Simultaneous determination of thymidylate and thymidine diphosphate by capillary electrophoresis as a rapid monitoring tool for thymidine kinase and thymidylate kinase activities

Abstract

A simple and rapid capillary electrophoretic method was developed for the simultaneous determination of thymidylate (TMP) and thymidine 5'-diphosphate (TDP) in enzyme assays without using radioactive-labeled substrates. Prior to electrophoretic separation, addition of acetonitrile and sodium chloride to the assay solution and brief centrifugation are recommended for the purpose of sample cleanup and sample stacking. The separation of micromolar TMP and TDP from millimolar adenosine 5'-triphosphate (ATP) was performed at 25 degrees C using sodium tetraborate as the background electrolyte. Under the optimal condition, a good separation with high efficiency was achieved in 6 min. Several parameters affecting the separation were studied, including the pH of electrolyte, the applied voltage, and acetonitrile-salt sample stacking. The fronting of the ATP peak resulting from the interference of magnesium ion in the enzyme assay buffer was suppressed by the addition of sodium ethylenediaminetetraacetate to the sample solution. Using deoxyadenylate as an internal standard, the linear range of the method was 5-200 microM, and the concentration limits of detection of TMP and TDP were 2.6 and 3.8 microM, respectively. Application of the proposed method for simultaneous determination of TMP and TDP in enzyme assays was demonstrated by the activity assays of thymidine kinase and thymidylate kinase from white spot syndrome virus. This is a sensitive, nonradioactive method for thymidine kinase and thymidylate kinase assays.

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 CE-based assay for the determination of TK and thymidylate kinase activity has recently been described [24]. However, the present study is the first report on a CE-based assay for the characterization of various, structurally diverse TK substrates and inhibitors.…”

“…Most recently, Tzeng and Hung published a CE-based assay for the determination of thymidine-5 -monophosphate (dTMP) and thymidine-5 -diphosphate (dTDP) [24] using uncoated fused-silica capillaries. In the present study, we have developed an optimized capillary electrophoresis method with increased sensitivity using coated capillaries, that allows to measure substrates and potential inhibitors for TK within a few minutes with high accuracy and reproducibility.…”

Liquid chromatographic methods for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy: A review

“…With their negative charge, this group of compounds may be easily resolved by capillary electrophoresis (CE) with a reduced analysis time. Recent studies highlight the promising potential of this alternative technique, and particularly when the mass detector was coupled [124][125][126][127]. Finally, nanoLC-MS could also constitute a future improvement of the nucleotide analysis offering a major advance in term of sensitivity and selectivity.…”

Characterization of tyrosine kinase and screening enzyme inhibitor by capillary electrophoresis with laser-induced fluoresce detector

“…Previously, CE has shown to be suitable for enzymatic assays, which are often used as the assay models for HTS [3][4][5][6]. By comparing with other conventional formats of assays, CE has demonstrated Abbreviations: HTS, high throughput screening; CE, capillary electrophoresis; TK, tyrosine kinase; ATP, adenosine triphosphate; ELISA, enzyme-linked immunosorbent assay; LIF, laser-induced fluorescence; MOPS, 3-(N-morpholino)-propanesulfonic acid; ADP, adenosine diphosphate; T, the transformation ratio of the substrate; HD, hydrodynamic; EK, electrokinetic; EOF, electro-osmotic flow.…”