Influence of long-chain free fatty acids on the binding of warfarin to bovine serum albumin

Abstract

The effects of the concentration of long-chain free fatty acids and bovine serum albumin on the binding characteristics of warfarin with albumin have been studied by adsorption and fluorescence spectroscopy using the method of ultrafiltration. The binding affinity of warfarin is higher in a concentrated than in a dilute albumin solution. In a dilute solution of serum albumin (55 μM or less), the binding constant of the warfarin-albumin complex is increased in the presence of 100 μM (or less) of either lauric or oleic acid or a mixture of these two acids, but higher concentrations of the fatty acids decrease such binding. A mixture of saturated and unsaturated fatty acids shows the same effects as those by either fatty acid. Simultaneous measurements of the fluorescence peak intensity and polarization of fluorescence of warfarin bound to albumin in a dilute solution indicate that low and high concentrations of free fatty acids induce different conformational states of the same warfarin-binding sites on the albumin molecule. Such a dualistic behavior of free fatty acids could be best interpreted in terms of allosteric interactions involving heterotropic effects. On the other hand, in a concentrated solution of serum albumin, the binding constant of warfarin is decreased in the presence of both low and high concentrations of free fatty acids. Such a decrease in warfarin binding with concentrated albumin in the presence of free fatty acids is not due simply to a displacement of warfarin by free fatty acids from the warfarin-binding sites, but also could result simultaneously from a further conformational change of warfarin-binding sites caused by the interaction of warfarin and free fatty acids (a negative heterotropic effect).