AN IMPROVED METHOD FOR THE QUANTITATIVE-DETERMINATION OF DEOXYRIBONUCLEOSIDE TRIPHOSPHATES IN CELL-EXTRACTS
AN IMPROVED METHOD FOR THE QUANTITATIVE-DETERMINATION OF DEOXYRIBONUCLEOSIDE TRIPHOSPHATES IN CELL-EXTRACTS
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DOI：
10.1016/0003-2697(84)90386-5
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发表时间：
1984-01-01
影响因子：
2.9
通讯作者：
WATAYA, Y
中科院分区：
文献类型：
--
作者：
TANAKA, K;YOSHIOKA, A;WATAYA, Y
通过高压液相色谱 [HPLC] 分析细胞提取物中的脱氧核糖核苷 5''-三磷酸 (dNTP)，需要事先选择性降解提取物中大量存在的核糖核苷 5''-三磷酸 (rNTP)。当该方法用于定量哺乳动物细胞提取物中的 dNTP（dATP、dCTP、dGTP、dTTP）时，在 HPLC 中观察到 dTTP 和 DATP 峰之间存在干扰峰。这个不需要的峰有时会与 dATP 的峰重叠，具体取决于洗脱液的 pH 值。给出该峰的物质是在甲胺存在下 rNTP 的高碘酸盐氧化过程中形成的，可以通过改变程序中试剂的添加顺序来去除它，即仅在过量的高碘酸盐分解后才添加甲胺，而不是像原始程序中那样将其与高碘酸盐一起添加。此外，向反应混合物中添加脱氧鸟苷可有效防止氧化过程中 dGTP 的部分损失。使用这种改进的方法，以准确且可重复的方式测量了[小鼠]艾氏腹水肿瘤细胞提取物的 dNTP 含量。该分析需要约 106 个细胞，从收获细胞开始，所有 4 种 dNTP 均可在 2.5 小时内定量。
The analysis of deoxyribonucleoside 5''-triphosphates (dNTP) in cell extracts by high-pressure liquid chromatography [HPLC] requires the prior, selective degradation of ribonucleoside 5''-triphosphates (rNTP) that are present in the extracts in large quantities. When this method was used for quantifying the dNTP (dATP, dCTP, dGTP, dTTP) in mammalian cell extracts, the presence of an interfering peak in the HPLC between the peaks for dTTP and DATP was observed. This unwanted peak sometimes overlapped with that of dATP, depending on the pH of the eluant. The material which gave this peak was formed during the periodate oxidation of rNTP in the presence of methylamine, and it could be removed by changing the order of addition of the reagents in the procedure, i.e., the methylamine was added only after the excess periodate was decomposed, instead of adding it together with periodate, as given in the original procedure. Furthermore, an addition of deoxyguanosine to the reaction mixture was effective in preventing the partial loss of dGTP in the oxidation procedure. Using this improved method, the dNTP contents of the extracts of [mouse] Ehrlich ascites tumor cells were measured in an accurate and reproducible manner. The analysis requires about 106 cells, and all 4 dNTP can be quantified in 2.5 h, starting from the harvest of the cells.