Preparative capillary zone electrophoresis in combination with off-line graphite furnace atomic absorption for the analysis of DNA complexes formed by a new aminocoumarine platinum (II) compound.
Calf thymus DNA was incubated in vitro with a new aminocoumarin platinum (II) complex in order to study its interaction with DNA. The platinated DNA was hydrolyzed enzymatically to the 5'-mononucleotide level using DNAase I and nuclease P1. Analysis of the DNA hydrolysate with capillary zone electrophoresis (CZE), using sample stacking, revealed the presence of unhydrolyzed oligonucleotides in the platinated DNA. A homemade system, using only some plastic pipet tips, was constructed to collect the oligonucleotide fraction during CZE analysis. The platinum content of this fraction was determined using graphite furnace atomic absorption with Zeeman background correction. This system proved to be a useful tool to detect platinated DNA species (with a quantifiable detection limit for the detection of platinum of 0.78 ng). Subsequent gel filtration experiments confirmed the presence of high molecular weight oligonucleotides that were platinated. This was proven by reversal of the platination using thiourea and subsequent enzymatic hydrolysis to 5'-mononucleotides.
Deforce
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DL
Laboratory for Pharmaceutical Biotechnology, University of Ghent, Belgium.
Kokotos
G
G
Esmans
E L
EL
De Leenheer
A
A
Van den Eeckhout
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EG
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D004247:DNA / Q000032:analysis* / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006868:Hydrolysis; D008970:Molecular Weight; D009944:Organoplatinum Compounds / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013890:Thiourea / Q000494:pharmacology
D009944:Organoplatinum Compounds; C117022:dichloro-di(6-aminocoumarin)platinum(II); D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013890:Thiourea