Subtle differences in the hydration of phospholipid aggregates as a function of temperature and structure may be studied by SP-EPR

The spin probe electron paramagnetic resonance (SP-EPR) method has been successfully used to study the physical properties of phospholipids and their aggregates (Cevc, 1993). Recently, by employing nonlinear least-squares spectral fitting, it has been possible to extract high precision EPR parameters, such as hyperfine coupling spacings and rotational times, from the fast motional EPR spectra. The hydrophobic spin probe 2,2,6,6-tetramethyl-piperidin-1-oxyl-4-yl-octadecanoate (TEMPO-stearate) was studied in a variety of fully hydrated phospholipid vesicles. The hyperfine coupling spacing is used to estimate the effective water concentration in the polar shell of vesicles, while the rotational correlation times give information on the motion of the spin probe. The experimental results show that the effective water concentration in the polar shell of negatively charged dimyristoyl-phosphatidylglycerol (DMPG) vesicles is about 4 M greater than in the polar shell of zwitterionic dimyristoyl-phosphatidylcholine (DMPC) vesicles. Studies of a series of phosphatidylcholine (PC) vesicles with phospholipids of different acyl chain lengths from 14 to 18 carbons and lipid PC having unsaturated 18 carbon acyl chains have shown that the effective water concentration decreases by about 0.5 M for an increase of two carbons in the chain, and increases noticeably with hydrocarbon chain unsaturation. The differences in effective water concentration in the polar shells of phospholipid vesicles measured by the SP-EPR method appear to be in good agreement with the values obtained by X-ray scattering and NMR.