Detection and quantitation of 8-hydroxydeoxyguanosine 5'-monophosphate in X-irradiated calf-thymus DNA by fluorescence postlabeling.
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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Department of Biophysics, Roswell Park Memorial Institute, Buffalo, New York 14263.
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D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000528:radiation effects*; D006150:Guanine Nucleotides / Q000032:analysis*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives / Q000032:analysis* / Q000138:chemical synthesis; D009838:Oligodeoxyribonucleotides / Q000528:radiation effects; D013950:Thymus Gland; D014965:X-Rays
D006150:Guanine Nucleotides; D009838:Oligodeoxyribonucleotides; C063477:8-hydroxydeoxyguanosine 5'-monophosphate; D006157:Guanosine Monophosphate; D004247:DNA