Bilayer Defects Facilitate DPPC Flip-Flop

Abstract

We measured the flip-flop rate of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in large unilamellar vesicles (LUVs) as a function of temperature. The transbilayer distribution of headgroup-protiated DPPC exchanged into the outer leaflet of headgroup-deuterated DPPC vesicles was monitored using 1H NMR spectroscopy coupled with the addition of a paramagnetic shift reagent, which allowed us to distinguish between inner and outer leaflet lipids. After careful determination of structure and thermotropic behavior of this system, we determined the fluid-phase flip-flop activation energy Ea to be 123 kJ/mol. This is the first report of probe-free fluid phase flip-flop kinetics for DPPC in freely floating vesicles. No flip-flop was observed over the course of 6 days when DPPC was incubated below the main transition temperature, and consequently gel phase thermodynamic values could not be determined in LUVs. This result is in contrast with previous studies using supported bilayers that reported fast flip-flop in the gel phase. To further investigate this discrepancy, we measured DPPC LUVs incubated within the main transition temperature, and found accelerated flip-flop (i.e., faster rates than were found just above the main transition temperature). We conclude that bilayer defects such as gel/fluid domain boundaries accelerate lipid flip-flop. We speculate that sample environment and measurement conditions that can alter the physical properties of bilayers have contributed to flip-flop discrepancies in literature.