Binding of alkaloid harmalol to DNA: Photophysical and calorimetric approach

•Harmalol exists in two forms-structure I protonated form at pH 6.8 and structure II deprotonated form at pH 9.2.•Structure I-DNA complexation show strong, intercalative mode of binding.•Structure II does not bind to DNA and in presence of high concentration of DNA is completely transformed to structure I.•Polyanionic phosphate backbone of DNA influences the formation of structure I from structure II.•The conversion is markedly suppressed with increasing salt concentration of the media.

Harmalol exhibits pH dependent structural equilibrium between protonated and deprotonated forms with a pKa of 7.8 as revealed from spectroscopic titration. The compound exists as protonated (structure I) and deprotonated (structure II) form in the pH range 1–7 and 9–12, respectively. The interaction of structure I and II to calf thymus DNA has been studied by different spectroscopic and calorimetric techniques in buffer of pH 6.8 and 9.2, respectively. The results show that structure I bind strongly to DNA showing a cooperative mode with a binding constant of 4.5 × 105 M−1 and a stoichiometry of 4.8 nucleotide phosphates. The alkaloid stabilized the DNA by 8 °C, the binding shows 40% quenching of fluorescence intensity, perturbation in circular dichroism spectra and enthalpy driven exothermic binding with a large hydrophobic contribution to the binding free energy. Furthermore, the alkaloid shows a prominent change of specific viscosity with sonicated linear DNA and unwinding–rewinding of covalently closed pUC 18 DNA, revealing intercalative binding. The deprotonated structure (structure II), on the other hand, in the presence of large amount of DNA concentration, converts back to a structure I-DNA complexation. This transition has been presumably induced by the polyanionic phosphate backbone of DNA at high concentration.

Graphical abstractThe aim of this work is to investigate the interaction of two structural forms of harmalol (I and II) with CT DNA at pH 6.8 and 9.2, and to correlate the structural differences with the energetics of the interaction. We reported for the first time that structure I of harmalol at pH 6.8 shows strong intercalative binding with the DNA whereas at pH 9.2 in presence of large concentration of DNA, structural form II of harmalol is completely converting back to structure I-DNA complexation i.e. it shifts the equilibrium to the left.Figure optionsDownload full-size imageDownload as PowerPoint slide