Thermodynamic investigation of the association of ethidium, propidium and bis-ethidium to DNA hairpins

Dionisios Rentzeperis, Miriam Medero, Luis A. Marky

Research output: Contribution to journalArticlepeer-review

28 Scopus citations


We have used a combination of calorimetric and spectroscopic techniques to investigate the association of the bis-intercalator ethidium homodimer (bis-ethidium) to short DNA hairpins with sequences: d(GCGCT5GCGC) and d(CGCGT5CGCG). The helix-coil transition of each hairpin, investigated by UV and calorimetric melting protocol, takes place in monomolecular two-state transitions with characteristic enthalpies of ∼37 kcal mol-1 for disrupting the four dG-dC base pairs of the hairpin stems. Deconvolution of the bis-ethidium-hairpin calorimetric titration curves indicate that each hairpin contains two distinct binding sites for the ligand: a high affinity site in the stem (Kb ∼107) that accommodates one bis-ethidium molecule and a lower affinity site (Kb ∼106) located probably at the loop that accommodates two bis-ethidium molecules. The overall stoichiometries of three ligands per hairpin are in agreement with those obtained in continuous variation experiments using visible spectroscopy. The interaction of bis-ethidium for each type of sites results in enthalpy driven reactions, with average binding enthalpies, ΔHb, of -13.1 and -12.1 kcal mol-1 for the stem and loop sites, respectively. Comparison to the thermodynamic profiles of ethidium and propidium binding reveals that the bis-ethidium binding to the stem site of each hairpin has a more favorable free energy term of -1.4 kcal mol-1 and more favorable enthalpy of -4.2 kcal mol-1. These suggest that only one phenanthridine ring of bis-ethidium intercalates in the stem, while the second planar ring is exposed to solvent or weakly associated to the surface of DNA. The Kbs for bis-ethidium binding to the loop sites are about two orders of magnitude larger than the mono-intercalators and correspond to a more favorable free energy of -2.0 kcal mol-1. The enthalpies of binding are also more favorable for bis-ethidium by -2 to -4 kcal mol-1. Overall, the magnitude of Kb for each ligand, and for each site, is in qualitative agreement with the electrostatic contribution from the actual number of positive charges of the ligand. The increased favorable enthalpic contributions of bis-ethidium are consistent with larger hydrophobic contributions, while the increase in unfavorable entropy contributions are consistent with the higher ordering of bis-ethidium in the stem and loop sites.

Original languageEnglish (US)
Pages (from-to)751-759
Number of pages9
JournalBioorganic and Medicinal Chemistry
Issue number6
StatePublished - Jun 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry


Dive into the research topics of 'Thermodynamic investigation of the association of ethidium, propidium and bis-ethidium to DNA hairpins'. Together they form a unique fingerprint.

Cite this