Shewach D S
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor 48109.
Anal Biochem. 1992 Oct;206(1):178-82. doi: 10.1016/s0003-2697(05)80030-2.
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.
本文描述了一种定量测定细胞脱氧核糖核苷5'-三磷酸的快速方法。首先通过在pH 8.9条件下的硼酸酯色谱法分离细胞提取物，该方法可从脱氧核糖核苷三磷酸（dNTP）中去除99%的核糖核苷三磷酸（rNTP）。所得的dNTP馏分通过使用强阴离子交换柱的梯度高压液相色谱法进行分析，该方法可将较小的rNTP峰与相应的dNTP分离。这个顺序过程每个样品的两个色谱步骤所需时间不到1小时，能从细胞提取物中定量回收超过98%的dNTP。核苷酸类似物，如1-β-D-阿拉伯呋喃糖基胞嘧啶-5'-三磷酸和5-溴-2'-脱氧尿苷-5'-三磷酸，也可通过该方法有效定量。
