Measurement of Spontaneous Transfer and Transbilayer Movement of BODIPY-Labeled Lipids in Lipid Vesicles

Abstract

An assay was developed to study the spontaneous transfer and transbilayer movement (flip−flop) of lipid analogs labeled with the fluorescent fatty acid, 5-(5,7-dimethyl BODIPY)-1-pentanoic acid (C₅-DMB-) in large unilamellar lipid vesicles comprised of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). The assay is based on the concentration-dependent changes in fluorescence intensity that occur when donor vesicles containing a C₅-DMB-lipid are mixed with nonfluorescent acceptor vesicles. A kinetic model was developed to describe the time-dependent changes in concentration of a lipid undergoing both spontaneous transfer between unilamellar vesicles and transbilayer movement within the vesicle membranes, and a mathematical solution was obtained. Data were obtained using C₅-DMB-labeled analogs of sphingomyelin (C₅-DMB-SM), ceramide (C₅-DMB-Cer), phosphatidylcholine (C₅-DMB-PC), and diacylglycerol (C₅-DMB-DAG), and kinetic parameters for each lipid were determined using a nonlinear least-squares fitting program. The half-times for interbilayer transfer of the lipids were C₅-DMB-SM (21 s) < C₅-DMB-PC (350 s) ≈ C₅-DMB-Cer (400 s) ≪ C₅-DMB-DAG (100 h). C₅-DMB-Cer (t_1/2 ≈ 22 min) and C₅-DMB-DAG (t_1/2 ≈ 70 ms) exhibited rapid spontaneous transbilayer movement, while C₅-DMB-SM (t_1/2 ≈ 3.3 h) and C₅-DMB-PC (t_1/2 ≈ 7.5 h) moved across the bilayer very slowly. These results provide a basis for interpreting the behavior of these lipid analogs in cells.