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(TM)-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 degrees 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.
Abreu
Magda S C
MS
Departamento de Química, Universidade de Coimbra, Portugal.
Estronca
Luís M B B
LM
Moreno
Maria João
MJ
Vaz
Winchil L C
WL
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005470:Fluorometry / Q000379:methods; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008560:Membrane Fluidity; D008823:Micelles; D008956:Models, Chemical; D011485:Protein Binding; D012235:Rhodamines / Q000737:chemistry* / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence / Q000379:methods; D013194:Staining and Labeling / Q000379:methods; D013501:Surface-Active Agents / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008823:Micelles; C465621:RG-tetradecylamine; D012235:Rhodamines; D013501:Surface-Active Agents; D014867:Water; D012710:Serum Albumin, Bovine