3 DISTINCT TYPES OF UNFROZEN WATER IN FULLY HYDRATED PHOSPHOLIPID-BILAYERS - A COMBINED H-2-NMR AND P-31-NMR STUDY

Combined H-2- and P-31-nuclear magnetic resonance (NMR) studies of six D2O/phospholipid systems with different headgroup are presented to id entify the molecular origin of unfrozen water detected in phospholipid membranes. When phospholipids are dispersed in excess water, NMR sign als of water molecules from the interbilayer space at subzero temperat ures are identifiable because their spin-lattice relaxation time (T-1) are relatively short in comparison with those from bulk ice. Three ty pes of interbilayer unfrozen water are then revealed by studying the t emperature-dependent behavior of isotropic H-2-NMR unfrozen D2O signal with T-1 values in the ms range for fully hydrated D2O/phospholipid b ilayers. The first type is the supercooled water in D2O/phosphatidylet hanolamine and D2O/phosphatidic acid. The unfrozen water of these syst ems can only be detected from -20 to -35 degrees C and will freeze upo n reaching the homogeneous nucleation temperature of ice formation for D2O, i.e., -35 degrees C. The second type is the perturbed water in D 2O/phosphatidylcholine and D2O/sphingomyelin. The isotropic H-2-NMR si gnals of these systems broaden with the decreasing temperature from -2 0 to -70 degrees C. The third type is the bound water in D2O/phosphati dylserine and D2O/phosphatidylinositol. The H-2-NMR signals of these s ystems remain unchanged in terms of their signal intensity and linewid th with decreasing temperature even at the lowest studied temperature of -70 degrees C. The P-31-NMR spectra obtained in all hydrated phosph olipid systems at -40 degrees C show an axially asymmetric powder patt ern similar to those obtained from dry lipids at room temperature sugg esting that the rotational motion of phosphorous group is frozen at -4 0 degrees C. We conclude that molecular groups attached to the phospha te segment in hydrated phospholipid systems are mainly responsible for the unfrozen water detected by NMR.