Different phase behaviour of the sn-1 and sn-3 stereoisomers of the glycolipid di-tetradecyl-beta-D-galactosylglycerol.

The phase behaviour of a synthetic, stereochemically pure glycolipid 2,3-di-O-tetradecyl-1-O-beta-D-galactosyl-sn-glycerol (14-2,3-Gal) in excess water has been characterized by differential scanning calorimetry and time-resolved X-ray diffraction and compared with that of the previously studied sn-3 stereoisomer, 1,2-di-O-tetradecyl-3-O-beta-D-galactosyl-sn-glycerol (14-1,2-Gal), and 1,2-di-O-tetradecyl-3-O-beta-D-glucosyl-sn-glycerol (14-1,2-Glc). The properties of 14-1,2-Gal and 14-2,3-Gal are completely different with respect to phase sequences, metastable behaviour, transition temperatures and enthalpies, but there is a rather close similarity between the phase patterns of 14-2,3-Gal and 14-1,2-Glc. The sn-3 stereoisomer, 14-1,2-Gal, exhibits a direct lamellar crystalline to inverted hexagonal phase transition (Lc-->HII) on heating and a HII-->L alpha (metastable)-->L beta(metastable)-->Lc phase sequence in subsequent cooling, while both 14-2,3-Gal and 14-1,2-Glc are characterized by an Lc-->L alpha-->HII sequence in first heating, and reversible L beta<-->L alpha<-->HII phase sequences in subsequent heating and cooling scans. The peak areas and temperatures of the L beta-->L alpha transitions are practically identical, while the Lc-->L alpha and L alpha-->HII temperatures for 14-2,3-Gal are about 5 degrees C higher than the corresponding temperatures for 14-1,2-Glc. These data are interpreted in terms of a significantly greater stability and faster formation kinetics of the lamellar crystalline Lc phase of 14-1,2-Gal.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and phase behavior of a charged glycolipid (1,2-O-dialkyl-3-O-beta-D-glucuronosyl-sn-glycerol).

In order to investigate the effects of a net surface charge on the properties of glycolipid membranes, we have synthesized a glyceroglycolipid, 1,2-O-dialkyl-3-O-beta-D-glucuronosyl- sn-glycerol (GlcUA lipid), with saturated alkyl chains of varying length (14, 16, and 18 carbon atoms; 14-, 16-, and 18-GlcUA, respectively) and glucuronic acid with an ionizable 6-carboxyl group as polar residue. Aqueous dispersions of GlcUA lipids have been characterized by differential scanning calorimetry, densitometry, and X-ray diffraction methods as a function of pH. The carboxyl group deprotonation of apparent pK about 5.5 leads to a decrease of the melting temperatures by about 7 degrees C for all three compounds and to a chain-length-dependent reduction of the transition enthalpies by 0, 7, and 14% for 14-, 16-, and 18-GlcUA, respectively. The decrease of the transition temperature is consistent with current electrostatic concepts and models of charged membrane interfaces, but the chain-length-specific dependence of the enthalpy decrease with an increase of pH shows that the pH effects in GlcUA lipids are not of purely electrostatic origin. However, these effects appear to be simpler in some instances than corresponding effects in phospholipids with multiply ionizable head groups. For this reason, the lipids with the glucuronic acid head group appear to represent an appropriate model system for studies of net electric charge effects on the membrane properties.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of glycerol on the phase behaviour of hydrated distearoylphosphatidylethanolamine and its possible relation to the mode of action of cryoprotectants.

A phase diagram for 1,2-distearoylphosphatidylethanolamine (DSPE) dispersed in glycerol/water mixtures was constructed using data obtained from differential scanning calorimetry and time-resolved X-ray diffraction measurements. The phase sequence seen on heating the lipid remains the same for samples containing up to 70 wt% glycerol. Depending on the hydration conditions, the samples are either in a metastable lamellar gel (L beta) or one or other of two possible sub-gel phases (Lc and Lc') at low temperatures. These phases convert first to a lamellar liquid crystalline (L alpha) and then to an inverted hexagonal (HII) phase on heating. On cooling, the samples revert first to the L alpha and then to the L beta phase. Although the phase sequence is preserved, marked changes are seen in the transition temperatures between the different phases. The temperature of the transition between the L alpha and the HII phases decreases strongly with increasing glycerol concentration while that of the Lc and Lc' phases to L alpha, and to a lesser extent that of the L beta to L alpha transition, increases. Substantial changes in phase behaviour are seen if the glycerol concentration is increased above 70 wt%. Under these conditions, the Lc and Lc' phases transform directly into the HII phase on heating (a similar direct transition from the L beta to the HII phase is seen above 80 wt% glycerol). An exothermic transition from the L beta phase to the Lc' phase is observed and there is also an increasing tendency for the samples to revert to the Lc or Lc' phases on storage. These changes in relative stability of the different phases are discussed in terms of a possible membrane Hofmeister effect and their relevance to the mode of action of cryoprotectants is explored.

