Phase equilibria of the ternary system 1-palmitoyl-sn-glycero-3-phosphocholine/oleic acid/water studied by NMR

Part of a phase diagram for the system 1-palmitoyl-sn-glycero-3-phosphocholine (PamGroPCho)/oleic acid/water has been constructed from mainly 31P-NMR data and a previous determination of the phase equilibria of the binary PamGroPCHo/water system. It was found that the appearance of the phase diagram is very similar to those found for several simple soap/fatty acid/water or soap/long-chain alcohol/water systems. The most striking features observed are: (1) the lamellar phase can swell towards very high water contents (2) vesicles are formed after sonication and (3) the cubic liquid crystalline phase disappears upon addition of very small amounts of oleic acid. The self-association of the amphiphiles and the shape of the aggregates are discussed in terms of existing first-order approximative theories.     

Inhibition of arachidonic acid incorporation into erythrocyte phospholipids by peracetic acid and other peroxides. Role of arachidonoyl-CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase

To explore possible mechanisms of the arachidonic acid deficiency of the red blood cell membrane in alcoholics, we compared the effect of ethanol and its oxidized products, acetaldehyde and peracetic acid, with other peroxides on the accumulation of [14C]arachidonate into RBC membrane lipids in vitro. Incubation of erythrocytes with 50 mM ethanol or 3 mM acetaldehyde had no effect on arachidonate incorporation. Pretreatment of erythrocytes with 10 mM hydrogen peroxide, 0.1 mM cumene hydroperoxide or 0.1 mM t-butyl hydroperoxide had little effect on [14C]arachidonate incorporation in the absence of azide. However, pretreatment of cells with N-ethylmaleimide, 0.1 mM peracetic acid or performic acid, with or without azide, inhibited arachidonate incorporation into phospholipids but not neutral lipids. In chase experiments, peracetate also inhibited transfer of arachidonate from neutral lipids to phospholipids. To investigate a possible site of this inhibition of arachidonate transfer into phospholipids by percarboxylic acids, we assayed a repair enzyme, arachidonoyl CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase (EC 2.3.1.23). As in intact cells, phospholipid biosynthesis was inhibited more by N-ethylmalemide and peracetic acid than by hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. Peracetic acid was the only active inhibitor among ethanol and its oxidized products studied and may deserve further examination in ethanol toxicity.     

Biosynthesis of pulmonary surfactant: Comparison of 1-palmitoyl-sn-glycero-3-phosphocholine and palmitate as precursors of dipalmitoyl-sn-glycero-3-phosphocholine in adenoma alveolar type II cells

Urethan-induced pulmonary adenomas of mice are composed of cells that appear to be morphologically identical to alveolar type II cells and synthesize disaturated diacyl-sn-glycero-3-phosphocholine, the major component of pulmonary surfactant. 1-[1-¹⁴C]Palmitoyl-sn-glycero-3-phosphocholine and [1-¹⁴C]palmitic acid were compared as precursors of disaturated diacyl-sn-glycero-3-phosphocholine in the adenoma type II cells by incubating both substrates with whole adenomas. When the precursors were compared at equal concentrations (100 μm) in the presence of albumin (1 mg/ml), the rates of incorporation of 1-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine and [1-¹⁴C]palmitic acid into diacyl-sn-glycero-3-phosphocholine were 5.2 and 2.9 nmol/min · g tissue, respectively. The concentration of monoacyl-sn-glycero-3-phosphocholine (lysolecithin) in the blood plasma of BALB/c mice was 150 μm. In short-term labeling experiments, the label in disaturated diacyl-sn-glycero-3-phosphocholine was equally distributed between the sn-1 and sn-2 positions when 1-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine was the precursor, whereas 75 to 80% was in the sn-2 position when [1-¹⁴C]palmitic acid was the precursor. The ratios are consistent with incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine via the lysolecithin:lysolecithin transacylase reaction and incorporation of palmitate via acylation of 1-palmitoyl-sn-glycero-3-phosphocholine by acyl-CoA:lysolecithin acyltransferase. 1-[1-¹⁴C]Palmitoyl-sn-glycero-3-phospho-[³H-methyl]choline was incorporated into total cellular diacyl-sn-glycero-3-phosphocholine with an isotope ratio similar to that of the precursor; the disaturated species was more enriched in ¹⁴C. These findings indicate the cells take up intact monoacyl-sn-glycero-3-phosphocholine and incorporate it into diacyl-sn-glycero-3-phosphocholine. The ability of the cells to utilize intact lysophosphoglycerides for synthesis of cellular lipids was further demonstrated by showing that ether analogs, 1-alkyl-sn-glycero-3-phosphocholine and 1-alkyl-sn-glycero-3-phosphoethanolamine, are taken up and acylated by the cells. Activities of lysolecithin:lysolecithin transacylase and acyl-CoA:lysolecithin acyltransferase were measured in subcellular fractions of the adenoma type II cells; the specific activities of the enzymes were 2.1 nmol/min · mg soluble protein and 21 nmol/min · mg microsomal protein, respectively. The total activity of the acyltransferase in the cell fractions was about four-fold higher than the activity of the transacylase. Characteristics of the two enzymes were studied and are discussed. The findings indicate that exogenous 1-palmitoyl-sn-glycero-3-phosphocholine and palmitic acid both serve as efficient precursors of disaturated diacyl-sn-glycero-3-phosphocholine in the adenoma alveolar type II cells.     

