Measurement of spontaneous transfer and transbilayer movement of BODIPY-labeled lipids in lipid vesicles

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 (C-5-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 C-5-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 C-5-DMB-labeled analogs of sphingomyelin (C-5-DMB-SM), ceramide (C-5-DMB-Cer), phosphatidylcholine (C-5-DMB-PC), and diacylglycerol (C-5-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 C-5-DMB-SM (21 s) < C-5-DMB-PC (350 s) approximate to C-5-DMB-Cer (400 s) much less than C-5-DMB-DAG (100 h). C-5-DMB-Cer(t(1/2) approximate to 22 min) and C-5-DMB-DAG (t(1/2) approximate to 70 ms) exhibited rapid spontaneous transbilayer movement, while C-5-DMB-SM (t(1/2) approximate to 3.3 h) and C-5-DMB-PC (t(1/2) approximate to 7.5 h) moved across the bilayer very slowly, These results provide a basis for interpreting the behavior of these lipid analogs in cells.