pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids.
A central, unresolved question in cell physiology is how fatty acids move across cell membranes and whether protein(s) are required to facilitate transbilayer movement. We have developed a method for monitoring movement of fatty acids across protein-free model membranes (phospholipid bilayers). Pyranin, a water-soluble, pH-sensitive fluorescent molecule, was trapped inside well-sealed phosphatidylcholine vesicles (with or without cholesterol) in Hepes buffer (pH 7.4). Upon addition of a long-chain fatty acid (e.g., oleic acid) to the external buffer (also Hepes, pH 7.4), a decrease in fluorescence of pyranin was observed immediately (within 10 sec). This acidification of the internal volume was the result of the "flip" of un-ionized fatty acids to the inner leaflet, followed by a release of protons from approximately 50% of these fatty acid molecules (apparent pKa in the bilayer = 7.6). The proton gradient thus generated dissipated slowly because of slow cyclic proton transfer by fatty acids. Addition of bovine serum albumin to vesicles with fatty acids instantly removed the pH gradient, indicating complete removal of fatty acids, which requires rapid "flop" of fatty acids from the inner to the outer monolayer layer. Using a four-state kinetic diagram of fatty acids in membranes, we conclude that un-ionized fatty acid flip-flops rapidly (t1/2 < or = 2 sec) whereas ionized fatty acid flip-flops slowly (t1/2 of minutes). Since fatty acids move across phosphatidylcholine bilayers spontaneously and rapidly, complex mechanisms (e.g., transport proteins) may not be required for translocation of fatty acids in biological membranes. The proton movement accompanying fatty acid flip-flop is an important consideration for fatty acid metabolism in normal physiology and in disease states such as cardiac ischemia.
Kamp
F
F
Department of Biophysics, Housman Medical Research Center, Boston University School of Medicine, MA 02118-2394.
Hamilton
J A
JA
D001692:Biological Transport; D005227:Fatty Acids / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008567:Membranes, Artificial; D009038:Motion; D010713:Phosphatidylcholines / Q000737:chemistry; D010743:Phospholipids / Q000502:physiology*
D005227:Fatty Acids; D007477:Ions; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010743:Phospholipids