USING CAPILLARY ELECTROPHORESIS TO FOLLOW THE ACETYLATION OF THE AMINO-GROUPS OF INSULIN AND TO ESTIMATE THEIR BASICITIES
USING CAPILLARY ELECTROPHORESIS TO FOLLOW THE ACETYLATION OF THE AMINO-GROUPS OF INSULIN AND TO ESTIMATE THEIR BASICITIES
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DOI：
10.1021/ac00114a001
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发表时间：
1995-09-15
影响因子：
7.4
通讯作者：
WHITESIDES, GM
中科院分区：
文献类型：
--
作者：
GAO, JM;MRKSICH, M;WHITESIDES, GM
毛细管电泳（CE）是一种分析方法，可用于研究改变蛋白质电荷的过程，本文探讨了毛细管电泳的能力，合理的电荷和电泳迁移率的一个简单的蛋白质-胰岛素及其酰化衍生物-作为一个功能的pH值。胰岛素是一种肽激素（MW = 5700），其具有两个α-氨基（G（α）和F-alpha）和一个ε-氨基（K-alpha）。用乙酸酐处理胰岛素得到七种衍生物，它们在三个氨基的乙酰化位点上不同。对这些衍生物的电泳迁移率的pH依赖性的分析给出了两个N-末端铵基团的pK（a）值：pK（a），（G（α））= 8.4; pK（a）（F-α）= 7.1。在pH 5.5 - 9.5范围内，从电泳迁移率估计的胰岛素总电荷值与从其可电离基团的pK（a）值估计的胰岛素总电荷值相差小于0.5单位（牛和人胰岛素）。胰岛素电泳迁移率的浓度依赖性分析得出K-D大于或等于6 × 10（3）M（-1）（25 mM tris和192 mM Gly，pH 8.4）的胰岛素二聚体缔合常数下限。电泳迁移率作为一个函数的研究。pH值和氨基乙酰化程度使胰岛素的电荷详细合理化。由于毛细管电泳对电荷的敏感性，可以定量研究溶液中蛋白质的静电性质。胰岛素是一种有用的小分子蛋白质模型，可以用来研究电泳现象。
Capillary electrophoresis (CE) is an analytical method that is useful for investigating processes that modify the charge of proteins, This paper explores the ability of CE to rationalize charges and electrophoretic mobilities of a simple protein-insulin and its acylated derivatives-as a function of pH. Insulin is a peptide hormone (MW = 5700) that has two alpha-amino groups (G(alpha) and F-alpha) and one epsilon-amino group (K-epsilon). Treatment of insulin with acetic anhydride affords seven derivatives that differ in the sites of acetylation of the three amino groups. Analysis of the pH dependence of the electrophoretic mobilities of these derivatives gives pK(a) values for the two N-terminal ammonium groups: pK(a), (G(alpha)) = 8.4; pK(a) (F-alpha) = 7.1. Values of the total charge of insulin estimated from electrophoretic mobility differ from those estimated from values of pK(a) for its ionizable groups by less than 0.5 unit for both bovine and human insulins over the range of pH from 5.5 to 9.5. Analysis of the concentration dependence of the electrophoretic mobility of insulin yields a lower limit for the association constant for dimerization of insulin of K-D greater than or equal to 6 x 10(3) M(-1) (25 mM tris and 192 mM Gly, pH 8.4). Studies of electrophoretic mobility as a function of. pH and extent of acetylation of amino groups rationalize the charge of insulin in detail. The sensitivity of CE to charge permits the quantitative study of electrostatic properties of proteins in solution, Insulin is a useful small-protein model with which to investigate phenomena in electrophoresis.