Phase transitions in 1,2-and 1,3-diacyl-sn-glycerol-3-phosphocholine monolayers at the oil/water interface

Moving the phosphatidylcholine group from the 3-to the 2-position in monolayers of $\text{distearoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ at the oil/water interface expands the surface pressure-area isotherm and markedly increases the surface pressure at which phase separation occurs with only a slight change in the monolayer surface density at the onset of the transition. This is interpreted in terms of a change in an ordering parameter in the solid-condensed state.     

Phase equilibria and molecular packing in the N,N-dimethyldodecylamine oxide/gramicidin D/water system studied by 2H nuclear magnetic resonance spectroscopy.

A partial phase diagram of the system N,N-dimethyldodecylamine oxide (DDAO)/water/gramicidin D was determined by 2H-NMR. Both 2H2O and perdeuterated DDAO (DDAO-d31) were studied by solid state NMR techniques. Addition of gramicidin D to the micellar (L1), normal hexagonal (HI) and cubic (I) phases of DDAO induces phase separations, giving two-phase regions, which all contain a lamellar (L alpha) phase. The L alpha phase containing gramicidin is characterized by larger order parameters for DDAO-d31 compared with the corresponding order parameters in the L alpha and HI phases of DDAO-d31/H2O. The L alpha phase may stay in equilibrium with any other phase in the phase diagram. The DDAO exchange between the coexisting phases is slow on the NMR timescale, which is why the recorded NMR spectrum consists of superimposed spectra from the different phases occurring in the sample. Gramicidin D can be solubilized in appreciable quantities only in the lamellar phase of DDAO-d31. Increasing amounts of gramicidin in the liquid crystalline phases result in a continuous increase in the molecular ordering up to about 5 mol% gramicidin, where a plateau is reached. This is consistent with a recent theoretical model describing the influence on the ordering of lipids by a membrane protein with larger hydrophobic thickness than the lipid bilayer. The solvent used for dissolving gramicidin at the incorporation of the peptide in the lipid aggregates has no effect on the 2H-NMR lineshapes of DDAO-d31. It is concluded that gramicidin is solubilized in the L alpha phase and that it always adopts the channel conformation independent of a particular solvent. The channel conformation is also supported by CD studies. In some of the samples, macroscopic orientation of the lipid aggregates is observed. It is concluded that DDAO-d31 in the binary system favors an orientation with the long axis of the hydrocarbon chain perpendicular to the magnetic field, whereas when gramicidin D is present the hydrocarbon chain orients parallel to the magnetic field. This is explained by the fact that gramicidin aligns with its helical axis parallel to the magnetic field, thereby forcing also the DDAO-d31 molecules to obtain such an orientation.     

Effect of chain-linkage on the structure of phosphatidyl choline bilayers. Hydration studies of 1-hexadecyl 2-palmitoyl-sn-glycero-3-phosphocholine.

