色谱

色谱 ›› 2013, Vol. 31 ›› Issue (1): 59-63.DOI: 10.3724/SP.J.1123.2012.07041

• 研究论文 • 上一篇    下一篇

毛细管区带电泳分析五步蛇毒蛋白C激活剂和蛋白C的相互作用

孙瑶1,2, 包鹏举3, 张根葆1,2*   

  1. 1. 皖南医学院蛇毒研究所, 安徽 芜湖 241002; 2. 皖南医学院病理生理学教研室, 安徽 芜湖 241002; 3. 皖南医学院生理学教研室, 安徽 芜湖 241002
  • 收稿日期:2012-07-26 修回日期:2012-09-16 出版日期:2013-01-28 发布日期:2013-01-21
  • 通讯作者: 张根葆
  • 基金资助:

    安徽省高等学校自然科学研究基金项目(KJ2007B022);皖南医学院中青年科研基金项目(WK201208).

Application of capillary zone electrophoresis in the interaction analysis of protein C with protein C activator from Agkistrodon acutus venom

SUN Yao1,2, BAO Pengju3, ZHANG Genbao1,2*   

  1. 1. Institute of Snake Venom of Wannan Medical College, Wuhu 241002, China; 2. Department of Pathophysiology of Wannan Medical College, Wuhu 241002, China; 3. Department of Physiology of Wannan Medical College, Wuhu 241002, China
  • Received:2012-07-26 Revised:2012-09-16 Online:2013-01-28 Published:2013-01-21

PDF

193

被引次数

9

摘要： 建立了毛细管区带电泳分析五步蛇毒蛋白C激活剂(protein C activator, PCA)与血浆蛋白C(protein C, PC)的相互作用。以未涂层毛细管(60.2 cm(有效长度50 cm)×75 μm)为分离柱,50 mmol/L Tris-HCl(pH 7.4)为运行缓冲液,于198 nm波长下检测。对影响五步蛇毒PCA分离的因素(如缓冲液、离子浓度)及在37.5 ℃下孵育不同时间的五步蛇毒PCA与PC的相互作用进行考察。方法的检出限(以信噪比为3计)为3 mg/L,线性范围为10～300 mg/L。五步蛇毒PCA迁移时间和峰面积的相对标准偏差分别为0.56%和3.8%(n=6)。等体积的五步蛇毒PCA(200 mg/L)与PC(60 mg/L)孵育5 min,其结合率达到最大,且谱图中未见有PC水解的肽链。该五步蛇毒PCA可改变PC空间构象直接激活PC。该方法简单,灵敏度和分辨率高,分析结果为今后快速检测五步蛇毒PCA及其活性提供了重要的理论依据。

关键词: 蛋白C, 蛋白C激活剂, 毛细管区带电泳, 五步蛇毒, 相互作用

Abstract: A new capillary zone electrophoresis method (CZE) has been established for the interaction analysis of protein C (PC) with a protein C activator (PCA) from Agkistrodon acutus venom. The analysis was performed on an uncoated fused-silica capillary with 75 μm i.d. and a total length of 60.2 cm (50 cm to the detector) with a buffer solution of 50 mmol/L Tris-HCl (pH 7.4) and 198 nm of wavelength. The factors which influence the separation of the PCA, such as buffer solution and ion concentration, and the interaction between the PCA and PC incubated for different times at 37.5 ℃ were studied. The linear range was from 10 to 300 mg/L. The limit of detection was 3 mg/L (S/N=3). The relative standard deviation (RSD) for the migration time of the PCA was 0.56%. The RSD for the peak area was 3.8% (n=6). The equal volumes of the PCA (200 mg/L) and PC (60 mg/L) were incubated for five minutes, at which their binding rate reached the maximum. And no hydrolyzed peptide chain from PC was found in the electropherogram. The PCA from Agkistrodon acutus venom could activate PC directly through changing the space conformation of PC. The method is simple, and highly sensitive with high resolution, and will provide important theoretical basis for the rapid detection of venom proteins and their activities in the future.

Key words: Agkistrodon acutus venom, interaction, protein C (PC), protein C activator (PCA), capillary zone electrophoresis (CZE)