Analysis of the Ethidium Bromide Bound to DNA by Photoacoustic and FTIR Spectroscopy¶

Abstract

Under physiological conditions B-form DNA is an exceedingly stable structure. However, experimental evidences obtained through nuclear magnetic resonance and fluorescence anisotropy suggest that the structure of the double helix fluctuates substantially. We describe photoacoustic phase modulation frequency 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 different 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 titration, this value falls to about 2.0 ms. This result suggests a strong coupling between the phenantridinium ring of the ethidium and the base pairs because of the stacking movement of the DNA molecule under salt effect. This effect may be understood considering DNA as a polyelectrolyte. The counterions in the solution shield the phosphate groups, reducing the electrostatic repulsion force between them, hence compacting the DNA molecule. The results from Fourier transform infrared demonstrated two important bands: 3187 cm-1 corresponding to the symmetric stretching of the NH group of the bases and 1225 cm-1 corresponding to the asymmetric stretching of phosphate groups shifted toward higher wavenumbers, suggesting a proximity between the intercalant and base pairs and a modification of the DNA backbone state, both induced by salt accretion.