Estimation of binding constants between ristocetin and teicoplanin and peptides using on-column ligand derivatization coupled to affinity capillary electrophoresis.
Estimation of binding constants between ristocetin and teicoplanin and peptides using on-column ligand derivatization coupled to affinity capillary electrophoresis.
复制标题
使用柱上配体衍生化与亲和毛细管电泳结合来估计瑞斯托菌素和替考拉宁与肽之间的结合常数。
DOI：
10.1007/s00216-004-2574-5
复制
发表时间：
2004
影响因子：
4.3
通讯作者：
Gomez,FrankA
中科院分区：
文献类型：
--
作者：
Azad,Maryam;Brown,Abby;Silva,Isba;Gomez,FrankA
本工作利用柱上配基合成和亲和毛细管电泳法(ACE)测定了9-茂甲氧基甲酰基(FMOC)-氨基酸衍生物与糖肽类抗生素瑞斯托菌素(Rist)和替考拉宁(Teic)的结合常数(Kb)。在这项技术中，样品的两个单独的塞子被注入毛细管柱并进行电泳法。初始样品塞含有ad-Ala-d-Ala端肽和一个或两个非相互作用的标准品(S)。第二个塞子含有Fmoc-氨基酸-N-羟基琥珀酰亚胺(NHS)酯。然后在运行缓冲液中增加Rist或TEIC的浓度进行电泳。经电泳后，初始丙氨酸-d-丙氨酸与Fmoc-氨基酸反应生成新的Fmoc-氨基酸-d-丙氨酸-d-丙氨酸。连续电泳法可使Rist或Teic与Fmoc-氨基酸-d-丙氨酸-d-丙氨酸多肽衍生物结合。分析Fmoc-氨基酸-d-丙氨酸-d-丙氨酸多肽衍生物相对于非相互作用标准的相对迁移时间比(RMTR)或电泳迁移率(μ)的变化，作为Rist和Teic浓度的函数，得到Kb值。这些发现证明了将柱上配体合成与ACE偶联用于估计抗生素与配体之间的结合参数的优势。
This work utilizes on-column ligand synthesis and affinity capillary electrophoresis (ACE) to determine binding constants (Kb) of 9-flourenylmethyloxy carbonyl (Fmoc)-amino acid derivatives to the glycopeptide antibiotics ristocetin (Rist) and teicoplanin (Teic). In this technique, two separate plugs of sample are injected on to the capillary column and electrophoresed. The initial sample plug contains ad-Ala-d-Ala terminus peptide and either one or two non-interacting standard(s). The second plug contains a Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester. The electrophoresis is then carried out with an increasing concentration of Rist or Teic in the running buffer. Upon electrophoresis the initiald-Ala-d-Ala peptide reacts with the Fmoc-amino acid yielding a new Fmoc-amino acid-d-Ala-d-Ala peptide derivative. Continued electrophoresis results in the binding of Rist or Teic to the Fmoc-amino acid-d-Ala-d-Ala peptide derivatives. Analysis of the change in the relative migration time ratio (RMTR) or electrophoretic mobility (μ) of the Fmoc-amino acid-d-Ala-d-Ala peptide derivatives relative to the non-interacting standards, as a function of the concentration of Rist and Teic, yields a value forKb. These findings demonstrate the advantage of coupling on-column ligand synthesis to ACE for estimating binding parameters between antibiotics and ligands.