonds, native HWTX-V was multi-proteolytically digested. The resulting peptides were identified by matrix assisted laser desorption/ionization time of flight mass spectrometry, and it indicated the presence of one disulfide bond Cys9-Cys21. The partially reduced peptides by using Tris- (2-carboxyethyl) -phosphine at pH 3. 0 over 12 minutes were purified by reverse phase high-performance liquid chromatography, and then one -free thiod and two-free thiod fractions were collected. The free thiods were carboxamidomethlate by iodoacetamide at the concentration of 0.5mol/L. The locations of disulfide bond Cys2-Cys16 and Cys15-Cys28 were proved by comparing N-terminal sequencing analysis these partially reduced and alkylated HWTX-V with that of the intact peptide. Finally, the three disulfide linkage of HWTX-V could be assigned as Cys2-Cys16, Cys9-Cys21 and Cys15-Cys28.

关 键 词：虎纹捕鸟蛛毒素V 二硫键定位 三羧甲基磷酸(TCEP) 

分 类 号：O6[理学—化学] Q5[生物学—生物化学]