Interaction of ethidium bromide and caffeine with DNA in aqueous solution

Two component (ethidium bromide–caffeine, ethidium bromide–DNA) and three component (ethidium bromide–caffeine–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)⋅103 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.