LIGAND-BINDING AT MEMBRANE MIMETIC INTERFACES - HUMAN SERUM-ALBUMIN IN REVERSE MICELLES

The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenylbutazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2-ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane-water interface. Upon micellar incorporation, the dansylsarcosine-albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin-albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 x 10(4) M-1 cm-1. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged (K(a) almost-equal-to 1.0 x 10(5) M-1), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant. These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344 nm in buffer to 327 nm in sodium bis(2-ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N-bromosuccinimide, indicate perturbations of the sole tryptophan-214 microenvironment. However, the distance between the indole residue and tyrosine-411 does not seem substantially modified by the 15% decrease affecting the alpha-helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane-like interface which generates two protein subpopulations differing in their membrane-surface and ligand affinities. Overall and local conformational changes, originating from this surface-induced effect, may thus constitute a ligand-release facilitating mechanism acting at cellular membrane levels.