Precision parameters from spin-probe studies of membranes using a partitioning technique. application to two model membrane vesicles.

A new version of the ESR spin probe partitioning method is developed and applied to the study of hydration properties of dimyristoyl-phosphatidylglycerol (DMPG) and dimyristoyl-phosphatidylcholine (DMPC) vesicles as functions of salt concentration and temperature above the lipid phase transition. The small spin probe di-tert-butyl nitroxide (DTBN) is used in order to achieve motionally narrowed Electron Spin Resonance (ESR) spectra which may be analyzed with high precision. The new method relies on the use of the second harmonic display of the ESR spectrum followed by spectral line fitting. Spectral fitting yields precise ESR parameters giving detailed information on the surroundings of the spin probe in both phospholipid and aqueous phases. The nitrogen hyperfine coupling constant of DTBN arising from those probes occupying the vesicles is used to study the hydration of the vesicle surface. The hydration properties of the negatively charged vesicle surface of DMPG vesicles are affected by the addition of salt at all temperatures. In contrast, the hydration of DMPC vesicles does not change with salt concentration at the low temperatures. However, at higher temperatures the hydration properties of DMPC vesicle are affected by salt which is interpreted to be due to the faster motion of the phospholipid molecules. The partitioning of the spin probe increases with salt concentration for both DMPG and DMPC vesicles, while water penetration decreases simultaneously. The spin probe in the phospholipid bilayer exhibits anisotropic motion and the extent of the anisotropy is increased at the higher salt concentrations.