Multiple-injection affinity capillary electrophoresis to estimate binding constants of receptors to ligands.
Multiple-injection affinity capillary electrophoresis to estimate binding constants of receptors to ligands.
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多次注射亲和毛细管电泳来估计受体与配体的结合常数。
DOI：
10.1007/s00216-005-0047-0
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发表时间：
2005
影响因子：
4.3
通讯作者：
Gomez,FrankA
中科院分区：
文献类型：
--
作者：
Chinchilla,Dinora;Zavaleta,Jose;Martinez,Karla;Gomez,FrankA
采用多次注射亲和毛细管电泳（MIACE）测定受体与配体之间的结合常数（Kb），分别以东方链霉菌（streptomyces orientalisisandactinoplanes teichomyceticus）中的万古霉素（vancomycin）和teicoplanin为模型体系，以及它们与d - ala - d - ala肽和碳酸酐酶B （CAB）的结合。EC 4.2.1.1)和后者与芳基磺酰胺的结合。首先注入含有非相互作用标准物的样品塞，然后注入含有受体的多个样品塞，然后最后注入含有第二种标准物的样品。在每次注射样品之间，注入一个小的缓冲塞，其中含有浓度不断增加的配体，以实现多次注射样品之间的分离。然后在运行缓冲液中增加配体浓度进行电泳。继续电泳会导致受体在与各自的配体结合后在样品塞中的迁移时间发生变化。通过分析相对于非相互作用标准物的受体-配体复合物的相对迁移时间比（RMTR）或电泳迁移率（μ）作为配体浓度的函数的变化，可以得到kb的值。MIACE技术是对ACE方法的改进，它允许在比标准ACE更快的时间尺度上估计生物相互作用之间的结合亲和力。此外，与传统的ACE分析相比，该技术的样本量要求降低了。这些发现证明了使用MIACE来估计受体和配体之间结合参数的优势。
Multiple-injection affinity capillary electrophoresis (MIACE) is used to determine binding constants (Kb) between receptors and ligands using as model systems vancomycin and teicoplanin fromStreptomyces orientalisandActinoplanes teichomyceticus, respectively, and their binding toD-Ala-D-Ala peptides and carbonic anhydrase B (CAB. EC 4.2.1.1) and the binding of the latter to arylsulfonamides. A sample plug containing a non-interacting standard is first injected followed by multiple plugs of sample containing the receptor and then a final injection of sample containing a second standard. Between each injection of sample, a small plug of buffer is injected which contains an increasing concentration of ligand to effect separation between the multiple injections of sample. Electrophoresis is then carried out in an increasing concentration of ligand in the running buffer. Continued electrophoresis results in a shift in the migration time of the receptor in the sample plugs upon binding to their respective ligand. Analysis of the change in the relative migration time ratio (RMTR) or electrophoretic mobility (μ) of the resultant receptor–ligand complex relative to the non-interacting standards, as a function of the concentration of ligand yields a value forKb. The MIACE technique is a modification in the ACE method that allows for the estimation of binding affinities between biological interactions on a timescale faster than that found for standard ACE. In addition sample volume requirements for the technique are reduced compared to traditional ACE assays. These findings demonstrate the advantage of using MIACE to estimate binding parameters between receptors and ligands.