Effect of trehalose on the phase properties of hydrated and lyophilized dipalmitoylphosphatidylcholine multilayers.

The structure and thermal behavior of hydrated and lyophilized dipalmitoylphosphatidylcholine (DPPC) multilayers in the presence of trehalose were investigated by differential scanning calorimetry and X-ray diffraction methods. Trehalose enters the aqueous space between hydrated bilayers and increases the interbilayer separation (from 0.36 to 1.37 nm in the different DPPC phases at 1 M trehalose). It does not affect the lipid chain packing and also the slow isothermal conversion at 4 degrees C of the metastable L beta' phase into the equilibrium crystalline Lc phase. Addition of trehalose leads to a slight upward shift (about 1 degrees C at 1 M trehalose) of the three phase transitions (sub-, pre-, and main transition) in fully hydrated DPPC while their other properties (enthalpy, excess specific heat, and transition width) remain unchanged. The effect of trehalose on the thermal behavior of DPPC multilayers freeze-dried from an initially completely hydrated state is qualitatively similar to that of water. These data support the "water replacement" hypothesis about trehalose action. It is suggested that trehalose prevents the formation of direct interbilayer hydrogen bonds in states of low hydration.

Structure and phase behavior of hydrated mixtures of L-dipalmitoylphosphatidylcholine and palmitic acid. Correlations between structural rearrangements, specific volume changes and endothermic events.

Several new features of the phase diagram of L-dipalmitoylphosphatidylcholine (DPPC)/palmitic acid mixtures in excess water were established by means of static and time-resolved X-ray diffraction, densitometry and differential scanning calorimetry (DSC). At low temperatures, palmitic acid has a biphasic effect on the lamellar subgel phases: at concentrations below 5-6 mol%, it prevents formation of the DPPC subgel phase (Lc), while at higher contents (between about 40 and 90 mol%) another subgel phase (Lccom) is formed as a result of lipid co-crystallization at 1 DPPC: 2 palmitic acid stoichiometry. A crystalline palmitic acid phase separates from Lccom above 70-80 mol% of fatty acid. The Lccomphase transforms into a lamellar gel phase (L beta) in an endothermic transition centered at 38 degrees C. At high temperatures, the mixtures form hexagonal liquid-crystalline phase (HII) in the region of 60-70 mol% and an isotropic phase (I) at 90-100 mol% of palmitic acid. No coexistence of HII phase with the fluid lamellar phase of DPPC was observed at intermediate compositions (20 and 50 mol% of palmitic acid) but rather formation of a complex phase with non-periodic geometry characterized by molten chains and a broad, continuous small-angle scattering band. No evidence for fluid phase coexistence was found also at compositions between HII and I phases. The L beta--HII transition at 60-70 mol% of palmitic acids is readily reversible and two-state in both heating and cooling modes. It is characterized by the coexistence of initial and final phases with no detectable intermediates by time-resolved and static X-ray diffraction. The crystalline-isotropic transition in palmitic acid is two-state only in heating direction. On cooling, it is characterized by strong undercooling and gradually relaxing lamellar crystalline structures. The slowly reversible Lccom--L beta transition proceeds continuously through intermediate states. Although clearly discernible by both DSC and X-ray diffraction, it is not accompanied by specific volume changes.

Lamellar gel-lamellar liquid crystal phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from aqueous trehalose solutions. A real-time X-ray diffraction study.

