Multiple-injection affinity capillary electrophoresis to examine binding constants between glycopeptide antibiotics and peptides.
Multiple-injection affinity capillary electrophoresis to examine binding constants between glycopeptide antibiotics and peptides.
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DOI：
10.1016/j.chroma.2005.10.062
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发表时间：
2006-02
期刊：
影响因子：
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通讯作者：
J. Zavaleta;Dinora B Chinchilla;Karla Martinez;F. Gomez
中科院分区：
文献类型：
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作者：
J. Zavaleta;Dinora B Chinchilla;Karla Martinez;F. Gomez
采用多次进样亲和毛细管电泳法（MIACE）测定了分别来自东方链霉菌、鲁里达诺卡氏菌和替考拉宁的万古霉素、瑞斯托霉素和替考拉宁与芴甲氧羰基（Fmoc）-（Gly、Ala、瓦尔和Phe）-d-Ala-d-Ala肽之间的结合常数（Kb）。在该技术中，将含有非相互作用标准品、肽一、缓冲液和肽二的单独样品塞注射到毛细管柱中并进行柱层析。肽以相似的电泳迁移率迁移通过柱，但由于肽之间的缓冲塞而保持为不同的区域。然后在运行缓冲液中增加抗生素浓度进行电泳。连续电泳导致肽与抗生素结合后的迁移时间发生变化。作为抗生素浓度的函数，分析所得复合物相对于非相互作用标准品的相对迁移时间比（RMTR）的变化，得出Kb值。MIACE是一种通用的技术，可用于测量相似的相对分子质量和电荷的配体之间的亲和常数，而不需要单独的结合实验。本文描述的发现证明了使用MIACE来估计配体和受体之间的结合参数的优点。
Multiple-injection affinity capillary electrophoresis (MIACE) was used to determine binding constants (Kb) between vancomycin, ristocetin, and teicoplanin from Streptomyces orientalis, Nocardia lurida, and Actinoplanes teichomyceticus, respectively, and fluorenylmethoxycarbonyl (Fmoc)-(Gly, Ala, Val, and Phe)-d-Ala-d-Ala peptides. In this technique, separate plugs of sample containing non-interacting standards, peptide one, buffer, and peptide two, were injected into the capillary column and electrophoresed. Peptides migrate through the column at similar electrophoretic mobilities but remain as distinct zones due to the buffer plug between peptides. The electrophoresis is then carried out in an increasing concentration of antibiotic in the running buffer. Continued electrophoresis results in a shift in the migration time of the peptides upon binding to the antibiotic. Analysis of the change in the relative migration time ratio (RMTR) of the resultant complexes relative to the non-interacting standards, as a function of the concentration of antibiotic yields a value for Kb. MIACE is a versatile technique that can be used to measure affinity constants between ligands of similar relative molecular mass and charge without the need of separate binding experiments. The findings described, herein, demonstrate the advantages of using MIACE to estimate binding parameters between ligands and receptors.