Evaluation of association constants between drug enantiomers and human α1-acid glycoprotein by applying a partial-filling technique in affinity capillary electrophoresis
Evaluation of association constants between drug enantiomers and human α1-acid glycoprotein by applying a partial-filling technique in affinity capillary electrophoresis
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DOI：
10.1021/ac970766q
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发表时间：
1998-04-01
影响因子：
7.4
通讯作者：
Westerlund, D
中科院分区：
文献类型：
--
作者：
Amini, A;Westerlund, D
本文以α(1)-酸性糖蛋白(AGP)为例，介绍了部分填充毛细管区带电泳法测定药物对映体与蛋白质条件结合常数的原理。在部分填充技术中，只在毛细管的第一部分填充选择器，并且可以通过在恒压下在不同时间引入选择器溶液来改变选择器区域(塞子)的长度。该技术的一个重要特征是选择剂溶液的低消耗。在本研究中，每次只使用40-290 nL，当选择剂的可用性有限时尤其重要，而且在其昂贵的情况下也是如此。选择的条件是使蛋白质具有净负电荷并向阳极迁移，而分析物向阴极侧的检测器迁移。对异丙吡胺和雷米普利对映体的分离表明，分离度与有效塞子长度呈线性关系。可以计算有效塞长，这是应用这一技术测定缔合常数的基础。测定的缔合常数表明，AGP与溶质对映体之间的缔合随温度的升高而变化。研究发现，对映体与AGP之间的结合在25℃时最强，在较低和较高温度下都会降低。这一意想不到的发现可能表明蛋白质的构象随着温度的变化而变化。
The principles for evaluation of conditional association constants between drug enantiomers and proteins, exemplified here by alpha(1)-acid glycoprotein (AGP), using capillary zone electrophoresis employing a partial filling technique, is presented. In the partial filling technique only the first part of the capillary is filled with the selector, and this selector zone (plug) length can be varied by introducing the selector solution at different times at constant pressure. An important feature of the technique is the low consumption of selector solution in this study only 40-290 nL is used per run, of special importance when the availability of the selector is limited, and also in case it is expensive. Conditions are chosen so that the protein has a net negative charge and migrates toward the anode, while the analytes migrate toward the detector at the cathodic side. The resolution is linearly related to the effective plug length, as shown in separations of the enantiomers of disopyramide and remoxipride. The effective plug length can be calculated, which forms the basis to apply this technique for determinations of association constants. The association between the enantiomers of the solutes and AGP varied with increasing temperature, as shown by determined association constants. It was found that the association between the enantiomers and AGP was strongest at 25 degrees C and decreased at both lower and higher temperatures. This unexpected finding may indicate conformational changes of the protein with temperature variations.