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有机化学
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有机化学 ›› 2006, Vol. 26 ›› Issue (05): 707-710. 上一篇    下一篇

研究简报

胶束电动色谱法测定蛋白质表面自由氨基

毛文学1，苏勇*,1，康经武2   

  1. (1华东理工大学生物工程学院 上海 200237)
    (2中国科学院上海有机化学研究所 上海 200032)
  • 收稿日期:1900-01-01 修回日期:1900-01-01 发布日期:2006-04-28

Determination of Free Amino Groups in Protein by Micellar Elec-trokinetic Chromatography

MAO Wen-Xue1,SU Yong*,1,KANG Jing-Wu2   

  1. (1 Institute of Bioengineering, East China University of Science and Technology, Shanghai 200237)
    (2 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032)
  • Received:1900-01-01 Revised:1900-01-01 Published:2006-04-28

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建立了一种毛细管电泳测定蛋白质分子表面自由氨基的方法. 配制蛋白质样品溶液, 分取5等份, 分别加入过量的不同摩尔数的芴甲氧羰酰琥珀酰亚胺(FMOC-OSu)溶液, 室温下反应30 min后, 用胶束电动色谱测定蛋白质溶液中剩余的FMOC-OSu的峰面积. 以FMOC-OSu的峰面积对加入的FMOC-OSu的摩尔数做校准曲线. 该直线外推到峰面积为0时所对应的FMOC-OSu摩尔数即为参加反应的蛋白质表面的氨基的摩尔数. 因此可以计算出蛋白质表面的氨基数目. 用甘氨酸检验了本方法的可靠性. 以牛血清白蛋白为蛋白质模型, 测得其表面氨基数为32, 和文献报道值一致. 该方法简单易行并且反应条件温和, 可用于测定蛋白质修饰过程中的平均修饰度

关键词: 蛋白质自由氨基, 氨基的测定, 胶束电动色谱

A method for determining the free amino groups on the surface of proteins by capillary electrophoresis is described. A protein solution was equally divided into 5 vials. Different amount of excess 9-fluorenylmethoxycarbonylsuccinimide (FMOC-OSu) solution was then added to each vial, followed by mixing and keeping at room temperature for 30 min. The unreacted FMOC-OSu was separated from the protein by a micellar electrokinetic chromatography (MEKC). The peak area of the unreacted FMOC-OSu was measure and the calibration curve was established between the molar numbers of added FMOC-OSu against the corresponding peak areas of unreacted FMOC-OSu. The molar numbers of amino groups on the surface of protein should be equal to that of the reacted FMOC-OSu, which could be gained by extending the calibration curve to cross the x-axis, where the peak area of unreacted FMOC-OSu was zero implying that all added FMOC-OSu reacted with the amino groups. Glycine solution was used to validate the method. Bovine serum albumin (BSA) was selected as a model protein and the number of free amino groups on the surface of the BSA was determined as 32, which is consistent with the reported value. The method can be potentially used to determine the average modification degree of the PEGylation proteins.

Key words: free amino groups of protein, determination of the amino group, micellar electrokinetic chromatography