Human serum albumin (HSA) interacts with a vast array
of chemically diverse ligands at specific binding sites.
To pinpoint the essential structural elements for the
formation of the warfarin binding site on human serum
albumin, a defined set of five recombinant proteins comprising
combinations of domains and/or subdomains of the N-terminal
part were prepared and characterized by biochemical standard
procedures, tryptophanyl fluorescence, and circular dichroic
measurements, indicating well-preserved secondary and tertiary
structures. Affinity constants for binding to warfarin
were estimated by fluorescence titration experiments and
found to be highest for HSA-DOM I-II and HSA, followed
by HSA-DOM IB-II, HSA-DOM II, and HSA-DOM I-IIA. In addition,
ultraviolet difference spectroscopy and induced circular
dichroism experiments were carried out to get an in depth
understanding of the binding mechanism of warfarin to the
fragments as stand-alone proteins. This systematic study
indicates that the primary warfarin binding site is centered
in subdomain IIA with indispensable structural contributions
of subdomain IIB and domain I, while domain III is not
involved in this binding site, underlining the great potential
that lies in the use of combinations of recombinant fragments
for the study and accurate localization of ligand binding
sites on HSA.