93 Spectroscopic investigation of methylene blue binding to DNA

Abstract

The interaction of methylene blue (MB) with DNA has been investigated by UV absorption spectra, Fluorescence spectra and UV-melting method. Analysis of the results of the melting experiments shows that melting temperature (T m) of the complexes increases with the [total ligand]: DNA ratio (r) at two concentrations of Na+ (2 mM Na+ and 20 mM Na+) providing support for conclusion that MB is a stabilizer of DNA helix structure. By contrast, the shapes of dependences of width of transition (ΔT) on r at low and high [Na+] are different which points to the existence of different types of binding modes of MB with DNA. UV-spectroscopy experiments and fluorescence spectra indicated that the binding modes of MB with DNA depended on r. At high r (r > 0.25), remarkable hypochromic effect with no shift of λ max in the absorption spectra of MB was observed. The fluorescence of MB was quenched which indicated that MB was bound to phosphate groups of DNA by electrostatic interaction. At low r ratios (r < 0.2), the absorption spectra of MB upon increasing the concentration of DNA showed gradually decrease in the peak intensities with a red shift. This phenomenon is usually associated with molecular intercalation into the base stack of the ds-DNA. Using the Scatchard’s model, the complex formation constants for MB with DNA were determined: the binding constant K ≈ 6.5 × 105 and binding site size n ≈ 4. Obtained data are not typical for intercalation model of ligands to DNA. Moreover, comparison between these data and our early experimental results of interaction of ethidium bromide with DNA made it possible to suggest that this binding type of MB is, more probably, semi-intercalation mode (Vardevanyan et al., 2003). This conclusion is in accordance with the analysis of the model structures of MB–DNA complexes which clearly shows the importance of solvent contributions in suggested structural form (Tong et al., 2010).