Abstract

The binding of phenylbutazone to human serum albumin was investigated by UV difference-spectroscopy. From these measurements at two different wavelengths an association constant of 6.5 x 10⁵ M^-1 at 27.5° was calculated. The intrinsic tryptophan fluorescence of human serum albumin was quenched by binding of phenylbutazone. This quenching was not due to absorption or nonradiative energy transfer, but to a direct perturbation of the molecular environment of the fluorophore. Fluorescence measurements allowed us to calculate the binding parameters of the phenylbutazone-albumin interaction and to evaluate the thermodynamics of the binding. The association constant varied from 6.2 to 9.5 x 10⁵ M^-1 at temperatures from 18° to 45° corresponding with free energy changes from -7.7 to -8.7 kcal/mole. The reaction is endothermic (ΔH = +3.0 kcal/mole) and strongly entropy-driven (ΔS = +36.8 e.u.). This is characteristic for a hydrophobic interaction with solvent perturbation. Spectrophotometric and fluorescence measurements both suggested that upon binding, phenylbutazone forms a π-π complex with the tryptophan residue of the protein. This points toward the presence of that residue in the high affinity binding site. Yet the alternative hypothesis of a conformational change in the protein causing the optical changes could not be definitely excluded.