The mechanism of the phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from fully hydrated gel phase (L beta') in the presence of trehalose has been investigated by real-time X-ray diffraction methods. Sequential diffraction patterns were recorded with an accumulation time of 3 s during heating and 1.2 s during cooling between about 20 and 80 degrees C. A transition is observed in the range 47-53 degrees C that involves structural events typical of a lamellar gel-lamellar liquid-crystal (L beta--L alpha) transformation. This transition is completely reversible with a temperature hysteresis of 2-3 degrees C and thereby resembles the main phase transition of fully hydrated dipalmitoylphosphatidylcholine multilayers. The mechanism of the transition from L beta to L alpha as seen in the wide-angle scattering profiles show that the sharp peak at about 0.41 nm, characteristic of the gel phase, broadens and shifts progressively to about 0.44 nm towards the end of the transition. A temperature jump of 6C degrees/s through the phase transition region of a freeze-dried dipalmitoylphosphatidylcholine: trehalose mixture (molar ratio 1:1) showed that the phase transition had a relaxation time of about 2 s which is similar to that of the main transition in the fully hydrated lipid. X-ray diffraction studies of the melting of dipalmitoylphosphatidylcholine freeze-dried from the lamellar-gel phase in the absence of trehalose showed a transition at above 70 degrees C. The low-angle diffraction data of phospholipid/trehalose mixtures are consistent with an arrangement of trehalose molecules in a loosely packed 'monolayer' separating bilayers of phospholipid. Trehalose appears to reduce the direct interbilayer hydrogen bond coupling thereby modifying the thermal stability and the phase transition mechanism of the bilayers.

Influence of head-group interactions on the miscibility of synthetic, stereochemically pure glycolipids and phospholipids.

Phase diagrams of binary mixtures of the glycoglycerolipids 1,2-di-O-tetradecyl-3-O-beta-D-galactosyl-sn-glycerol (14-Gal) and 1,2-di-O-tetradecyl-3-O-beta-D-glucosyl-sn-glycerol (14-Glc) with the phospholipids L-dimyristoylphosphatidylcholine (DMPC) and L-dimyristoylphosphatidylethanolamine (DMPE) were recorded by high-sensitivity differential scanning calorimetry and used for determination of the glycolipid-phospholipid miscibility in solid and liquid-crystalline states. As a consequence of a metastable behavior of both glycolipids and DMPE, the solid-state glycolipid/phospholipid miscibility was strongly dependent on the temperature prehistory of the samples. While DMPC and 14-Glc mix continuously, the other three binaries display extended regions of solid-solid-phase separation in the equilibrium low-temperature states. The DMPE/glycolipid phase diagrams were of clearly expressed eutectic type. Continuous solutions were formed in the liquid-crystalline and in the metastable solid phases of the mixtures. Simulations of the shape of the phase diagrams using the Bragg-Williams approximation showed certain deviations from ideal mixing in the liquid-crystalline continuous solutions. Since both glycolipids and phospholipids contain fully saturated fatty acids of equal chain length, their mixing properties were predominantly determined by the interactions between the lipid polar moieties, assuming the influence of ester or either linkages of the alkyl chains on the mixing parameters to be negligible. The clearly expressed differences in the mixing of 14-Glc and 14-Gal with phospholipids are most probably due to different hydrogen-bond networks formed by the glucosyl and galactosyl residues.

Effect of lipid admixtures on the L-dipalmitoylphosphatidylcholine subtransition.

The effect of lipid admixtures on the properties of the L-dipalmitoylphosphatidylcholine (L-DPPC) subtransition is investigated by using high-sensitivity differential scanning calorimetry. The four admixtures used are D-DPPC, L-dipalmitoylphosphatidylethanolamine (L-DPPE), cholesterol, and palmitic acid. In all cases the subtransition decreases in enthalpy until disappearance with increase of the admixture concentrations. About 5-7 mol% of D-DPPC or palmitic acid are sufficient for abolishment (without position shifts) of the subtransition, while, on addition of L-DPPE or cholesterol, it persists up to about 20 mol% of the admixture and its disappearance is accompanied by a slight shift to higher temperatures. These data are tentatively interpreted in terms of lateral mixing of L-DPPC and admixture as indicating compound formation with D-DPPC and palmitic acid, and clustering of L-DPPE and cholesterol.

A probability concept about size distributions of sonicated lipid vesicles.

