Mechanism of binding of warfarin enantiomers to recombinant domains of human albumin.

Domain fragments of human serum albumin corresponding to domains 1 and 2 (D12) and domains 2 and 3 (D23) were expressed in yeast. The kinetics of warfarin binding to these fragments were investigated using stopped-flow fluorescence spectroscopy. Binding can be characterized by a two-step process, a rapid diffusion-controlled step and a slower rate-limiting step in which a stable drug-protein complex is formed. The equilibrium constant for step 1 is greater for both D12 and D23 than for albumin, probably as a result of reduced steric hindrance offered by the domain fragments. Binding step 2, thought to be the result of a conformational change as warfarin is accommodated by the protein, is faster for D12 and D23. Albumin and the domain fragments show an increased preference for the R enantiomer, but the preference is particularly enhanced for domain fragment D12. These preferences can largely be explained by the domains having different rates for step 2 of the binding process.