Determination by fluorescence quenching of the environment of DNA crosslinks made by malondialdehyde.

DNA crosslinks made by malondialdehyde are fluorescent. The fluorescence is quenched by collision or intercalation. A 3.3-fold higher concentration of the external (collision) quencher KI was required to cause 50% quenching of the fluorescence of the interstrand-DNA crosslinks than to cause 50% quenching of the fluorescence of the model compounds Val2MDA and the malondialdehyde-crosslinked heterodimer of GMP and CMP. Thus, the crosslinked nucleotide dimers in the DNA were shown to be 70% shielded from the solvent. Similarly, DNA-protein crosslinks made by malondialdehyde were shown to be 55% shielded. The internal (intercalation) quencher Ag+ enhanced the fluorescence of the DNA crosslinks at concentrations below 0.3 mM; higher concentrations quenched the fluorescence. Concentrations of Ag+ below 10 mM did not affect the fluorescence of the model compounds. The calculated dissociation constant for Ag+ was much less at pH 5 than at pH 7 or 9. The observed binding of Ag+ and its pH dependence suggest that pi-stacking of adjacent bases strengthens the binding of Ag+ to the crosslinks. These results indicate that the crosslinks are in the interior of the DNA, so they may not easily be recognized by a repair system.