While hydrated dipalmitoyl phosphatidylcholine (DPPC) forms tilted chain L beta' bilayers in the gel phase, the ether-linked analogue dihexadecyl phosphatidylcholine (DHPC) exhibits gel phase polymorphism. At low hydration DHPC forms L beta' phases but at greater than 30% H2O a chain-interdigitated gel phase is observed (Ruocco, M. J., D. S. Siminovitch, and R. G. Griffin. 1985. Biochemistry. 24:2406-2411; Kim, J.T., J. Mattai, and G.G. Shipley. 1987. Biochemistry. 26:6599-6603). In this study we report the behavior of a phosphatidylcholine (PC) with both types of chain linkage, 1-hexadecyl-2-palmitoyl-sn-glycero-3-phosphocholine (HPPC). HPPC has been investigated as a function of hydration using differential scanning calorimetry (DSC) and x-ray diffraction. By DSC, over the hydration range 5. 1-70.3 wt% H2O, HPPC exhibits two reversible transitions. The reversible main chain-melting transition decreases from 69 degrees C, reaching a limiting value of 40 degrees C at full hydration. X-ray diffraction patterns of hydrated HPPC have been recorded as a function of hydration at 20 degrees and 50 degrees C. At 50 degrees C, melted-chain L alpha bilayer phases are observed at all hydrations. At 20 degrees C, at low hydrations (less than 34 wt% H2O) HPPC exhibits diffraction patterns characteristic of bilayer gel phases similar to those of the gel phase of DPPC. In contrast, at greater than or equal to 34 wt% H2O, HPPC shows a much reduced bilayer periodicity, d = 47 A, and a single sharp reflection at 4.0 A in the wide angle region. This diffraction pattern is identical to that exhibited by the interdigitated phase of DHPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase transitions in 1,2- and 1,3-diacyl-sn-glycero-3-phosphocholine monolayers at the oil/water interface.

Moving the phosphatidylcholine group from the 3- to the 2-position in monolayers of distearoyl-sn-glycero-3-phosphocholine at the oil/water interface expands the surface pressure-area isotherm and markedly increases the surface pressure at which phase separation occurs with only a slight change in the monolayer surface density at the onset of the transition. This is interpreted in terms of a change in an ordering parameter in the solid-condensed state.     

On the surface properties of oleate micelles and oleic acid/oleate vesicles studied by spin labeling

Dilute aqueous systems composed of sodium oleate micelles and sodium oleate/oleic acid vesicles were investigated as a function of pH by electron spin resonance spectroscopy with TEMPO-stearate TEMPO-stearamide as well as with a positively charged water soluble spin label, TEMPO-choline. The dynamics of the three TEMPO-spin labels were found to be sensitive to changes in the interfacial region of the aggregates as a function of pH. The results obtained are consistent with the formation of a hydrogen bond network (RCOO⁻ ↔ HOOCR) at the surface of the sodium oleate/oleic acid system in the course of the transformation of micelles into the closed bilayers (vesicles). Vesicles formation below pH 10 was determined independently with a spin labeled glucose derivative.     

Phase equilibria in four lysophosphatidylcholine/water systems. Exceptional behaviour of 1-palmitoyl-glycerophosphocholine

The phase equilibria in four lysophosphatidylcholine/water systems were investigated at different temperatures. Each of the 1-palmitoyl-, 1-stearoyl-, 1-oleoyl- and 1-linoleoyl-sn-glycero-3-phosphocholines was dispersed in heavy water at different concentrations. The phase structures were determined by 2H-, 14N- and 31P-NMR, polarization microscopy and low-angle X-ray diffraction. The phase diagrams of the oleoyl and linoleoyl systems were quite similar. At room temperature and with decreasing water content the isotropic micellar solution was followed by a hexagonal phase and then a cubic phase. Finally the lamellar phase appeared before the region of hydrated crystals. The same sequence of phases was observed in the stearoyl system at elevated temperatures. The palmitoyl system differed from the others: here a cubic phase followed after the micellar solution, then came a hexagonal phase and after this a lamellar phase. In general the lysophosphatidylcholines seem to behave similarly to the many soaps and detergents as they show the same sequence of isotropic micellar solution, hexagonal phase, lamellar phase with interspersed cubic phases. The presently established phase diagrams demonstrate that the major lysophosphatidylcholines which may be generated by phospholipase A2 in mammalian cell membranes, viz. 1-palmitoyl- and 1-stearoyl-glycerophosphocholines differ greatly in their packing properties. The extraordinary ability of 1-palmitoyl-glycerophosphocholine to form a cubic phase in equilibrium with a micellar solution is of particular interest with regard to the possible occurrence of cubic structures in biomembranes during the process of fusion.     

