Iqbal Jamshed, Scapozza Leonardo, Folkers Gerd, Müller Christa E
Pharmaceutical Institute, Department of Pharmaceutical Chemistry Poppelsdorf, University of Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany.
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.
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.
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。
