Journal of Fluorescence

, Volume 23, Issue 4, pp 745–752 | Cite as

Fluorescence Spectroscopic and Calorimetry Based Approaches to Characterize the Mode of Interaction of Small Molecules with DNA



Ethidium bromide displacement assay by fluorescence is frequently used as a diagnostic tool to identify the intercalation ability of DNA binding small molecules. Here we have demonstrated that the method has pitfalls. We have employed fluorescence, absorbance and label free technique such as isothermal titration calorimetry to probe the limitations. Ethidium bromide, a non-specific intercalator, netropsin, a (A-T) specific minor groove binder, and sanguinarine, a (G-C) specific intercalator, have been used in our experiments to study the association of a ligand with DNA in presence of a competing ligand. Here we have shown that netropsin quenches the fluorescence intensity of an equilibrium mixture of ethidium bromide - calf thymus DNA via displacement of ethidium bromide. Isothermal titration calorimetry results question the accepted interpretation of the observed decrease in fluorescence of bound ethidium bromide in terms of competitive binding of two ligands to DNA. Furthermore, isothermal titration calorimetry experiments and absorbance measurements indicate that the fluorescence change might be due to formation of ternary complex and not displacement of one ligand by another.


Ethidium bromide displacement assay DNA Isothermal titration calorimetry Competitive binding Ternary complex 



Ethidium bromide


Isothermal titration calorimetry



ct DNA

Calf thymus DNA





This work was funded by CBAUNP project of Intramural Funding from Department of Atomic Energy, Govt. of India. Jasdeep Singh did this work as a part of his project work in the M.S.(Pharm.), Pharmacoinformatics program of NIPER-Kolkata, Govt. of India.


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Amrita Banerjee
    • 1
  • Jasdeep Singh
    • 2
  • Dipak Dasgupta
    • 1
  1. 1.Biophysics DivisionSaha Institute of Nuclear PhysicsKolkataIndia
  2. 2.Kusuma School of Biological SciencesIndian Institute of Technology-DelhiNew DelhiIndia

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