Interaction of beta-lactoglobulin with small hydrophobic ligands as monitored by fluorometry and equilibrium dialysis: Nonlinear quenching effects related to protein-protein association

Although a thorough characterization of binding parameters is essential for application of beta -lactoglobulin as a carrier for a variety of small hyd rophobic ligands, the binding parameters derived in various studies using v arious techniques are inconsistent. The bindings of several small ligands a s detected by fluorometry and equilibrium dialysis were compared. Fluoresce nce spectroscopy showed that beta -ionone, retinol, and fatty acid lactones all bound in the vicinity of a tryptophan residue. Retinol and fatty acid lactone competed for the same binding site. Exclusively for ligands that qu ench the beta -lactoglobulin fluorescence through a resonance energy transf er mechanism, fluorometry yielded a systematically higher binding affinity than equilibrium dialysis. The binding overestimation in fluorometric measu rements can be explained by oligomer formation of protein, together with an underestimation of the limiting quenching level at saturating ligand conce ntrations due to the use of a limited set of data points.