| 2334905 |
Smith RA, Williamson DS, Cerny RL, Cohen SM: Detection of 1,N6-propanodeoxyadenosine in acrolein-modified polydeoxyadenylic acid and DNA by 32P postlabeling. Cancer Res. 1990 May 15;50(10):3005-12.
The interaction of acrolein, an alpha,beta-unsaturated aldehyde, with polydeoxyadenylic acid and DNA has been investigated using 32P-postlabeling analysis. In preliminary experiments, polydeoxyadenylic acid was incubated with excess acrolein and then digested to 3' monophosphates prior to transfer of 32P from [gamma-32P] ATP with T4 polynucleotide kinase. The 3',5'-bisphosphates were 3'-dephosphorylated prior to two-dimensional thin layer chromatography on polyethyleneimine-cellulose layers. Autoradiography provided evidence for the formation of one extra spot of radioactivity, compared to the control. To determine the adduct structure, deoxyadenosine-5'-monophosphate was incubated with a 3-fold excess of acrolein. This material was mixed with a 32P-labeled digest of acrolein-polydeoxyadenylic acid, and the sample was analyzed by ion-pair high performance liquid chromatography. The spot of 32P observed by thin layer chromatography co-eluted with the major product of the acrolein nucleotide reaction mixture, which was purified by ion-pair high performance liquid chromatography. Two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry showed the adduct to be 3-(2'-deoxyribosyl-5'-monophosphatyl)-7,8,9-trihydro-9-hydro xy- pyrimido [2,3-i] purine (1,N6-propanodeoxyadenosine-5'-monophosphate) . High performance liquid chromatography was used to fractionate digests of acrolein-modified DNA prior to detection of this exocyclic adduct by 32P-postlabeling. |
81(1,1,1,1) |