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We are using time-dependent and steady state measurements of fluorescence to study the physical interaction between DNA and carcinogenic hydrocarbons. The (±)7,8-diol-benzo[a]pyrene(7,8-diolBaP) is used as a model compound that forms a physical complex with DNA by intercalating between the DNA base pairs. An exact emission spectrum can be obtained by measuring the decay-associated emission spectra. In this technique the time-dependent fluorescence decay is measured using time-correlated single-photon counting detection. The data is fit to a sum of exponentials using a weighted non-linear regression program to obtain amplitudes and lifetimes of the emitting species at various wavelengths. Plotting the normalized intensities of a particular lifetime as a function of wavelength yields the emission spectrum associated with the lifetime. The measured fluorescence lifetime is 26.7 nsec for the free 7,8-diolBaP in solution and 5.0 nsec for the bound 7,8-diolBaP. The time resolved emission spectrum of bound hydrocarbon is red-shifted by 6 nm, characteristic of an intercalated complex. Fluorescence quenching experiments with iodide, an external quencher of DNA, gave a bimolecular dynamic quenching constant, 1.4 (10)9 (Msec)-1, for the free hydrocarbon and a constant lifetime and amplitude for the bound 7,8-diolBaP. Fluorescence quenching with low concentrations of silver ions, which bind predominantly to guanine sites of DNA, results in reduced fluorescence intensity of the bound hydrocarbon, with the lifetime remaining unchanged. These quenching studies indicate that at least part of the fluorescence emitted is from 7,8-diolBaP bound at guanine sites. In addition, results of quantum yield experiments have been used to calculate the fraction of bound hydrocarbon which is totally quenched (52%) and not totally quenched (25%). The remainder is free in solution. Quenching experiments with mercury ions suggest that the totally quenched hydrocarbon is bound at adenine-containing sites.
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