Probing the structure of the warfarin-binding site on human serum albumin using site-directed mutagenesis.
The binding of warfarin to the following human serum albumin (HSA) mutants was examined: K195M, K199M, F211V, W214L, R218M, R222M, H242V, and R257M. Warfarin bound to human serum albumin (HSA) exhibits an intrinsic fluorescence that is approximately 10-fold greater than the corresponding signal for warfarin in aqueous solution. This property of the warfarin/HSA complex has been widely used to determine the dissociation constant for the interaction. In the present study, such a technique was used to show that specific substitutions in subdomain 2A altered the affinity of HSA for warfarin. The fluorescence of warfarin/mutant HSA complexes varied widely from the fluorescence of the warfarin/wild-type HSA complex at pH = 7.4, suggesting changes in the structure of the complex resulting from specific substitutions. The fluorescence of the warfarin/wild-type HSA complex increases about twofold as the pH is increased from 6.0 to 9.0 due to the neutral-to-base (N-B) transition, a conformational change that occurs in HSA as a function of pH. Changes in the fluorescence of warfarin/mutant HSA complexes as a function of pH suggests novel behavior for most HSA species examined. For the HSA mutants F211V and H242V, the midpoint of the N-B transition shifts from a wild-type pH of 7.8 to a pH value of 7.1-7.2.
Petersen
Charles E
CE
Department of Biochemistry and Biophysics, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822, USA.
Ha
Chung-Eun
CE
Curry
Stephen
S
Bhagavan
Nadhipuram V
NV
D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012709:Serum Albumin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014859:Warfarin / Q000737:chemistry / Q000378:metabolism*
D012709:Serum Albumin; D014859:Warfarin