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

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 × 50 μm), electrokinetic injection for 30 s, 50 mM phosphate buffer at pH 6.5, constant current of −60 μA, 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 μM for dTMP and 0.86 μM for GMP. The method was used to study enzyme kinetics, and to investigate alternative substrates and inhibitors.