Probing the structure of the warfarin-binding site on human serum albumin using site-directed mutagenesis

Proteins. 2002 May 1;47(2):116-25. doi: 10.1002/prot.10068.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Crystallography, X-Ray
  • Humans
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Serum Albumin / chemistry*
  • Serum Albumin / genetics*
  • Serum Albumin / metabolism
  • Spectrometry, Fluorescence
  • Warfarin / chemistry
  • Warfarin / metabolism*

Substances

  • Serum Albumin
  • Warfarin