Interaction of two prolamins with 1-13C oleic acid by 13C NMR

In this paper, we analyzed the interaction of Z19 prolamin from a BR451 maize variety and pennisetin from a BRS1501 pearl millet variety with 1-(13)C-enriched oleic acid (OA) by (13)C NMR in solution. In both proteins, we identified the presence of free fatty acids by NMR in solid state and solution. The interactions were analyzed at the protein/OA molar ratios of 1:1 and 1:4. In the Z19/OA 1:1 mixture in 70% ethanol and 30% D(2)O, the chemical shift of OA C1 was 182.9 ppm, about 3 ppm above that of the pure OA in the same solvent. In contrast, upon addition of OA to the pennisetin (1:1), the chemical-shift value slightly decreased by less than 1 ppm. The chemical-shift titration curve of OA C1 in an apparent pH range of 5.5-7.3 shifted by approximately 0.3 pH units toward higher pH values in the pennisetin/OA 1:1 complex relative to the pure OA. The results obtained for the pennisetin/OA 1:4 mixture were similar to the complexes at a 1:1 molar ratio. A significant difference was observed between the 1:1 and 1:4 curves for Z19. The titration curve for Z19/OA 1:1 suggested specific binding at the sites with electrostatic interaction.     

CoA-independent transfer of arachidonic acid from 1,2-diacyl-sn-glycero-3-phosphocholine to 1-O-alkyl-sn-glycero-3-phosphocholine (lyso platelet-activating factor) by macrophage microsomes

Macrophage microsomes catalyzed the transfer of arachidonic acid (20:4) from 1,2-diacyl-glycerophosphocholine (GPC) to 1-alkyl-GPC (lyso platelet-activating factor). This enzyme reaction did not require the presence of cofactors such as Co A. Free arachidonic acid or linoleic acid-labeled phospholipids failed to act as the acyl donor. These results suggest that the reaction is a CoA-independent direct transfer of arachidonic acid. This arachidonoyl transacylation system may play a very important role in the metabolism of lyso platelet-activating factor and also in the elimination or release of arachidonic acid from diacyl-GPC.     

Ionization and binding equilibria of papaverine in ionic micelles studied by 1H NMR and optical absorption spectroscopy

The binding of the vasodilator drug papaverine (PAV) to micelles of zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS), cationic cetyltrimethylammonium chloride (CTAC) and anionic sodium dodecylsulfate (SDS) in aqueous solution was studied by 1H NMR and electronic absorption spectroscopy. In the presence of HPS or CTAC, the apparent pK(a) of PAV decreased by about 2 units, while it increased by about 2 units upon binding to SDS. However, the chemical shift patterns of both protonated (PAVH+) and deprotonated (PAV0) forms of PAV are not sensitive to the type of surfactant. The association constants were estimated as 5 +/- 2 M(-1) for PAVH+-CTAC, 8 +/- 3 M(-1) for PAVH+-HPS, (7 +/- 2) x 10(5) M(-1) for PAVH+-SDS, and 1.5 x 10(3) to 3.0 x 10(3) M(-1) for the complexes of PAV0 with all three types of micelles. Using these data, an electrostatic potential difference on the micelle-water interface was calculated as 150 +/- 10 mV for CTAC, 140 +/- 10 mV for HPS and - 140 +/- 10 mV for SDS. The results suggest that PAV aromatic rings are located in the hydrophobic part of the micelle. The electrostatic attraction or repulsion of the protonated quinoline nitrogen and surfactant headgroups changes the affinity of PAV to micelles and, thus, shifts the ionization equilibrium of PAV. The electrostatic potential of HPS micellar surface is determined by the cationic dimethylammonium headgroup fragment, whereas the anionic sulfate fragment attenuates the effective charge of HPS headgroup.     

