Resolution and Retention of Proteins near Isoelectric Points in Ion-Exchange Chromatography. Molecular Recognition in Electrostatic Interaction Chromatography
Resolution and Retention of Proteins near Isoelectric Points in Ion-Exchange Chromatography. Molecular Recognition in Electrostatic Interaction Chromatography
复制标题
离子交换色谱中等电点附近蛋白质的分离和保留。
DOI：
10.1081/ss-100102489
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发表时间：
2000
影响因子：
2.8
通讯作者：
T. Ishihara
中科院分区：
文献类型：
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作者：
Shuichi Yamamoto;T. Ishihara
分子识别和转运现象在蛋白质的超高分辨率离子交换（静电相互作用）色谱 (IEC) 中发挥着重要作用。描述了一种快速而简单的方法，用于从线性梯度洗脱实验（盐浓度在固定流动相 pH 值下线性增加）中获取有关分子识别、峰保留（和结合位点数量）和蛋白质峰分辨率的重要信息。使用β-乳球蛋白A和B（LgA、LgB）作为模型蛋白验证了所提出的方法。梯度洗脱实验数据是在各种流动相 pH 值范围内使用各种类型的 IEC 介质获得的。随着流动相 pH 值接近等电点 pI (= 5.1–5.2)，参与保留（吸附）的结合位点数量减少。然而，即使在 pH 5.2 下，LgA 和 LgB 仍保留在阴离子和阳离子交换色谱柱上。当阴离子交换色谱柱的 pH 值接近等电点时，LgA 和 LgB 的分离（分辨率）变得更好，其中吸附位点值的数量较少（约 2-3）。即使使用高效的阳离子交换层析柱也无法分离这两种蛋白质。与 pH 6.0 时相比，等电点 (pH 5.2) 附近的分辨率和保留值并不显着依赖于样品上样量。
Both molecular recognition and transport phenomena play important roles in very high resolution ion-exchange (electrostatic interaction) chromatography (IEC) of proteins. A fast and simple method for obtaining important information on molecular recognition, peak retention (and the number of binding sites), and peak resolution of proteins from linear gradient elution experiments (salt concentration is increased linearly at a fixed mobile phase pH) is described. The proposed method was verified with β-lactoglobulin A and B (LgA, LgB) as model proteins. The gradient elution experimental data were obtained over a wide range of mobile phase pH with various types of IEC media. The number of binding sites involved in the retention (adsorption) decreased as the mobile phase pH approached the isoelectric points pI (= 5.1–5.2). However, even at pH 5.2 both LgA and LgB were retained on anion- and cation-exchange chromatography columns. The separation (resolution) of LgA and LgB became better when the pH approached the pI in anion-exchange chromatography columns where the number of adsorption site values are small(ca. 2–3). The two proteins were not separated even with efficient cation-exchange chromatography columns. The resolution and the retention near the pI (pH 5.2) did not significantly depend on sample loading compared with those at pH 6.0.