Premium
Probing the structure of the warfarin‐binding site on human serum albumin using site‐directed mutagenesis
Author(s) -
Petersen Charles E.,
Ha ChungEun,
Curry Stephen,
Bhagavan Nadhipuram V.
Publication year - 2002
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.10068
Subject(s) - human serum albumin , chemistry , binding site , mutant , warfarin , fluorescence , dissociation constant , conformational change , albumin , biochemistry , stereochemistry , receptor , gene , medicine , physics , quantum mechanics , cardiology , atrial fibrillation
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. Proteins 2002;47:116–125. © 2002 Wiley‐Liss, Inc.