High-affinity binding of two molecules of warfarin and phenprocoumon to human serum albumin

Binding equilibria of warfarin, 3-(α-acetonylbenzyl)-4-hydroxycoumarin, and phenprocoumon, 3-(α-ethylbenzyl)-4hydroxycoumarin, to defatted human serum albumin (Kabi Vitrum) were studied by equilibrium dialysis in a 33 mM sodium phosphate buffer (pH 7.4) at 37°C. The binding data were analysed in terms of several acceptable sets of binding constants using a computerized curve fitting procedure. The findings were consistent with binding of two warfarin or two phenprocoumon molecules with high affinity and additional molecules bound with lower affinity. The binding of warfarin or phenprocoumon was explained by a model with two independent and equal high-affinity binding sites besides several independent weak sites in the albumin molecule (p < 0.01, by F-test). The findings were not consistent with binding of warfarin or phenprocoumon to a single high-affinity site besides several weak sites. A model of sequential binding of several ligand molecules to one locus is proposed.