Perturbation of the conformational equilibria in Ras by selective mutations as studied by 31P NMR spectroscopy

Ras regulates a variety of different signal transduction pathways acting as molecular switch. It was shown by liquid and solid-state (31)P NMR spectroscopy that Ras exists in the guanosine-5'-(beta,gamma-imido)triphosphate bound form in at least two conformational states interconverting in millisecond time scale. The relative population between the two conformational states affects drastically the affinity of Ras to its effectors. (31)P NMR spectroscopy shows that the conformational equilibrium can be shifted specifically by point mutations, including mutations with oncogenic potential, thus modifying the effector interactions and their coupling to dynamic properties of the protein.     

Hydration water molecules of nucleotide-free RNase T1 studied by NMR spectroscopy in solution

The hydration of uncomplexed RNase T₁ was investigated by NMR spectroscopy at pH 5.5 and 313 K. Two-dimensional heteronuclear NOE and ROE difference experiments were employed to determine the spatial proximity and the residence times of water molecules at distinct sites of the protein. Backbone carbonyl oxygens involved in intermolecular hydrogen bonds to water molecules were identified based on¹ J coupling constants. These coupling constants were determined from 2D-H(CA)CO and ¹⁵N-HSQC experiments with selective decoupling of the ^13CC nuclei during the t₁ evolution time. Our results support the existence of a chain of water molecules with increased residence times in the interior of the protein which is observed in several crystal structures with different inhibitor molecules and serves as a space filler between the -helix and the central -sheet. The analysis of¹ J_NC' coupling constants demonstrates that some of the water molecules seen in crystal structures are not involved in hydrogen bonds to backbone carbonyls as suggested by crystal structures. This is especially true for a water molecule, which is probably hydrogen bonded by the protonated carboxylate group of D76 and the hydroxyl group of T93 in solution, and for a water molecule, which was reported to connect four different amino acid residues in the core of the protein by intermolecular hydrogen bonds.     

Magic-angle spinning NMR studies of molecular organization in multibilayers formed by 1-octadecanoyl-2-decanoyl-sn-glycero-3-phosphocholine.

Magic-angle spinning 1H and 13C nuclear magnetic resonance (NMR) have been employed to study 50%-by-weight aqueous dispersions of 1-octadecanoyl-2-decanoyl-sn-glycero-3-phosphocholine (C[18]:C[10]PC) and 1-octadecanoyl-2-d19-decanoyl-PC (C[18]:C[10]PC-d19), mixed-chain phospholipids which can form interdigitated multibilayers. The 1H NMR linewidth for methyl protons of the choline headgroup has been used to monitor the liquid crystalline-to-gel (LC-to-G) phase transition and confirm variations between freezing and melting temperatures. Both 1H and 13C spin-lattice relaxation times indicate unusual restrictions on segmental reorientation at megahertz frequencies for C(18):C(10)PC as compared with symmetric-chain species in the LC state; nevertheless each chemical moiety of the mixed-chain phospholipid exhibits motional behavior that may be classified as liquidlike. Two-dimensional nuclear Overhauser spectroscopy (NOESY) on C(18):C(10)PC and C(18):C(10)PC-d19 reveals cross-peaks between the omega-methyl protons of the C18 chain and the N-methyl protons of the phosphocholine headgroup, and several experimental and theoretical considerations argue against an interpretation based on spin diffusion. Using NMR relaxation times and NOESY connectivities along with a computational formalism for four-spin systems (Keepers, J. W., and T. L. James. 1984. J. Magn. Reson. 57:404-426), an estimate of 3.5 A is obtained for the average distance between the omega-methyl protons of the C18 chain and the N-methyl protons of the phosphocholine headgroup. This finding is consistent with a degree of interdigitation similar to that proposed for organized assemblies of gel-state phosphatidylcholine molecules with widely disparate acyl-chain lengths (Hui, S. W., and C.-H. Huang. 1986. Biochemistry. 25:1330-1335); however, acyl-chain bendback or other intermolecular interactions may also contribute to the NOESY results. For multibilayers of C(18):C(10)PC in the gel phase, 13C chemical-shift measurements indicate that trans conformers predominate along both acyl chains. 13C Spin-lattice relaxation times confirm the unusual motional restrictions noted in the LC state; nevertheless, 13C and 1H rotating-frame relaxation times indicate that the interdigitated arrangement enhances chain or bilayer motions which occur at mid-kilohertz frequencies.     

