Noncovalent polycationic coatings for capillaries in capillary electrophoresis of proteins
Noncovalent polycationic coatings for capillaries in capillary electrophoresis of proteins
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DOI：
10.1021/ac960432l
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发表时间：
1997-04-01
影响因子：
7.4
通讯作者：
Whitesides, GM
中科院分区：
文献类型：
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作者：
Cordova, E;Gao, JM;Whitesides, GM
熔融石英毛细管壁上净正电荷（pI>pH）的蛋白质的吸附是毛细管电泳分析蛋白质中的常见问题，本文探索使用聚阳离子聚合物作为非共价涂层来限制这个问题，使用未涂层和涂层毛细管比较了三组蛋白质的行为：（i）通过溶菌酶乙酰化获得的蛋白质电荷梯（EC 3.2.1.17）； (ii)通过碳酸酐酶II (EC 4.2.1.1)的乙酰化获得的蛋白质电荷梯； (iii) 具有一系列分子量和 PI 值的蛋白质测试组。检查了四种聚阳离子聚合物：聚乙烯亚胺 (PEI；MW(av) = 15 000)、聚凝胺 (MW(av) = 25 000)、聚(甲氧基乙氧基乙基)乙烯亚胺 (MW(av) = 64 000) 和聚(二烯丙基二甲基氯化铵) (MW(av) = 10 000)。这些聚阳离子聚合物涂层中的三种，但不具有聚(二烯丙基二甲基氯化铵)。对这些涂层稳定性的检查表明它们是稳健的：使用涂有 PEI 或聚凝胺的毛细管，进行 25 次相同分离的重复，电渗的变化现在小于 10%。这项研究表明，通过溶菌酶乙酰化获得的电荷梯是测试聚阳离子涂层效率的良好模型。对该电荷梯成员在 pH 8.3 下的电泳迁移率的研究确定了有效电荷溶菌酶 (Z(p)(O) = +7.6 +/- 0.1) 并确定了溶菌酶 α-铵基团的酸度 (pK(a), = 7.8 +/- 0.1)。蛋白质测试组的结果表明蛋白质吸附主要由静电相互作用驱动。
The adsorption of proteins with net positive charges (pI > pH) on the walls of fused-silica capillaries is a common problem in the analysis of proteins by capillary electrophoresis, This paper explores the use of polycationic polymers as noncovalent coatings to limit this problem, The behavior of three sets of proteins was compared using uncoated and coated capillaries: (i) a protein charge ladder obtained by acetylation of lysozyme (EC 3.2.1.17); (ii) a protein charge ladder obtained by acetylation of carbonic anhydrase II (EC 4.2.1.1); (iii) a test panel of proteins with a range of values of molecular weight and pi. Four polycationic polymers were examined: polyethylenimine (PEI; MW(av) = 15 000), Polybrene (MW(av) = 25 000), poly(methoxyethoxyethyl)ethylenimine (MW(av) = 64 000), and poly(diallyldimethylammonium chloride) (MW(av) = 10 000), Detection of proteins with high pr was readily achieved using the first three of these polycationic polymer coatings but not with the poly(diallyldimethylammonium chloride). Examination of the stability of these coatings indicates that they are robust: the change in electroosmotic now was less than 10% for 25 replications of the same separations, using capillaries coated with PEI or Polybrene, This study demonstrates that the charge ladder obtained by acetylation of lysozyme is a good model with which to test the efficiency of polycationic coatings, A study of the electrophoretic mobilities of the members of this charge ladder at pH 8.3 determined the effective charge of lysozyme (Z(p)(O) = +7.6 +/- 0.1) and established the acidity of the alpha-ammonium group of lysozyme (pK(a), = 7.8 +/- 0.1). Results from the test panel of proteins suggest that protein adsorption is mainly driven by electrostatic interactions.