MEASUREMENT OF SPONTANEOUS TRANSFER AND TRANSBILAYER MOVEMENT OF BODIPY-LABELED LIPIDS IN LIPID VESICLES

An assay was developed to study the spontaneous transfer and transbila yer movement (flip-flop) of lipid analogs labeled with the fluorescent fatty acid, 5-(5,7-dimethyl BODIPY)-1-pentanoic acid (C-5-DMB-) in la rge unilamellar lipid vesicles comprised of 1-palmitoyl-2-oleoyl phosp hatidylcholine (POPC). The assay is based on the concentration-depende nt changes in fluorescence intensity that occur when donor vesicles co ntaining a C-5-DMB-lipid are mixed with nonfluorescent acceptor vesicl es, A kinetic model was developed to describe the time-dependent chang es in concentration of a lipid undergoing both spontaneous transfer be tween unilamellar vesicles and transbilayer movement within the vesicl e membranes, and a mathematical solution was obtained. Data were obtai ned using C-5-DMB-labeled analogs of sphingomyelin (C-5-DMB-SM), ceram ide (C-5-DMB-Cer), phosphatidylcholine (C-5-DMB-PC), and diacylglycero l (C-5-DMB-DAG), and kinetic parameters for each lipid were determined using a nonlinear least-squares fitting program, The half-times for i nterbilayer 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 m ovement, 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, T hese results provide a basis for interpreting the behavior of these li pid analogs in cells.