Binding of a Fluorescent Lipid Amphiphile to Albumin and its Transfer to Lipid Bilayer Membranes

Kinetics and thermodynamics of the binding of a fluorescent lipid amphiphile, Rhodamine Green™-tetradecylamide (RG-C 14:0), to bovine serum albumin were characterized in an equilibrium titration and by stopped-flow fluorimetry. The binding equilibrium of RG-C 14:0 to albumin was then used to reduce its concentration in the aqueous phase to a value below its critical micelle concentration. Under these conditions, the only two species of RG-C 14:0 in the system were the monomer in aqueous solution in equilibrium with the protein-bound species. After previous determination of the kinetic and thermodynamic parameters for association of RG-C 14:0 with albumin, the kinetics of insertion of the amphiphile into and desorption off lipid bilayer membranes in different phases (solid, liquid-ordered, and liquid-disordered phases, presented as large unilamellar vesicles) were studied by stopped-flow fluorimetry at 30°C. Insertion and desorption rate constants for association of the RG-C 14:0 monomer with the lipid bilayers were used to obtain lipid/water equilibrium partition coefficients for this fluorescent amphiphile. The direct measurement of these partition coefficients is shown to provide a new method for the indirect determination of the equilibrium partition coefficient of similar molecules between two defined lipid phases if they coexist in the same membrane.