Human serum albumin (HSA) is the most abundant plasma protein in circulation. The three most important drug-binding sites on HSA are Sudlow's Site I (subdomain IIA), Sudlow's Site II (subdomain IIIA), and Heme site (subdomain IB). Heme site and Site I are allosterically coupled; therefore, their ligands may be able to allosterically modulate the binding affinity of each other. In this study, the effects of four Heme site ligands (bilirubin, biliverdin, hemin, and methyl orange) on the interaction of the Site I ligand warfarin with HSA were tested, employing fluorescence spectroscopic, ultrafiltration, and ultracentrifugation studies. Our major results/conclusions are the following. (1) Quenching studies indicated no relevant interaction, while the other fluorescent model used suggested that each Heme site ligand strongly decreases the albumin binding of warfarin. (2) Ultrafiltration and ultracentrifugation studies demonstrated the complex modulation of warfarin-HSA interaction by the different Heme site markers; for example, bilirubin strongly decreased while methyl orange considerably increased the bound fraction of warfarin. (3) Fluorescence spectroscopic studies showed misleading results in these diligand-albumin interactions. (4) Different Heme site ligands can increase or decrease the albumin binding of warfarin and the outcome can even be concentration dependent (e.g., biliverdin and hemin).

Department of Biophysics Medical School University of Pécs Szigeti út 12 H 7624 Pécs Hungary

Department of Pharmacology Faculty of Pharmacy University of Pécs Rókus u 2 H 7624 Pécs Hungary

Green Chemistry Research Group János Szentágothai Research Centre University of Pécs Ifjúság útja 20 H 7624 Pécs Hungary

Lab on a Chip Research Group János Szentágothai Research Centre University of Pécs Ifjúság útja 20 H 7624 Pécs Hungary

The Department of Pharmacognosy and Pharmaceutical Botany Faculty of Pharmacy in Hradec Králové Charles University Akademika Heyrovského 1203 500 05 Hradec Králové Czech Republic

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