The sonication procedure of preparation of small unilamellar vesicles is modelled as a process of uniform random fragmentation of the lipid aggregates. The vesicle size distribution evolving in this process is shown to be identical with the Weibull extremal probability distribution. Size histograms of sonicated small vesicles of various phospholipid composition were obtained by using electron microscopy (negative staining). Their successful simulation with Weibull curves shows that theory agrees with experiment. A similarly good agreement is found also with size histograms obtained by freeze-fracture of phosphatidylcholine-cholesterol vesicles (Van Venetië, R., Leunissen-Bijvelt, J., Verkleij, A.J. and Ververgaert, P.H.J.T. (1980) J. Microsc. 118, 401-408). This analysis allows a refinement of some earlier conclusions about the effect of cholesterol on the size of the sonicated vesicles. It follows from the theoretical model that the only intrinsic characteristic of the sonicated vesicles is the lower limit of their size. The other characteristics of the size distribution such as expectancy, dispersion, position and height of the maximum depend on the intensity of fragmentation. It is concluded that the size distribution of sonicated small vesicles is completely determined by the procedure of their preparation and, therefore, the condition of thermodynamic equilibrium between aggregated and monomeric lipid is irrelevant in this case.

The effect of nonideal lateral mixing on the transmembrane lipid asymmetry.

It is shown that the equilibrium transmembrane lipid asymmetry strongly depends on the degree of nonideality in the lateral mixing of the lipid components. In two-component bilayers the effect of nonideal lateral mixing is maximal for a given component at mole fractions of this component between 0.35 and 0.4. For asymmetry creating factors about 3 kT correcting for lateral nonidealities typical for lipids can increase as much as three times the transmembrane asymmetry. The relationship between lateral nonideality and transbilayer asymmetry is analysed in detail in the case of electrostatically induced asymmetry by using the Gouy-Chapman theory of electric double layers and the Bragg-Williams (regular solutions) approximation of nonideal lateral mixing. Two representative models are studied: (a) a single flat bilayer with a transmembrane electric potential difference applied on it; (b) two parallel membranes at short separation. In case (a), for transmembrane potentials of about 50-100 mV the introduction of nonideality corrections increases up to 40% the transmembrane asymmetry. In case (b), at physiological electrolyte concentrations the lipid asymmetry and, consequently, the effect of lateral nonideality become significant only at unrealistically small separations between the membranes. The surprisingly great influence of the lateral nonideality on the equilibrium transmembrane asymmetry suggests a significant role for this effect in determining the membrane molecular organization. A restricted lateral lipid miscibility might serve as a peculiar, but rather strong 'amplifier' of the transmembrane asymmetry. The qualitatively different asymmetries found in small unilamellar phosphatidylcholine-phosphatidylethanolamine vesicles of different fatty acid composition (Lentz, B.R. and Litman, B.J. (1978) Biochemistry 17, 5537-5543) can be reasonably well explained as an effect of the lateral nonideality. A hypothesis considering the transmembrane distributions of the major phospholipid species in erythrocytes as evolving from their lateral miscibilities is proposed.

Effect of ion concentration on phosphatidylethanolamine distribution in mixed vesicles.

The membrane of mixed phosphatidylcholine-phosphatidylethanolamine vesicles was found to be impermeable to the fluorescent label fluorescamine. Added to the vesicle solution, fluorescamine labels only the phosphatidylethanolamine molecules in the outer layer. The separation of labeled and free phospholipid by thin-layer chromatography permits the determination of the inner to outer phosphatidylethanolamine ratio. This ratio can be independently obtained by multiple sonication and the addition of fluorescamine, which results in a progressive increase of the labeled phosphatidylethanolamine. These two methods give identical results for the phospholipid distribution. The ratio of the outer to the total phosphatidylethanolamine decreases with the increase in the mole fraction of phosphatidylethanolamine, in agreement with the results published by Litman (Litman, B.J. (1973) Biochemistry 12, 2545-2554). It was also found that the lipid distribution is sensitive to changes of the electrolyte concentration in the aqueous phase. At high concentrations the distribution is close to the symmetrical one; a decrease in ionic strength results in preferential localisation of phosphatidylethanolamine molecules in the inner vesicle layer.