The binding of the known DNA intercalators methylene blue and quinacrine with four sequence specific polynucleotides, viz. poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT), have been compared using absorbance, fluorescence, competition dialysis and thermal melting and the thermodynamic aspects of the interaction studied. In all the cases, non-cooperative binding phenomena obeying neighbor exclusion principle was observed though the affinity was remarkably higher for quinacrine and the nature of the binding was characterized to be true intercalation. The data on the salt dependence of binding derived from the plot of log Kvs. log[Na⁺ ] revealed a slope of around 1.0, consistent with the values predicted by the theories for the binding of monovalent cations, and contained contributions from polyelectrolytic and non-polyelectrolytic forces. The bindings were characterized by strong stabilization of the polynucleotides against thermal strand separation in both optical melting as well as differential scanning calorimetry studies. The data analyzed from the thermal melting and isothermal titration calorimetry studies were in close proximity to those obtained from absorption spectral titration data. Isothermal titration calorimetry results revealed the bindings to poly(dG-dC).poly(dG-dC), poly(dG).poly(dC) and poly(dA-dT).poly(dA-dT) to be exothermic and favoured by both negative enthalpy and large favourable positive entropy changes, while that to poly(dA).poly(dT) was endothermic and entropy driven. The heat capacity changes obtained from temperature dependence of enthalpy gave negative values to all polynucleotides. New insights on the molecular aspects of interaction of these molecules to DNA have emerged from these studies.

There is compelling evidence that cellular DNA is the target of many anticancer agents. Consequently, elucidation of the molecular nature governing the interaction of small molecules to DNA is paramount to the progression of rational drug design strategies. In this study, we have compared the binding and thermodynamic aspects of two known DNA-binding agents, quinacrine (QNA) and methylene blue (MB), with calf thymus (CT) DNA. The study revealed noncooperative binding phenomena for both the drugs to DNA with an affinity one order higher for QNA compared to MB as observed from diverse techniques, but both bindings obeyed neighbor exclusion principle. The data of the salt dependence of QNA and MB from the plot of log K versus log [Na ⁺ ] revealed a slope of 1.06 and 0.93 consistent with the values predicted by theories for the binding of monovalent cations, and have been analyzed for contributions from polyelectrolytic and nonpolyelectrolytic forces. The binding of both drugs was further characterized by strong stabilization of DNA against thermal strand separation in both optical melting and differential scanning calorimetry studies. The binding data analyzed from the thermal denaturation and from isothermal titration calorimetry (ITC) were in close proximity to those obtained from spectral titration data. ITC results revealed the binding to be exothermic and favored by both negative enthalpy and positive entropy changes. The heat capacity changes obtained from temperature dependence of enthalpy indicated −146 and −78 cal/(mol·K), respectively, for the binding of QNA and MB to CT DNA. Circular dichroism study further characterized the structural changes on DNA upon intercalation of these molecules. Molecular aspects of interaction of these molecules to DNA are discussed.

Berberine, palmatine and coralyne, the isoquinoline alkaloids distributed in many botanical families, are extensively investigated due to their potential therapeutic actions and clinical utilities. In this work, their binding characteristics to hemoglobin (Hb) were studied by UV-vis absorption spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, isothermal calorimetric titration and differential scanning calorimetric techniques. The results indicated that all the three alkaloids caused strong fluorescence quenching of Hb by the static quenching mechanism, but with differing quenching efficiencies. There was a single binding site on Hb for these alkaloids. According to the theory of Förster resonance energy transfer, the binding distances between β-Trp37 of Hb and berberine, palmatine and coralyne were evaluated to be 2.78 nm, 2.64 nm and 3.29 nm, respectively. The result of synchronous fluorescence, circular dichroism and 3D fluorescence revealed that the polarity around Trp residues experienced a significant increase in the presence of alkaloids. The binding was favoured by enthalpy and entropy changes. Results of circular dichroism, 3D and synchronous fluorescence studies confirmed that the binding of the alkaloids significantly changed the secondary structure of Hb. The studies revealed that berberine and palmatine bound to a site near to the α1β2 interface on Hb different than coralyne but the affinity of coralyne was one order higher than that of berberine and palmatine.

Interaction of the iminium and alkanolamine forms of sanguinarine with bovine serum albumin (BSA) was characterized by spectroscopic and calorimetric techniques. Formation of strong complexes of BSA with both iminium and alkanolamine forms was revealed from fluorescence quenching of sanguinarine. Binding parameters calculated from Stern-Volmer quenching method revealed that the neutral alkanolamine had higher affinity to BSA compared to the charged iminium form. Specific binding distances of 3.37 and 2.38 nm between Trp 212 (donor) and iminium and alkanolamine forms (acceptor), respectively, were obtained from Forster resonance energy transfer studies. Competitive binding using the site markers warfarin and ibuprofen, having definite binding sites, demonstrated that both forms of sanguinarine bind to site I (subdomain IIA) on BSA. Sanguinarine binding alters protein conformation by reducing the α-helical organization and increasing the coiled structure, indicating a small but definitive partial unfolding of the protein. Thermodynamic parameters evaluated from isothermal titration calorimetry suggested that the binding was enthalpy driven for the iminium form but favoured by negative enthalpy and strong favourable entropy contributions for the alkanolamine form, revealing the involvement of different molecular forces in the complexation. The results suggest that the neutral alkanolamine form binds to the protein more favourably compared to the charged iminium, in stark contrast to the reported DNA binding preference of sanguinarine.