Multivariate extension of the continuous variation and mole-ratio methods for the study of the interaction of intercalators with polynucleotides Original Research Article

A recently proposed approach for the study of the intercalation equilibria of drugs and polynucleotides is applied to ethidium bromide and poly(inosinic)–poly(cytidylic) acid. The procedure consists of the extension of the continuous variation and mole-ratio methods by recording all the UV–VIS absorption, fluorescence and circular dichroism spectra for a set of solutions containing a range of polynucleotide:dye concentration ratios. The whole set of spectroscopic data matrices was simultaneously analyzed by a multivariate curve resolution method based on alternating least squares. This procedure allowed the detection of the intercalation complex, the recovery of the concentration profiles and pure spectra for each species and the calculation of the polynucleotide:dye ratio in the complex and the apparent equilibrium constant. The intercalation sites occur every three base pairs and the value for the logarithm of the apparent equilibrium constant at 37°C in 0.26 M NaCl was 4.6±0.1 M−1.