Detection and quantification of adducts formed upon interaction of diamminedichloroplatinum (II) with DNA, by anion-exchange chromatography after enzymatic degradation

Abstract

A method has been developed to determine the adducts formed upon interaction of cis - and trans -diamminedichloroplatinum(II) ( cis - and trans -DDP) with DNA. After 5 h at 50°C in the dark, the amount of cis -DDP bound to salmon sperm DNA was larger than the amount of the trans -isomer. After enzymatic degradation with deoxyribonucleases to nucleotides and Pt-containing (oligo)nucleotides, the various products were separated by DEAE chromatography and analyzed for Pt by flameless AAS. Indications were obtained for the presence of nucleotides containing monofunctionally bound Pt and of adducts originating from interstrand DNA crosslinks. DEAE chromatography of digests of cis -DDP-treated DNA yielded a product with overall charge −1, which was identified with NMR and CD as cis -[Pt(NH ₃ ) ₂ -d(pGpG)], the oligonucleotide derived from intrastrand crosslinks between two adjacent guanines. Another major peak contained Pt-oligonucleotides with overall charge −2, which could be derived from intrastrand crosslinks between two guanines at sites with pGpXpG (X=T,C,A or G) base sequences.