Possible overestimation of the site size in fluorescent dye/nucleic acid systems when using the McGhee and von Hippel equation

In the analysis of the binding of small molecules (D) to polynucleotides (P) to give a complex PD, the evaluation of the site size (defined as the number of monomer units of the polymer involved in the binding of one dye molecule under conditions of complete saturation) is of primary interest. For such systems, wide use of spectrophotometric and spectrofluorometric measurements is made. In particular, titration data, plotted according to Scatchard representations, are commonly analysed by means of the McGhee and von Hippel equation to evaluate the binding constant (K) and the site-size (n). We have performed spectrofluorometric titrations of the intercalating dye of the cyanine family Cyan40 with natural DNA, at different initial dye concentration (CD). It is found that the McGhee and von Hippel analysis has to be done with care as the very low extent of monomer units occupation obtained with the low dye amounts generally used in fluorescence measurements can yield misleading overestimated results as concerns the site size. This should be a constant parameter characteristic of the analysed system; instead, the apparent value of n estimated is found to depend on CD with n increasing as the dye loading is decreased. The reliability of the analysis can be improved by increasing the polymer saturation, i.e. using considerably higher dye concentrations (tendency to a constant value for the highest CD in). However, under such conditions, positively charged dye self-aggregation on the negative polymer backbone can occur. A method is proposed that, from a set of titrations at different total dye concentration, estimates the parameters K and n, together with the aggregation constant of the dye on the polynucleotide (KD).