Stereoselective kinetics of warfarin binding to human serum albumin: effect of an allosteric interaction.
Kinetic and equilibrium binding studies were performed on the interaction of warfarin enantiomers with human serum albumin (HSA) in the absence and presence of lorazepam acetate (LoAc) enantiomers. Binding kinetics were followed by recording changes in the fluorescence of warfarin upon binding to HSA using the stopped-flow technique. The binding of (R)-warfarin displayed an exponentially increasing fluorescence, satisfying the two-step mechanism reported previously for the racemate, i.e., a diffusion controlled pre-equilibrium is followed by a slower rearrangement of the complex. In the case of (S)-warfarin, the signal was biphasic: a fast fluorescence enhancement was followed by a slow decline. The different kinetic features indicate that the equilibrium conformations of the [(S)-warfarin-HSA] and [(R)-warfarin-HSA] complexes are achieved via different mechanisms. The phenomenon was seen in buffers of different pH and compositions. Equilibrium binding measurements indicated significantly lower molar intrinsic fluorescence for the (S)-warfarin complex, suggesting differences in the microenvironments of the bound enantiomers. In the presence of (S)-LoAc, the allosterically enhanced binding of (S)-warfarin manifested itself in accelerated relaxation kinetics. In accordance with the low molar intrinsic fluorescence determined for the stable ternary complex, the amplitude of the decline in fluorescence became larger.
Fitos
Ilona
I
Department of Molecular Pharmacology, Institute of Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary. fitosi@chemres.hu
Visy
Júlia
J
Kardos
Julianna
J
D000494:Allosteric Regulation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008140:Lorazepam / Q000031:analogs & derivatives* / Q000494:pharmacology; D046911:Macromolecular Substances; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D014859:Warfarin / Q000737:chemistry* / Q000378:metabolism*
D046911:Macromolecular Substances; D012709:Serum Albumin; D014859:Warfarin; D008140:Lorazepam; C028245:lorazepam acetate