Development and validation of a capillary electrophoresis method for the characterization of herpes simplex virus type 1 (HSV-1) thymidine kinase substrates and inhibitors.
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.
Iqbal
Jamshed
J
Pharmaceutical Institute, Department of Pharmaceutical Chemistry Poppelsdorf, University of Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany.
Scapozza
Leonardo
L
Folkers
Gerd
G
Müller
Christa E
CE
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*
D004791:Enzyme Inhibitors; D013937:Thymidine Kinase