虎纹捕鸟蛛毒素-Ⅴ去折叠过程的色谱分析  

Reversed-Phase High Performance Liquid Chromatographic Behavior of Unfolding Procedure of Huwentoxin-Ⅴ

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,湖南长沙410008 [2]湖南师范大学生命科学学院教育部蛋白质化学与鱼类发育生物学重点实验室,湖南长沙410081

出　　处：《色谱》2004年第4期390-393,共4页Chinese Journal of Chromatography

基　　金：国家自然科学基金重大项目资助 (No .3 9990 60 0 )

摘　　要：将纯化的天然虎纹捕鸟蛛毒素 Ⅴ经盐酸胍变性 30min后 ,在pH 3 0、反应温度为 37℃的条件下与三羧甲基磷酸 (TCEP)反应 12min ,用反相高效液相色谱分离得到其全部去折叠中间体 ,通过基体辅助激光解吸电离飞行时间质谱 (MALDI TOFMS)进行鉴定 ,并利用烷基化反应对这些去折叠中间体予以进一步确证。根据其保留时间 ,分析虎纹毒素 Ⅴ各去折叠中间体的色谱行为 ,初步探讨了多肽或蛋白质构象异构体反相色谱行为的多样性。Huwentoxin-Ⅴ (HWTX-Ⅴ) is an insecticidal peptide purified from the venom of spider Selnocosmia huwena. HWTX-Ⅴ contains 33 amino acid residues, including six cysteine residues that form three pairs of disulfide bond. A method for measuring unfolding intermediates of HWTX-Ⅴ by reversed-phase high performance liquid chromatography (HPLC) has been established. HWTX-Ⅴ was first denatured over 30 min in the guanidine hydrochloride, and then reduced by using tris-(2-carboxyethyl)-phosphine at pH 3.0 over 12 min. All intermediates were separated on a C_(18) column (4.6 mm i.d.×390 mm) with a linear gradient elution of acetonitrile containing 0.1% (v/v) trifluoroacetic acid, and identified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Finally, these intermediates were carboxamidomethylated by iodoacetamide at the concentration of 0.5 mol/L and verified by MALDI-TOF MS. The diverse chromatographic retention behaviors for intermediates of Huwentoxin-Ⅴ are discussed. This method is helpful to reveal the conformation changes in the procedures of proteins/peptides unfolding.

关 键 词：反相高效液相色谱 基体辅助激光解吸电离-飞行时间质谱 三羧甲基磷酸 去折叠 虎纹捕鸟蛛毒素-V 

分 类 号：O658[理学—分析化学]