【摘要】 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物质的量的芴甲氧羰酰琥珀酰亚胺(FMOC-OSu)溶液,室温下反应30min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的物质的量做校准曲线.该直线外推到峰面积为0时所对应的FMOC-OSu物质的量即为参加反应的蛋白质表面的氨基的物质的量.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.更多还原

【Abstract】 A method for determining the free amino groups on the surface of proteins by capillary electro- phoresis was established. 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.更多还原