Spectroscopic studies on the binding interaction of phenothiazinium dyes toluidine blue O, azure A and azure B to DNA

Spectrochim Acta A Mol Biomol Spectrosc. 2013 Apr 15;107:303-10. doi: 10.1016/j.saa.2013.01.063. Epub 2013 Feb 1.


In this study a detailed characterization of the binding aspects of three phenothiazinium dyes, toluidine blue O (TBO), azure A and azure B with herring testes DNA is presented employing spectroscopic techniques. The absorbance and fluorescence properties of these dyes have been remarkably modified upon binding with DNA and the interaction is manifested through noncooperative binding as revealed form non-linear Scatchard plots with negative slopes at all binding ratios. The binding clearly revealed the high preference of TBO to DNA followed by the other two dyes azure A and azure B. The affinity of TBO was higher by about two times than that of the azures. From the series of studies using absorption, steady-state emission, the effect of ferrocyanide ion-induced steady-state fluorescence quenching, fluorescence polarization anisotropy, circular dichroism, the mode of binding of these dyes to the DNA double helix has been substantiated to be principally intercalative in nature. The stoichiometry of the association of these dyes to DNA was determined by the continuous variation analysis of Job from fluorescence data. The conformational aspects of the interaction was delineated from circular dichroism studies wherein higher perturbation was observed with TBO. Hydrodynamic study using viscosity measurements of linear rod like DNA confirmed that the binding was intercalative and strongest for TBO and weaker for azure A and azure B. The utility of the present work lies in exploring the potential binding applicability of these dyes to DNA for their development as effective therapeutic agents.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Azure Stains / metabolism*
  • Binding Sites
  • Circular Dichroism
  • Coloring Agents / metabolism*
  • DNA / chemistry
  • DNA / metabolism*
  • Fishes
  • Intercalating Agents / metabolism*
  • Models, Molecular
  • Nucleic Acid Conformation / drug effects
  • Spectrometry, Fluorescence
  • Spectrophotometry
  • Tolonium Chloride / metabolism*


  • Azure Stains
  • Coloring Agents
  • Intercalating Agents
  • Tolonium Chloride
  • Azure B
  • DNA
  • Azure A