Interaction between methylene blue and calf thymus deoxyribonucleic acid by spectroscopic technologies

Characterization of the interaction between methylene blue (MB) and calf thymus deoxyribonucleic acid (ctDNA) was investigated by UV absorption spectra, fluorescence spectra, fluorescence polarization and fluorescence quenching experiments by ferrocyanide. The above results indicated that the binding modes of MB to ctDNA were relative to the molar ratio gamma (gamma=[DNA]/[MB]). At low gamma ratios (gamma < 4), remarkable hypochromic effect with no shift of lambda(max) in the absorption spectra of MB was observed in the presence of increasing amounts of ctDNA, the fluorescence of MB was efficiently quenched by the ctDNA bases and the fluorescence polarization of MB was slightly increased, which indicated that MB cations bound to phosphate groups of ctDNA by electrostatic interaction and then stacked on the surface of ctDNA helix. While at high gamma ratios (gamma > 6), besides the fluorescence of MB was quenched efficiently by the ctDNA bases, a red shift (about 3 nm) in the absorption spectra of MB was observed and the fluorescence polarization of MB was obviously increased, which indicated the intercalation binding that MB molecules were intercalated into the space of two neighbouring DNA base pairs was the preferred mode. Effects of K(4)Fe(CN)(6) on the fluorescence quenching of the MB-ctDNA system at low and high gamma ratios were also performed. The results showed that at gamma = 1.7, the quenching effect by ferrocyanide was higher than that of pure MB, while at gamma = 13.6 a decreased quenching of the fluorescence intensity was observed as compared with that of pure MB, which further proved the above conclusion. In addition, the mechanisms of the hypochromic effect and the fluorescence quenching were also discussed in detail.