Development and validation of a capillary electrophoresis method for the characterization of herpes simplex virus type 1 (HSV-1) thymidine kinase substrates and inhibitors |
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Authors: | Iqbal Jamshed Scapozza Leonardo Folkers Gerd Müller Christa E |
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Institution: | Pharmaceutical Institute, Department of Pharmaceutical Chemistry Poppelsdorf, University of Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany. |
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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. |
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