Complexes between Fluorescent Cholic Acid Derivatives and Human Serum Albumin. A Photophysical Approach To Investigate the Binding Behavior

• Published in: The journal of physical chemistry. B 2010-04, Vol.114 (13), p.4710-4716
• Main Authors: Rohacova, Jana, Marin, M. Luisa, Miranda, Miguel A
• Format: Article
• Language: English
• Subjects: Anti-Inflammatory Agents, Non-Steroidal - chemistry; Anticoagulants - chemistry; B: Biophysical Chemistry; Binding Sites; Cholic Acid - chemistry; Fluorescence Resonance Energy Transfer; Fluorescent Dyes - chemistry; Humans; Ibuprofen - chemistry; Protein Binding; Serum Albumin - chemistry; Spectrometry, Fluorescence; Warfarin - chemistry
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/jp911114n

Interaction between bile acids and plasma proteins has attracted considerable attention over past decades. In fact, binding of bile acids to human serum albumin (HSA) determines their level in plasma, a value that can be used as a test for liver function. However, very little is known about the role that bile acids−HSA complexes play in hepatic uptake. In the present paper, we report on the utility of the singlet excited state properties of 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorescent derivatives of cholic acid (ChA); namely, 3α-NBD-ChA, 3β-NBD-ChA, 3β-NBD-ChTau, 7α-NBD-ChA, and 7β-NBD-ChA to clarify key aspects of bile acids−HSA interactions that remain poorly understood. On the basis of either absorption or emission measurements, formation of NBD-ChA@HSA complexes with 1:1 stoichiometry has been proven. Enhancement of the fluorescence emission upon addition of HSA has been used for determination of the binding constants, which are in the range of 104 M−1. Energy transfer from tryptophan to NBD-ChA occurs by a FRET mechanism; the donor−acceptor distances have been determined according to Förster’s theory. The estimated values (27−30 Å) are compatible with both site I and site II occupancy and do not provide sufficient information for a safe assignment; however, fluorescence titration using warfarin (site I probe) and ibuprofen (site II probe) for displacement clearly indicates that the employed cholic acid derivatives bind to HSA at site I.