Mourato L L, Beland F A, Marques M M
Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Chem Res Toxicol. 1999 Jul;12(7):661-9. doi: 10.1021/tx990038t.
32P-Postlabeling is an extremely powerful technique for the detection of DNA adducts. Typically, the quantitation of DNA adducts by (32)P-postlabeling is achieved by relative adduct labeling, via comparison of the radioactivity incorporated into the adducts to that associated with the normal nucleotides. This approach is based on a number of assumptions, the foremost being that normal and adducted nucleotide 3'-phosphates are converted to 3', 5'-bisphosphates with similar efficiencies. To evaluate labeling efficiencies for specific DNA adducts, we conducted a comparative study of the kinetics of phosphorylation by T(4) polynucleotide kinase using 2'-deoxyguanosine 3'-phosphate (dG3'p) and a series of N-(deoxyguanosin-8-yl)arylamine 3'-phosphate adduct standards (dG3'p-C8-Ar, Ar being 4-aminobiphenyl, 3- and 4-methylaniline, and 2,4- and 3,4-dimethylaniline). Phosphorylation of dG3'p and the dG3'p-C8-Ar adducts followed Michaelis-Menten kinetics. The apparent turnover numbers were 40-240-fold lower when labeling dG3'p-C8-Ar adducts compared to that when labeling dG3'p. The apparent specificity constant calculated for dG3'p-C8-4-aminobiphenyl (1.4 microM(-)(1) min(-)(1)) was approximately 4-fold lower than that (5. 4 microM(-)(1) min(-)(1)) found for dG3'p. Apparent specificity constants for the monoarylamine adducts were even lower (0.043-0.23 microM(-)(1) min(-)(1)) and decreased in the following order: 4-methylaniline > 3,4-dimethylaniline > 3-methylaniline > 2, 4-dimethylaniline. Similar experiments conducted with dG3'p-C8-Ar standards for 2-methylaniline and 2,3-dimethylaniline produced very poor and irreproducible labeling. These results indicate that (32)P-postlabeling of dG3'p-C8-Ar adducts is less efficient than that of dG3'p and suggest that normal nucleotides will be labeled preferentially to the arylamine adducts under kinetically controlled conditions. The data also indicate a further decrease in labeling efficiency upon substitution ortho to the amino group (e.g., 2, 4-dimethylaniline). In addition, the ATP concentrations required for optimal labeling were found to be substantially higher than those used in typical (32)P-postlabeling assays. Since the high specific activity of carrier-free [gamma-(32)P]ATP precludes increasing the ATP concentration to a significant extent, these data emphasize the need for using highly efficient adduct enrichment procedures when conducting (32)P-postlabeling analyses of DNA adducts.
32P后标记法是检测DNA加合物的一种极其强大的技术。通常，通过32P后标记法定量DNA加合物是通过相对加合物标记来实现的，即通过比较掺入加合物中的放射性与与正常核苷酸相关的放射性。这种方法基于一些假设，其中最重要的是正常和加合的核苷酸3'-磷酸以相似的效率转化为3',5'-二磷酸。为了评估特定DNA加合物的标记效率，我们使用2'-脱氧鸟苷3'-磷酸（dG3'p）和一系列N-（脱氧鸟苷-8-基）芳基胺3'-磷酸加合物标准品（dG3'p-C8-Ar，Ar为4-氨基联苯、3-和4-甲基苯胺以及2,4-和3,4-二甲基苯胺），对T(4)多核苷酸激酶的磷酸化动力学进行了比较研究。dG3'p和dG3'p-C8-Ar加合物的磷酸化遵循米氏动力学。与标记dG3'p相比，标记dG3'p-C8-Ar加合物时的表观转换数低40-240倍。为dG3'p-C8-4-氨基联苯计算的表观特异性常数（1.4 microM(-)(1) min(-)(1)）比dG3'p的表观特异性常数（5.4 microM(-)(1) min(-)(1)）低约4倍。单芳基胺加合物的表观特异性常数甚至更低（0.043-0.23 microM(-)(1) min(-)(1)），且按以下顺序降低：4-甲基苯胺>3,4-二甲基苯胺>3-甲基苯胺>2,4-二甲基苯胺。用2-甲基苯胺和2,3-二甲基苯胺的dG3'p-C8-Ar标准品进行的类似实验产生的标记效果很差且不可重复。这些结果表明，dG3'p-C8-Ar加合物的32P后标记效率低于dG3'p，并表明在动力学控制条件下，正常核苷酸将比芳基胺加合物优先被标记。数据还表明，在氨基邻位取代（如2,4-二甲基苯胺）后标记效率会进一步降低。此外，发现最佳标记所需的ATP浓度大大高于典型32P后标记分析中使用的浓度。由于无载体[γ-(32)P]ATP的高比活度使得无法在很大程度上提高ATP浓度，这些数据强调了在对DNA加合物进行32P后标记分析时使用高效加合物富集程序的必要性。
