Abstract

Binding of warfarin to human serum albumin results in a red shift of the UV absorption maximum, suggesting that the binding site is a hydrophobic area of the protein. The enhancement of the fluorescence of warfarin upon binding to human serum albumin was used to study the binding equilibrium and the kinetics of this drug-protein interaction. From equilibrium fluorescence measurements, contributions from free and bound warfarin could be evaluated. From the resulting Scatchard plots, equilibrium constants ranging from 4.2 X 10(5) to 3.5 X 10(5) M-1 for temperatures from 8 degrees to 37 degrees were calculated. The reaction is slightly exothermic (delta H = -1.2 kcal m mole-1) and strongly entropy-driven (delta S = +21 cal . mole-1 . K-1). The reaction rate constants of the warfarin-albumin interaction were determined by the stopped-flow technique. The association rate constant varies from 2.2 X 10(5) to 7.7 X 10(5) M-1 sec-1 from 10 degrees to 32 degrees. The corresponding activation enthalpy is 9.0 kcal . mole-1. These values are not consistent with a diffusion-controlled reaction. The dissociation of the complex was studied by making use of the direct competition between warfarin and phenylbutazone for the same binding site. The dissociation rate constant varies from 2.5 to 10.8 sec-1 in the same temperature range. Activation parameters obtained in the kinetic experiments correspond very well with the thermodynamic parameters calculated from the equilibrium study, validating the fluorescence approach to the equilibrium studies.