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.
Twine
S M
SM
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, UK.
Gore
M G
MG
Morton
P
P
Fish
B C
BC
Lee
A G
AG
East
J M
JM
D001665:Binding Sites; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012709:Serum Albumin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013237:Stereoisomerism; D014859:Warfarin / Q000737:chemistry*; D015003:Yeasts / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D014859:Warfarin