Fatty acid flip-flop in a model membrane is faster than desorption into the aqueous phase.
Fatty acids (FA) are known to diffuse (flip-flop) rapidly across protein-free phospholipid bilayers in their un-ionized form. However, whether flip-flop through the hydrophobic core of the bilayer or desorption from the membrane into the aqueous phase is the rate-limiting step in FA transport through membranes is still debated. The issue has remained unresolved in part by disagreements over whether some methods of adding FA create artifacts that lead to erroneous conclusions and in part by the lack of fluorescence methods to monitor each individual step. Here we study the kinetics of FA transfer from donors to phospholipid vesicles (small and large unilamellar vesicles) by a dual fluorescence approach that utilizes the probes fluorescein phosphatidylethanolamine (FPE) and pyranine. FPE detects the concentration of FA anions in the outer membrane leaflet, allowing a precise measurement of kinetics of FA adsorption or desorption. Our results showed that as soon as FPE detects adsorption of FA into the outer leaflet, pyranine detects its movement to the inner leaflet. We further demonstrated that (i) flip-flop for FA with 14-22 carbons is much faster than the rates of desorption and therefore cannot be the rate-limiting step of FA translocation across membranes; (ii) fluorescence changes detected by probes located on or in acceptor vesicles are dependent upon the method used to deliver the FA (i.e., uncomplexed, or complexed to albumin or phospholipid bilayers); however, (iii) transfer kinetics observed in the presence of different donors is rate-limited by the desorption of FA from the donor into the aqueous phase rather than by flip-flop.
Simard
Jeffrey R
JR
Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA.
Pillai
Biju K
BK
Hamilton
James A
JA
D001190:Arylsulfonates / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008566:Membranes / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D010714:Phosphatidylethanolamines / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000378:metabolism
D001190:Arylsulfonates; D005227:Fatty Acids; D010714:Phosphatidylethanolamines; D053835:Unilamellar Liposomes; C483858:phosphatidylethanolamine; C005047:pyranine