用于表征单纯疱疹病毒1型（HSV-1）胸苷激酶底物和抑制剂的毛细管电泳方法的开发与验证

Development and validation of a capillary electrophoresis method for the characterization of herpes simplex virus type 1 (HSV-1) thymidine kinase substrates and inhibitors.

作者信息

Iqbal Jamshed, Scapozza Leonardo, Folkers Gerd, Müller Christa E

机构信息

Pharmaceutical Institute, Department of Pharmaceutical Chemistry Poppelsdorf, University of Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany.

出版信息

J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Feb 1;846(1-2):281-90. doi: 10.1016/j.jchromb.2006.09.018. Epub 2006 Oct 4.

DOI:10.1016/j.jchromb.2006.09.018

PMID:17023224

Abstract

A fast, convenient capillary electrophoresis (CE) method was developed for monitoring the enzymatic reaction of herpes simplex virus type 1 thymidine kinase (HSV-1 TK). The reaction was performed in a test tube followed by quantitative analysis of the products. The optimized CE conditions were as follows: polyacrylamide-coated capillary (20 cm effective length x 50 microm), electrokinetic injection for 30s, 50 mM phosphate buffer at pH 6.5, constant current of -60 microA, UV detection at 210 nm, UMP or cAMP were used as internal standards. Phosphorylated products eluted within less than 7 min. The limits of detection were 0.36 microM for dTMP and 0.86 microM for GMP. The method was used to study enzyme kinetics, and to investigate alternative substrates and inhibitors.

摘要

开发了一种快速、便捷的毛细管电泳（CE）方法，用于监测单纯疱疹病毒1型胸苷激酶（HSV-1 TK）的酶促反应。反应在试管中进行，随后对产物进行定量分析。优化后的CE条件如下：聚丙烯酰胺涂层毛细管（有效长度20 cm×内径50μm），电动进样30 s，pH 6.5的50 mM磷酸盐缓冲液，-60μA的恒定电流，210 nm处紫外检测，UMP或cAMP用作内标。磷酸化产物在不到7分钟内洗脱。dTMP的检测限为0.36μM，GMP的检测限为0.86μM。该方法用于研究酶动力学，并研究替代底物和抑制剂。