Mechanism by which warfarin binds to human serum albumin. Stopped-flow kinetic experiments with two large fragments of albumin.

In order to obtain information about the kinetics of the process by which warfarin binds to human serum albumin at a molecular level, we performed stopped-flow kinetic experiments on albumin and on a large peptic fragment (residues 1-387) and a large tryptic fragment (residues 198-585) of albumin. From these experiments it can be concluded that the first interaction between warfarin and the proteins is almost certainly diffusion-controlled and is dependent on the net charges of the reactants. The next step in the binding process involves the formation of an activated warfarin-protein complex, whereafter the final complex is formed. The warfarin-albumin complex forms more slowly than the warfarin-fragment complexes, because the formation is sterically hindered by the albumin structure. We think it very unlikely that albumin has an oblate ellipsoid structure; it is much more likely to have a U-shaped structure, where the domains make contact with each other. If this hypothesis is correct, then this indicates that the domains do not act independently of each other. The formation of the activated warfarin-albumin complex is further influenced by the conformational state of the albumin molecule, i.e. the N-B transition. The possible role of this N-B transition in albumin-mediated transport of drugs through cellular membranes is discussed.