Electron spin resonance observations on the annealing process of low-water-content dipalmitoylphosphatidylcholine bilayers

We report on ESR spectra obtained from 2 wt % water content dipalmitoylphosphatidylcholine (DPPC) doped with various piperidine-derivative nitroxides, placed between parallel glass surfaces and maintained at 125°C for several hours to achieve uniform alignment of bilayers. Spectral changes as a function of time, orientation in the magnetic field of the ESR spectrometer, and temperature were followed. Two classes of ESR spectra, with a typical evolution as a function of temperature and orientation, are obtained. We associate these with two distinct sites: piperidine rings located within the hydrophobic-chain region of the bilayer and at the interface between adjacent bilayers. The nitroxide moiety of Tempamine belongs to the first group, and that of TBBP (a 4-butoxybenzoyl derivative of Tempamine) and stearamide to the second. Time dependence is only observed with POATP, a propionyl derivative of Tempamine and a homologue intermediate between Tempamine and stearamide. This molecule changes location in the course of the annealing process from the intra- to the interbilayer site, suggesting an annealing-enhanced collective ordering of the phospholipid chains and long-range cooperative orientation of the phospholipid head groups.