Ceramide drives cholesterol out of the ordered lipid bilayer phase into the crystal phase in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol/ceramide ternary mixtures

The effect of brain ceramide on the maximum solubility of cholesterol in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and ceramide was investigated at 37 degrees C by a cholesterol oxidase (COD) reaction rate assay and by optical microscopy. The COD reaction rate assay showed a sharp increase in cholesterol chemical potential as the cholesterol mole fraction approaches the solubility limit. A decline in the COD reaction rate was found after the formation of cholesterol crystals. The maximum solubility of brain ceramide in POPC bilayers was determined to be 68 +/- 2 mol % by microscopy. We found that ceramide has a much higher affinity for the ordered bilayers than cholesterol, and the maximum solubility of cholesterol decreases with the increase in ceramide content. More significantly, the displacement of cholesterol by ceramide follows a 1:1 relation. At the cholesterol solubility limit, adding one more ceramide molecule to the lipid bilayer drives one cholesterol out of the bilayer into the cholesterol crystal phase, and cholesterol is incapable of displacing ceramide from the bilayer phase. On the basis of these findings, a ternary phase diagram of the POPC/cholesterol/ceramide mixture was constructed. The behaviors of ceramide and cholesterol can be explained by the umbrella model. Both ceramide and cholesterol have small polar headgroups and relatively large nonpolar bodies. In a PC bilayer, ceramide and cholesterol compete for the coverage of the headgroups of neighboring PC to prevent the exposure of their nonpolar bodies to water. This competition results in the 1:1 displacement as well as the displacement of cholesterol by ceramide from lipid raft domains.     

Phase behavior of the monoerucin/water system

Monoerucin is a monoacylglycerol incorporating a cis monounsaturated fatty acid, 22 carbon atoms long, with the double bond at carbon number 13. The original temperature-composition phase diagram for the monoerucin/water system, constructed on the basis of ‘consistency’ and light microscopy, includes unexpectedly only the lamellar and fluid isotropic phases [Lutton, E.S., 1965. J. Am. Oil Chem. Soc. 42:1068–1070]. In separate studies using X-ray diffraction, the cubic phase [Larsson et al., 1978. J. Sci. Food Agric. 29:909–914] and the inverted hexagonal (H_II) phase [Caffrey, M., 1989. Biophys. J. 95:11–21] were shown to be the dominant phases. We have resorted to the traditional isoplethal diffraction method for constructing the equilibrium phase diagram of monoerucin in water with a view to resolving the controversy regarding the thermotropic and lyotropic properties of the system. The study confirms the presence of the pure H_II phase that extends from ca. 10 to 22% (w/w) water and from ca. 35 to 125°C, and its coexistence with excess water above ca. 24%(w/w) water in the same temperature range. The low temperature region of the diagram is dominated by the lamellar crystal (L_c) phase, with the existence of the lamellar liquid crystal (L) and cubic-Ia3d phases at intermediate temperatures and hydration levels. Differential scanning calorimetry and polarizing light microscopy were used to confirm the reported equilibrium phase behavior. Full structure characterization of the system and how phase microstructure depends on sample hydration and temperature are reported.     

The state of water in muscle as studied by pulsed NMR

Spin-lattice relaxation times for the water protons in rat gastronemius muscle are reported over the temperature range +37 to −70°C at six resonance frequencies ranging from 4.5 to 60.0 MHz. From −8 to −70°C, the bulk of the muscle water is frozen. The unfrozen part is termed the hydrated layer and amounts to 7–12% of the total water content. Its correlation time takes teh form of a log-Gaussaian distribution function. From +37 to −8°C, the spin-lattice relaxation time is explained by the exchange of water between the hydration layer and the rest of the water, which behaves like ordinary liquid water. The fact that the observed T₂ values are smaller than the calculated values is attributed to the inner field inhomogeneity of the heterogenous system and/or the modification of T₂ due to non-zero dipolar interaction.In the presence of perdeuterated dimethylsulfoxide, the freezing point of water decreases and the amount of non-freezable water increases. T₁ of water protons for muscle containing 10, 20, and 40% dimethylsulfoxide was calculated.