Analysis of the ethidium bromide bound to DNA by photoacoustic and FTIR spectroscopy

Under physiological conditions B-form DNA is an exceedingly stable structur e. However, experimental evidences obtained through nuclear magnetic resona nce and fluorescence anisotropy suggest that the structure of the double he lix fluctuates substantially. We describe photoacoustic phase modulation fr equency measurements of ethidium bromide (Eb) with calf thymus, DNA. As in fluorescence phase modulation measurements, we used an intercalating dye as a probe; however, we monitored the triplet excited state lifetime at diffe rent ionic strengths. The triplet lifetime of Eb varied from about 0.30 ms, with no DNA present, to 20 ms, (at a DNA:Eb molar ratio of 5). With salt t itration, this value falls, to about 2.0 ms. This result suggests, a strong coupling between the phenantridinium ring of the ethidium and the base pai rs because of the stacking movement of the DNA molecule under salt effect. This, effect may be understood considering DNA as a polyelectrolyte. The co unterions, in the solution shield the phosphate groups, reducing the electr ostatic repulsion force between them, hence compacting the DNA molecule. Th e results from Fourier transform infrared demonstrated two important bands: 3187 cm(-1) corresponding to the symmetric stretching of the NH group of t he bases, and 1225 cm(-1) corresponding to the asymmetric stretching of pho sphate groups shifted toward higher wavenumbers, suggesting a proximity bet ween the intercalant and base pairs and a modification of the DNA backbone state, both induced by salt accretion.