Inverse dependence of binding constants upon albumin concentration. Results for L-tryptophan and three anionic dyes

The interactions of methyl orange, bromocresol green, 2-(4'-hydroxybenzeneneazo)benzoic acid (HABA) and L-tryptophan with human albumin at pH 7.4 were investigated by equilibrium dialysis at 37 degrees C. Binding characteristics of each of the three dyes were studied by two approaches: (1) variation of total ligand concentration with a single albumin concentration and (2) variation of albumin concentration with a single total ligand concentration. Both approaches gave typical Scatchard plots with negative slope for methyl orange and bromocresol green, with good agreement between the two sets of data for each dye. In contrast, approach (2) gave Scatchard plots with a positive slope for HABA and L-tryptophan, indicating a decrease in the number of binding sites (n) and/or association constant (k) as the albumin concentration increased. This inverse dependence of nk upon albumin concentration for 2-(4'-hydroxybenzeneazo)benzoic acid was mainly due to changes in n which were still observed in the presence of inhibitory chloride ions at pH 5.75. Reasons for this type of binding behaviour are discussed together with general implications for binding studies. The results show 2-(4'-hydroxybenzeneazo)benzoic acid to be a useful ligand for investigation of this problem.