Five recombinant fragments of human serum albumin - Tools for the characterization of the warfarin binding site

Human serum albumin (HSA) interacts with a vast array of chemically diverse ligands at specific binding sites. To pinpoint the essential structural el ements for the formation of the warfarin binding site on human serum albumi n, a defined set of five recombinant proteins comprising combinations of do mains and/or subdomains of the N-terminal part were prepared and characteri zed by biochemical standard procedures, tryptophanyl fluorescence, and circ ular dichroic measurements, indicating well-preserved secondary and tertiar y 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 addi tion, ultraviolet difference spectroscopy and induced circular dichroism ex periments were carried out to get an in depth understanding of the binding mechanism of warfarin to the fragments as stand-alone proteins. This system atic 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, underl ining the great potential that lies in the use of combinations of recombina nt fragments for the study and accurate localization of ligand binding site s on HSA.