The binding of the polynuclear aromatic dyes acridine orange (AO) and proflavin (PF) to DNA in aqueous phosphate buffer solution at 25 ± 1°C has been studied by measuring the properties of the triplet excited states of these dyes. The triplet lifetimes can be measured either by triplet-triplet absorption flash photolysis techniques or by delayed fluorescence methods. The triplet lifetimes of AO vary from about 0.5 ms with no DNA present to 20-35 ms at DNA concentration above 10-3 M expressed in concentration of DNA phosphate [P], or at [P]/[D] ratios above 1000 ([D] is the dye concentration). At all DNA concentrations the decay profiles are exponential, except at high excitation intensities where nonexponentialities, attributed to triplet-triplet annihilation, become apparent. Similar results are observed with PF-DNA solutions. The exponentiality of the triplet decay at all DNA concentrations is attributed to rapid association and dissociation of the dye-DNA complexes on the time scales of the triplet lifetimes. A simplified one-step binding model is utilized to describe this effect. A dissociation rate of AO-DNA complexes ≳ 103 s-1 has been estimated from these results. It is shown that a detailed study of the triplet lifetime vs. DNA concentration provides a novel method for the estimation of the apparent equilibrium association constant K* for dye molecules in the triplet excited state and DNA. For AO, K* ≈ 105 M-1, while for PF it is ≈ 3 × 104 M-1. These values are of the same order of magnitude as the ground-state dye-DNA equilibrium association constants measured by others.
ASJC Scopus subject areas
- General Engineering
- Physical and Theoretical Chemistry