通过荧光后标记法检测和定量X射线辐照的小牛胸腺DNA中的8-羟基脱氧鸟苷5'-单磷酸。

Detection and quantitation of 8-hydroxydeoxyguanosine 5'-monophosphate in X-irradiated calf-thymus DNA by fluorescence postlabeling.

作者信息

Sharma M, Box H C, Paul C R

机构信息

Department of Biophysics, Roswell Park Memorial Institute, Buffalo, New York 14263.

出版信息

Biochem Biophys Res Commun. 1990 Mar 16;167(2):419-24. doi: 10.1016/0006-291x(90)92039-3.

DOI:10.1016/0006-291x(90)92039-3

PMID:2157405

Abstract

8-Hydroxydeoxyguanosine 5'-monophosphate (8-OH dGmp) was synthesized from deoxyguanosine 5'-monophosphate (dGmp) by ascorbic acid in the presence of hydrogen peroxide and labeled with dansyl chloride through a phosphoramidate linkage with ethylenediamine (EDA). A DNA model 8-OHd(TACG), isolated intact by high pressure liquid chromatography (HPLC) from x-irradiated d(TACG) and characterized by nmr, was digested enzymatically to 5'-mononucleotides. The modified nucleotide was enriched by HPLC and dansylated. Analysis of the dansylated product by HPLC, using a fluorescent detector, detected a peak with retention time corresponding to that of the dansyl labeled authentic marker. The same overall procedure was used to detect 8-OHdGmp from x-irradiated calf-thymus DNA. The content of 8-OHdGmp in the irradiated DNA increased linearly with increasing levels of x-irradiation in the dose range of 6-60 Gy.

摘要

8-羟基脱氧鸟苷5'-单磷酸（8-OH dGmp）由脱氧鸟苷5'-单磷酸（dGmp）在过氧化氢存在下通过抗坏血酸合成，并通过与乙二胺（EDA）形成磷酰胺键用丹磺酰氯标记。通过高压液相色谱（HPLC）从经X射线辐照的d（TACG）中完整分离出DNA模型8-OHd（TACG），并通过核磁共振进行表征，然后将其酶解为5'-单核苷酸。通过HPLC对修饰的核苷酸进行富集并进行丹磺酰化。使用荧光检测器通过HPLC分析丹磺酰化产物，检测到一个保留时间与丹磺酰标记的 authentic 标记物相对应的峰。使用相同的总体程序从经X射线辐照的小牛胸腺DNA中检测8-OHdGmp。在6-60 Gy的剂量范围内，辐照DNA中8-OHdGmp的含量随X射线辐照水平的增加呈线性增加。