Aggregation Features and Fluorescence of Hoechst 33258

The functionality of the bisbenzimide Hoechst 33258 in solution has been largely exploited in the quantification of DNA. Understanding of its behavior is essential to promote its widespread application and learning of biological processes. A detailed study of the dimerization process of the fluorescent blue dye Hoechst 33258 is carried out by isothermal titration calorimetry, absorbance, fluorescence, differential scanning calorimetry and T-jump kinetic measurements. The dimer/monomer ratio depends on the dye concentration and the ionic strength. The dimerization constant determined under physiological conditions (pH = 7.0; I = 0.10 M), KD = 3 × 104 M−1, conveys that only micromolar concentrations of the dye can ensure reasonably high amounts of the monomer species in solution. For instance, for 10 μM dye content, the dimer prevails for I > 0.08 M, whereas the monomer is observed at low ionic strength, a key issue to be elucidated as long as the dimer species is more fluorescent than the monomer and the fluorescence intensity strongly relies on the ionic strength and the dye concentration.