Molecular Motion in Frozen Phospholipid Bilayers in the Presence of Sucrose and Sorbitol Studied by the Spin-Echo EPR of Spin Labels

Electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR), was applied to spin-labeled stearic acids in phospholipid bilayers hydrated in the presence of sucrose and sorbitol, which are known for their cryoprotective action on biological membranes. The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Stearic acids were labeled by nitroxide 4,4-dimethyl-oxazolidine-1-oxyl (DOXYL) attached rigidly at either the 5th or 16th specific carbon positions. ESE detects fast stochastic small-angle restricted molecular rotations (stochastic molecular librations) with correlation times on the nanosecond timescale. These motions are believed to have the same nature as the anharmonic motions of hydrogen atoms in biological substances detected by neutron scattering and Mossbauer spectroscopy, which become active above 200 K. To ensure that the echo decays indeed originate from fast stochastic molecular librations, a three-pulse stimulated spin echo was employed. It was found that the presence of sucrose or sorbitol suppresses the observed molecular motions. The observed effect was nearly the same for both label positions, indicating that the motions are similarly suppressed near the bilayer surface and in the bilayer interior. This finding suggests non-specific interactions of sugars with bilayer surface, which are likely to influence only the bulk physical properties of hydrated membranes. The results obtained show the usefulness of spin-echo EPR of spin labels when applied to investigate the molecular mechanisms of action of cryoprotective agents on biological systems.