Indexed on: 18 Mar '10Published on: 18 Mar '10Published in: Journal of Physical Chemistry B
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, 3alpha-NBD-ChA, 3beta-NBD-ChA, 3beta-NBD-ChTau, 7alpha-NBD-ChA, and 7beta-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 A) 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.