1,2-diacyl-phosphatidylcholine flip-flop measured directly by sum-frequency vibrational spectroscopy

ABSTRACT Sum-frequency vibrational spectroscopy (SFVS) is used to measure the intrinsic rate of lipid flip-flop for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in planar-supported lipid bilayers (PSs). Asymmetric PSLBs were prepared using the Langmuir-Blodgett/Langmuir-Schaefer method by placing a perdeuterated lipid analog in one leaflet of the PSLB. SFVS was used to directly measure the asymmetric distribution of the native lipid within the membrane by measuring the decay in the CH3 vs intensity at 2875 cm1 with time and as a function of temperature. An average activation energy of 220 kJ/mol for the translocation of DMPC, DPPC, and DSPC was determined. A decrease in alkyl chain length resulted in a substantial increase in the rate of flip-flopmanifested as an increase in theArrhenius preexponential factor. The effect of lipid labelingwas investigated bymeasuring the exchange of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n,n-Dimethyl-n-(29,29,69,69-tetramethyl-49-piperidyl) (TEMPO-DPPC). The rate of TEMPO-DPPC flip-flop was an order-of-magnitude slower compared to DPPC. An activation energy of 79 kJ/ mol wasmeasured which is comparable to that previouslymeasured by electron spin resonance. The results of this study illustrate how SFVS can be used to directly measure lipid flip-flop without the need for a fluorescent or spin-labeled lipid probe, which can significantly alter the rate of lipid translocation