Two component (ethidium bromide-caffeine, ethidium bromide-DNA) and three component (ethidium bromidecaffeine-DNA) systems in aqueous saline (0.01 M NaCl) phosphate buffer solutions (pH 6.86, T=298 K) are studied spectrophotometrically. The equilibrium constants for dimerization of caffeine, K D = 1.22 ± 2 M -1 , and for heteroassociation of ethidium bromide with caffeine, K = 71 ± 8 M -1 , in ethidium bromide-caffeine systems are determined. When the concentration of caffeine is increased, the dynamic equilibrium of the solution shifts toward formation of heterocomplexes which are, presumably, stabilized by dispersive and hydrophobic interactions of chromophores. The equilibrium parameters for ethidium bromide complex formation with DNA are calculated: the coupling constant for the dye with the biopolymer, K 1 = (232 ± 16)⋅10 3 M -1 , and the number of base pairs of the biopolymer participating in bonding with the ligand, n 1 = 3.6 ± 0.2, are calculated. Given these values, it is suggested that under these experimental conditions there are two types of bonding between ethidium bromide and the nucleic acid -intercalation and "external" bonds. A McGhee-von Hippel model for a three component system and the numerical values of the parameters for molecular complex formation in two component systems are used to calculate the bonding constant for caffeine with DNA, K 2 = 127 ± 30 M -1 , and the number of base pairs of DNA which bond with caffeine, n 2 = 1.7 ± 0.2. The concentrations of ethidium bromide and caffeine in the composition of two and three component complexes are calculated as functions of the nucleic acid content in the solution. An analysis of the concentration dependences shows that heteroassociation of ligands has a significant effect on the reduction in the concentration of ethidium bromide-DNA complexes in a three component system for low DNA concentrations, while at high DNA concentrations the bonding of caffeine with the biopolymer has this effect.

We have studied self-association of aromatic molecules of the thiazine dye methylene blue in aqueous solution, using a dimer model. We have determined the dimerization equilibrium constant for the dye molecules K D = 3900 ± 800 M -1 at T = 293 K. We have decomposed the experimental spectrum into dimer and monomer components. Using the ratio of the molar absorption coefficients for two absorption bands of the dimer spectrum, we obtained the "average" value of the angle between the electronic transition moments of the molecules in the dimers, α = 48 o .We have studied heteroassociation of methylene blue (MB) and 1,3,7-trimethylxanthine (caffeine) molecules in aqueous solution. We have calculated the heteroassociation constant as 200 ± 34 M -1 . We conclude that heteroassociation of methylene blue and caffeine molecules leads to a lower effective dye concentration in solution, which hypothetically may affect its biological activity.We have determined the values of the Gibbs free energy, the enthalpy, and the entropy for dimerization of methylene blue molecules: ∆G 293 = -(20 ± 3) kJ/mol, ∆H = -(25 ± 9) kJ/mol, ∆S 293 = -(17 ± 6) J/mol⋅K; and for methylene blue-caffeine heteroassociation: ∆G 293 = -(13 ± 3) kJ/mol, ∆H = -(14 ± 10) kJ/mol, ∆S 293 = -(2.4 ± 0.2) J/mol⋅K, respectively. We have shown that the methylene blue aggregates and the heteroassociates with caffeine are predominantly stabilized by dispersion interactions between the chromophore molecules in the associates.

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We have used spectrophotometry to study self-association and complexation with DNA by organic heterocyclic compounds in the acridine and phenothiazine series: proflavin, thionine, and methylene blue. Based on the experimental concentration dependences of the molar absorption coefficient of the molecules in an aqueous buffer solution (0.01 M NaCl, 0.01 M Na 2 EDTA, 0.01 M Tris, pH 7.4, T = 298 K), we have determined the equilibrium dimerization constants for the dyes and the DNA complexation parameters using the Scatchard and McGhee-von Hippel models. The observed increase in the cooperativity parameters as the dimerization constants of the ligands increase allowed us to hypothesize that the same interactions occur between dye molecules adsorbed on DNA as in their self-association. The equilibrium DNA-binding constants for the ligands, obtained using the are (20.9 ± 2.7)⋅10 3 M -1 for proflavin and (33.8 ± 4.1)⋅10 3 M -1 for thionine. Using the Scatchard model, taking into account intercalation and "external" binding of ligands with DNA, we determined the DNA complexation constants for methylene blue: (26.4 ± 4.6)⋅10 3 and (96 ± 17)⋅10 3 M -1 respectively. Based on analysis of the data obtained, we hypothesized that the predominant type of binding with DNA is intercalation binding in the case of proflavin and thionine, and "external" binding with the DNA surface in the case of methylene blue.

We have used UV and visible spectrophotometry to study self-association of aromatic riboflavin molecules (RFN, vitamin B 2 ,7,isoalloxazine) in aqueous solution (pH 6.86) at T = 298 K, using a dimer model. We have determined the equilibrium dimerization constant for riboflavin, K dB = 125 ± 40 M −1 . We have studied heteroassociation in the system of molecules of 7,8-dimethyl-10-ribitylisoalloxazine with 1,3,7-trimethylxanthine (caffeine) and sodium salicylate (NAS) in aqueous solution (pH 6.86; T = 298 K). We have determined the heteroassociation constants for RFN-NAS and RFN-caffeine molecules in the absence and in the presence of urea in solutions using a modified Benesi-Hildebrand equation: 25 ± 4, 17 ± 3, and 74 ± 11, 53 ± 7 M −1 respectively. We have determined the dimerization constants for NAS (2.7 ± 0.5 M −1 ) and caffeine (17.0 ± 1.5 M −1 ). We conclude that heteroassociation of the aromatic molecules leads to a lower effective riboflavin concentration in solution, and the presence of urea in mixed solutions leads to an decrease in the complexation constants for the RFN-NAS and RFN-caffeine systems.