A SIMPLIFIED HPLC METHOD FOR SIMULTANEOUSLY QUANTIFYING RIBONUCLEOTIDES AND DEOXYRIBONUCLEOTIDES IN CELL-EXTRACTS OR FROZEN TISSUES
A SIMPLIFIED HPLC METHOD FOR SIMULTANEOUSLY QUANTIFYING RIBONUCLEOTIDES AND DEOXYRIBONUCLEOTIDES IN CELL-EXTRACTS OR FROZEN TISSUES
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DOI：
10.1111/j.1365-2184.1993.tb00328.x
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发表时间：
1993-07-01
影响因子：
8.5
通讯作者：
JAMES, SJ
中科院分区：
文献类型：
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作者：
CROSS, DR;MILLER, BJ;JAMES, SJ
诱导脱氧核糖核苷酸库改变的试剂和条件可以对DNA合成速率以及细胞周期进程具有重要的调节作用。提出了一种利用反相高效液相色谱-二极管阵列检测器分离核糖核苷三磷酸（NTP）和脱氧核糖核苷三磷酸（dNTP）的简化方法。同一细胞提取物内NTP和dNTP峰的同时分离有效地消除了对提取后步骤的需要，例如先前用于降解或从dNTP分离共洗脱NTP的高碘酸盐氧化和/或硼酸盐亲和色谱。根据经验发现两种核苷酸dGTP和ADP的分辨率随C18柱的效率而变化。高效柱（>90 000板/m）提供了良好的分离;然而，效率较低的柱导致dGTP和ADP的共洗脱。这些共洗脱的核苷酸可以准确地定量，如果需要的话，使用二极管阵列技术和数学表达式，其中包括摩尔峰系数和峰面积，通过在双波长监测获得。用三氯乙酸提取组织样品或单细胞悬液，将中和的提取物直接进样至色谱柱中，无需预先冻干。标准品的回收率大于或等于99%，样品内或样品间的重复提取具有高度重现性（SD
Agents and conditions that induce alterations in deoxyribonucleotide pools can have important regulatory effects on the rate of DNA synthesis as well as cell cycle progression. A simplified procedure for the separation of both ribonucleoside triphosphates (NTP) and deoxyribonucleoside triphosphates (dNTP) is presented which utilizes reversed phase high-performance liquid chromatography coupled with diode array detection. The simultaneous resolution of NTP and dNTP peaks within the same cell extract effectively eliminates the need for post-extraction steps such as periodate oxidation and/or boronate affinity chromatography previously used to degrade or isolate co-eluting NTP from dNTP. The resolution of two nucleotides, dGTP and ADP, was found empirically to vary with the efficiency of the C18 column. High efficiency columns (>90 000 plates/m) provided good separation; however, less efficient columns resulted in co-elution of dGTP and ADP. These co-eluting nucleotides can be accurately quantified, if necessary, using diode array technology and a mathematical expression which incorporates molar peak coefficients and peak areas obtained by monitoring at dual wavelengths. Tissue samples or single cell suspensions were extracted with trichloroacetic acid and the neutralized extract was injected directly into the column without prior lyophilization. The per cent recovery of standards was greater-than-or-equal-to 99% and replicate extractions within or between samples were highly reproducible (SD