Get access

Simultaneous determination of deoxycytidine diphosphate and deoxycytidine triphosphate by capillary electrophoresis with transient isotachophoretic stacking: A sensitive monitoring method for ribonucleotide reductase activity

Authors

  • Shi-Wei Huang,

    1. Department of Applied Chemistry, National Chi Nan University, Puli, Nantou County, Taiwan
    Search for more papers by this author
  • Huey-Fen Tzeng

    Corresponding author
    1. Department of Applied Chemistry, National Chi Nan University, Puli, Nantou County, Taiwan
    • Department of Applied Chemistry, National Chi Nan University, 1 University Road, Puli, Nantou County 54561, Taiwan Fax: +886-49-2917956
    Search for more papers by this author

Abstract

A simple and rapid capillary electrophoretic method was developed for simultaneous determination of sub-micromolar 2′-deoxycytidine 5′-diphosphate (dCDP) and 2′-deoxycytidine 5′-triphosphate (dCTP) levels in enzyme assays without using radioactively labeled substrates. The separation was performed at 25°C using MES in the BGE as the terminating ion, the chloride ions in the sample buffer as the leading ion, and PEG 4000 in the BGE as the EOF suppressor for sample stacking by transient isotachophoresis (tITP). Several parameters affecting the separation were investigated, including the pH of the BGE, the concentration of sodium chloride in the sample buffer, and the concentrations of MES and PEG 4000 in the running buffer. Good separation with high separation efficiency was achieved within 6 min under optimal conditions. In comparison with the simple CZE method, the present tITP-CZE method enabled a 150-fold increase in the injection time without any decrease in resolution and the sensitivity was enhanced up to two orders of magnitude with the new method. The linear range of the method was 0.1–10 μM for dCDP and dCTP. The limits of detection of dCDP and dCTP were 85 and 73 nM, respectively. The proposed method was successfully applied for the activity assay of ribonucleotide reductase from Hep G2 and Sf9 cells.

Ancillary