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

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, 3 alpha-NBD-ChA, 3 beta-NBD-ChA, 3 beta-NBD-ChTau, 7 alpha-NBD-ChA, and 7 beta-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 10(4) M-1. Energy transfer from tryptophan to NBD-ChA occurs by a FRET mechanism; the donor-acceptor distances have been determined according to Forster's theory. The estimated values (27-30 angstrom) are compatible with both site I and site 11 occupancy and do not provide sufficient information for a safe assignment; however, fluorescence titration using warfarin (site I probe) and ibuprofen (site 11 probe) for displacement clearly indicates that the employed cholic acid derivatives bind to HSA at site I.