Serologic assay of DNA base damage. I. 5-Hydroxymethyldeoxyuridine, a radiation product of thymidine.
Serologic assay of DNA base damage. I. 5-Hydroxymethyldeoxyuridine, a radiation product of thymidine.
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DNA 碱基损伤的血清学测定。
作者：
H. L. Lewis;D. Muhleman;J. Ward
我们建立了一种5-羟甲基脱氧尿苷（5-HMUdR）的血清学检测方法。制备针对5-羟甲基尿苷（5-HMUR）的牛血清白蛋白缀合物的抗体。使用噬菌体中和试验，可以检测到0.1纳摩尔的5-HMUR。抗血清与胸苷发生交叉反应，但不与其他天然存在的DNA成分发生交叉反应。与尿苷、6-甲基尿嘧啶及胸苷的卤代类似物有交叉反应。通过用过量胸苷饱和试验，消除了与胸苷交叉反应的干扰。在存在8 mM胸苷的情况下，可检测到3微摩尔（0.3 nmole/0.1 ml）5-HMUR。该测定法用于估计由..产生的5-HMUdR。伽马..在O/sub 2/中照射胸苷。产物形成的G值为0.05。在pH 14下照射大大提高了产率。当在pH 9下加热或用10/sup-3/ M亚铁离子处理辐照样品时，G值增加了3倍，辐照后程序将胸苷甲基过氧化物转化为5-HMUdR。这表明与过氧化物的交叉反应并不广泛。估计5-HMUdR在DNA照射O/分2/给出了一个G值大致相同的发现在照射的胸苷。该产率基于抗体与DNA中的5-HMU部分以及作为单核苷酸同等反应的假设。5-HMUdR检测为每3 × 10/sup 6/道尔顿DNA一个核苷。«少
We have developed a serologic assay for 5-hydroxymethyldeoxyuridine, (5-HMUdR) an ionizing radiation product of thymidine. Antibodies were prepared to a bovine serum albumin conjugate of 5-hydroxymethyluridine (5-HMUR). Using phage neutralization assay 0.1 nmole of 5-HMUR can be detected. The antiserum cross-reacted with thymidine but with no other naturally occurring DNA components. There was cross-reaction with uridine, 6-methyluracil, and the halogenated analogs of thymidine. Interference by cross-reaction with thymidine was eliminated by saturation of the assay with excess thymidine. Three micromolar (0.3 nmole/0.1 ml) 5-HMUR can be detected in the presence of 8 mM thymidine. The assay was applied to the estimation of 5-HMUdR produced by ..gamma.. irradiation of thymidine in O/sub 2/. The G value for product formation was 0.05. Irradiation at pH 14 greatly increased the yield. There was a threefold increase in G value when irradiated samples were heated at pH 9 or treated with 10/sup -3/ M ferrous ions, postirradiation procedures which convert thymidine methylperoxides to 5-HMUdR. This suggests that cross-reaction with the peroxide is not extensive. Estimation of 5-HMUdR in DNA irradiated in O/sub 2/ gave a G value roughly the same as that found in irradiation of thymidine. This yield is based on the assumptionmore » that antibody reacts equally with the 5-HMU moiety in DNA and as the mononucleoside. 5-HMUdR was detected as one nucleoside per 3 x 10/sup 6/ daltons of DNA.« less