Protein retention in ion-exchange chromatography:: effect of net charge and charge distribution
Protein retention in ion-exchange chromatography:: effect of net charge and charge distribution
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DOI：
10.1016/s0021-9673(00)00190-4
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发表时间：
2000-04-28
影响因子：
4.1
通讯作者：
Ståhlberg, J
中科院分区：
文献类型：
--
作者：
Hallgren, E;Kálmán, F;Ståhlberg, J
采用电荷调节板模型评价了葡萄球菌核酸酶A及其突变体在阳离子交换色谱中的保留对盐的依赖性。这项工作的一个重要特征是，蛋白质的净电荷以两种不同的方式变化：(a)通过改变洗脱液的pH值，使质子化产生电荷；(b)通过116位的点突变产生电荷。由于葡萄球菌核酸酶和突变体的结构是已知的，不同突变体保留数据的pH依赖性为保留机制提供了详细的见解。实验结果表明，淋洗液pH值的变化对盐依赖性的影响比对点突变的影响更大。这意味着116位氨基酸仅与固定相表面有中等强度的相互作用，而蛋白质表面一侧的一个斑块主要负责与表面的静电相互作用。(C) 2000 Elsevier Science B.V.版权所有
The charge regulated slab model is used to evaluate the salt dependence of the retention of Staphylococcal nuclease A and its mutants in cation-exchange chromatography. An important feature of this work is that the net charge of the proteins is varied in two different ways: (a) by changing the eluent pH so that the charges are created by protonation and (b) by point mutation at position 116. Since the structure of Staphylococcal nuclease and the mutants are known, the pH dependence of retention data of the different mutants gives detailed insights into the retention mechanism. Experimental results show that the salt dependence of retention is affected more strongly by changes of the eluent pH than by point mutations. This implies that the amino acid in position 116 has only a moderately strong interaction with the stationary phase surface and that a patch on one side of the protein surface is mainly responsible for the electrostatic interaction with the surface. (C) 2000 Elsevier Science B.V. All rights reserved.