通过高效液相色谱法对肽段特定残基生物素化的动力学分析

Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography.

作者信息

Kurosky A, Miller B T, Knock S L

机构信息

Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0645.

出版信息

J Chromatogr. 1993 Feb 12;631(1-2):281-7. doi: 10.1016/0021-9673(93)80534-f.

DOI:10.1016/0021-9673(93)80534-f

PMID:8450022

Abstract

A procedure employing C18 reversed-phase high-performance liquid chromatography (HPLC) is described for evaluating the kinetics of biotinylation of specific residues of peptides after reaction with N-hydroxysuccinimide esters of biotin. Utilizing this HPLC method, we determined the observed pseudo-first-order reaction rate constant (k'1) of biotinylation of lysyl residues in two model peptides, [biotinyl-Ser108]ProA-egg laying hormone (108-121) and pGlu-Lys-Trp-Ala-Pro, to be 1.22.10(-2)s-1 and 1.08.10(-2)s-1, respectively, in 0.05 M sodium phosphate buffer, pH 8.2, at 25 degrees C. The respective reaction half-lives of the two peptides were 57 s and 64 s. In addition, HPLC analytical methods were established for determining the time-course of hydrolysis of biotinylating reagents at acidic and alkaline pH and for evaluating biotin reagent homogeneity.

摘要

描述了一种采用C18反相高效液相色谱（HPLC）的方法，用于评估肽的特定残基与生物素的N-羟基琥珀酰亚胺酯反应后生物素化的动力学。利用这种HPLC方法，我们测定了两种模型肽[生物素化-Ser108]ProA-产卵激素（108-121）和pGlu-Lys-Trp-Ala-Pro中赖氨酸残基生物素化的表观一级反应速率常数（k'1），在25℃、pH 8.2的0.05 M磷酸钠缓冲液中，分别为1.22×10⁻² s⁻¹和1.08×10⁻² s⁻¹。这两种肽各自的反应半衰期分别为57秒和64秒。此外，还建立了HPLC分析方法，用于测定生物素化试剂在酸性和碱性pH下的水解时间进程以及评估生物素试剂的均一性。