RT Journal Article
SR Electronic
T1 Optical Studies into the Nature of the High Affinity Binding Site of Human Serum Albumin for Phenylbutazone
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 147
OP 153
VO 16
IS 1
A1 MAES, VIVIANE
A1 HOEBEKE, JOHAN
A1 VERCRUYSSE, ANTOINE
A1 KANAREK, LOUIS
YR 1979
UL http://molpharm.aspetjournals.org/content/16/1/147.abstract
AB 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.