Rendement quantique, temps de vie de fluorescence et heterogeneité des sites de fixation de la proflavine sur le DNA

Abstract

AbstractThe binding of Acridine Orange (AO) to DNA at a phosphorus:dye molar ratio higher than 60 results in an increase of the quantum yield of fluorescence of the bound dye with respect to the free dye. The binding of proflavine (Pro) under the same conditions results, on the contrary, in a large decrease of quantum yield. This difference is surprising in view of the common increase of quantum yield of Acridine Orange and proflavine on going from aqueous to glycerol solution, the latter being a solvent with higher viscosity and lower dielectric constant. A comparative study of the spectra, lifetime of fluorescence τ, and quantum yield of fluorescence ϕ of the free and bound dye can shed light on this specific interaction and discriminate between a uniform exaltation or quenching of the dye molecules and a heterogeneity in the binding sites. For AO, the ratio τ/ϕτ0, where τ0 is the “natural” lifetime calculated from absorption spectra, remains constant for free and bound dye, a test for uniform exaltation. For proflavine, τ remains constant (4.4 ± 0.4 × 10−9 sec.), while ϕ drops from 0.7 to 0.23. This result shows that two molecules out of three are bound on a site producing almost complete quenching while the third exhibits “normal” fluorescence, comparable to that of the free dye. It is suggested that intercalation between two adenine thymine base pairs could account for the quenching. Fluorescence techniques would be valuable to study the equilibrium between the binding sites as a function of ionic strength and DNA base composition. By taking advantage of the accuracy of the lifetime determination by a photon‐sampling technique, comparison of τ for direct and sensitized excitation of fluorescence of the bound dye has been performed. No difference has been observed, which indicates a transfer time faster than 0.1 × 10−9 sec., a result in agreement with singlet singlet transfer and with the very low quantum yield of fluorescence of DNA alone at room temperature.