title: 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 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.
authors.affiliation: Pharmaceutical Institute, Department of Pharmaceutical Chemistry Poppelsdorf, University of Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany.
authors.forename: Jamshed
authors.initials: J
authors.lastname: Iqbal
authors.forename: Leonardo
authors.initials: L
authors.lastname: Scapozza
authors.forename: Gerd
authors.initials: G
authors.lastname: Folkers
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis*; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D007700:Kinetics; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D013937:Thymidine Kinase