Thermodynamics of mucopolysaccharide–dye binding. I. Identification of free and bound dye via membrane filtration: Acridine orange–dermatan sulfate system

Stoichiometric mixtures of acridine orange with dermatan sulfate at total dye concentrations ⩽ 1 × 10−5 M show fluorescence maxima at 540 nm and 660 nm on excitation at 436 nm. By means of membrane filtration, it is directly demonstrated that the species emitting at 540 nm is due only to unbound dye whereas the 660‐nm emitting species is due to bound dye. It is, therefore, possible to differentiate unbound acridine orange from its dermatan sulfate complex solely by spectroscopic methods. Thermodynamic binding parameters can be calculated from rapid spectroscopic measurements without disturbing the system.