Detection and quantitation of 8-hydroxydeoxyguanosine 5′-monophosphate in x-irradiated calf-thymus DNA by fluorescence postlabeling

Add time:07/31/2019    Source:sciencedirect.com

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.

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