Fluorescence polarization study of DNA–proflavine complexes

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Abstract
The binding of proflavine to DNA has been studied by measuring the polarization and intensity of emission of DNA–dye complexes. Such measurements also permit the determination of the fluorescence of the bound dye as a function of the degree of binding. Techniques of emission spectroscopy permit the study of complexing at high phosphate to dye ratios, and we have examined complexes formed at up to 12,300:1 phosphates to dye. At high phosphate to dye ratios, we find that equilibrium plots of the binding data show only one type of binding. Reports in the literature of multiple binding constants are shown to be due to the incorrect assumption that the fluorescence of the bound dye is independent of the amount bound. The emission properties can be qualitatively accounted for by assuming that nearest‐neighbor interaction between bound dyes quenches the fluorescence. We report that, within experimental error, the binding constant is insensitive to the base content of the DNA. The DNA‐dye complexes show a temperature dependent depolarization, the cause of which is, as yet, unknown. Heat denaturation of the DNA–dye complex may be followed on a Perrin plot.