Adsorption of thionine on heparin studied by combining ultrafiltration and difference VIS spectroscopy

The combination of ultrafiltration and difference spectroscopy allows the quantitative determination of spectra of thionine bound to heparin. The spectra of the bound dye do not depend on the degree of coverage; this and the shape of the Scatchard plot show that “all-or-none” binding is valid. A calculus of variations based on a modification of the Hill plot shows that aggregates of seven thionine cations are bound. Tetrasaccharides with an average charge of two carboxylate and five sulfate groups are suggested to be the binding sites. The binding constant given for one mole thionine is 4.4 · 10⁵ M^?1. The Gibbs enthalpy for binding of one mole of thionine is ?31.7 kJ·M^?1 at 20°C.