Quantitation of deoxyribonucleoside 5'-triphosphates by a sequential boronate and anion-exchange high-pressure liquid chromatographic procedure.
A rapid method for the quantitative determination of cellular deoxyribonucleoside 5'-triphosphates is described. Cell extracts are first separated by boronate chromatography at pH 8.9, which removes 99% of the ribonucleoside triphosphate (rNTPs) from the deoxyribonucleoside triphosphates (dNTPs). The resulting dNTP fraction is analyzed by gradient high-pressure liquid chromatography utilizing a strong anion-exchange column, which can separate minor rNTP peaks from the corresponding dNTPs. This sequential procedure, which requires less than 1 h per sample for both chromatographic steps, results in the quantitative recovery of greater than 98% of the dNTPs from cell extracts. Nucleotide analogs, such as 1-beta-D-arabinofuranosylcytosine-5'-triphosphate and 5-bromo-2'-deoxyuridine-5'-triphosphate, can also be quantitated efficiently by this method.
Shewach
D S
DS
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor 48109.
D001897:Boronic Acids; D002021:Buffers; D002457:Cell Extracts / Q000737:chemistry; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003854:Deoxyribonucleotides / Q000032:analysis* / Q000302:isolation & purification; D005910:Glioma / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008214:Lymphocytes / Q000737:chemistry; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D001897:Boronic Acids; D002021:Buffers; D002457:Cell Extracts; D003854:Deoxyribonucleotides