Simultaneous determination of deoxycytidine diphosphate and deoxycytidine triphosphate by capillary electrophoresis with transient isotachophoretic stacking: a sensitive monitoring method for ribonucleotide reductase activity.
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.
Department of Applied Chemistry, National Chi Nan University, Puli, Nantou County, Taiwan.
Shi-Wei
SW
Huang
Huey-Fen
HF
Tzeng
D000818:Animals; D002461:Cell Line, Transformed; D003845:Deoxycytosine Nucleotides / Q000032:analysis* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D056945:Hep G2 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057625:Isotachophoresis / Q000379:methods*; D016014:Linear Models; D011092:Polyethylene Glycols; D015203:Reproducibility of Results; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012965:Sodium Chloride; D018411:Spodoptera
D003845:Deoxycytosine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D011092:Polyethylene Glycols; D012965:Sodium Chloride; C045813:2'-deoxycytidine diphosphate; D012264:Ribonucleotide Reductases