CONDITIONS INFLUENCING YIELD AND ANALYSIS OF 8-HYDROXY-2'-DEOXYGUANOSINE IN OXIDATIVELY DAMAGED DNA
CONDITIONS INFLUENCING YIELD AND ANALYSIS OF 8-HYDROXY-2'-DEOXYGUANOSINE IN OXIDATIVELY DAMAGED DNA
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DOI：
10.1016/0003-2697(90)90544-j
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发表时间：
1990-07-01
影响因子：
2.9
通讯作者：
HOGSETT, WE
中科院分区：
文献类型：
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作者：
FLOYD, RA;WEST, MS;HOGSETT, WE
我们进行了研究，以获得有关氧化损伤DNA中8-羟基-2-脱氧鸟苷（8-OHdG）含量的稳定性，消化和分析测定的实用知识。利用H2O2 +紫外光形成氧化损伤DNA，我们发现DNA在-20度下储存。在碱性条件下，8-OHdG损失显著，而在-20℃条件下，8-OHdG损失显著。中性或酸性pH下的C可防止8-OHdG的损失。DNA中的8-OHdG在100度时是稳定的。C至少15分钟。当使用低水平的磷酸盐缓冲液时，使用紫外光和H2O2作为羟基自由基生成系统在DNA内形成8-OHdG的产量最高；但使用Tris或柠檬酸缓冲液会导致8-OHdG的产量降低，因为这些缓冲液起到了清除羟基自由基的作用。通过使用水杨酸捕获对羟基自由基通量进行独立评估，可以评估竞争性自由基反应。在DNA酶解之前，对塑料微管进行乙醇洗涤，提高了8-OHdG的产量，减少了样品之间的差异。利用DNA酶I、核酸内切酶、磷酸二酯酶和碱性磷酸酶对氧化损伤的DNA进行消化，可产生最高产量的8-OHdG。
We have conducted studies to obtain practical knowlege regarding the stability, digestion, and analytical determination of the content of 8-hydroxy-2-deoxyguanosine (8-OHdG) in oxidatively damaged DNA. Utilizing H2O2 plus uv light to form oxidatively damaged DNA, we found that storage of the DNA at -20.degree. C at alkaline pH caused a significant loss of 8-OHdG, whereas storage at -20.degree. C at neutral or acidic pH prevented loss of 8-OHdG. The 8-OHdG within DNA is stable at 100.degree. C for at least 15 min. Formation of 8-OHdG within DNA using uv light and H2O2 as a hydroxyl free radical-generating sysem yields the highest amounts when low levels of phosphate buffer are used; but the use of Tris or citrate buffers causes a lower yield of 8-OHdG because these buffers act as scavengers for the hydroxyl free radicals. Independent assessment of hydroxyl free radical flux by the use of salicylate trapping allows assessment of competitive radical reactions. Ethanol washing of plastic microfuge tubes prior to DNA enzymatic digestion improved the yield of 8-OHdG and reduced the variability between samples. Digestion of the oxidatively damaged DNA by the use of a method involving DNase I, endonuclease, phosphodiesterase, and alkaline phosphatase produced the highest yield of 8-OHdG.