Effect of lamellarity and size on calorimetric phase transitions in single component phosphatidylcholine vesicles

Nano-differential scanning calorimetry (nano-DSC) is a powerful tool in the investigation of unilamellar (small unilamellar, SUVs, or large unilamellar, LUVs) vesicles, as well as lipids on supported bilayers, since it measures the main gel-to-liquid phase transition temperature (T_m), enthalpies and entropies. In order to assign these transitions in single component systems, where T_m often occurred as a doublet, nano-DSC, dynamic light scattering and cryo-transmission electron microscopy (cryo-TEM) data were compared. The two T_ms were not attributable to decoupled phase transitions between the two leaflets of the bilayer, i.e. nano-DSC measurements were not able to distinguish between the outer and inner leaflets of the vesicle bilayers. Instead, the two T_ms were attributed to mixtures of oligolamellar and unilamellar vesicles, as confirmed by cryo-TEM images. T_m for the oligolamellar vesicles was assigned to the peak closest to that of the parent multilamellar vesicle (MLV) peak. The other transition was higher than that of the parent MLVs for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and increased in temperature as the vesicle size decreased, while it was lower in temperature than that of the parent MLVs for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and decreased as the vesicle size decreased. These subtle shifts arose due to small differences in the values of ΔH and ΔS, since T_m is determined by their ratio (ΔH/ΔS). It was not possible to completely eliminate oligolamellar structures for MLVs extruded with the 200 nm pore size filter, even after 120 passes, while these structures were eliminated for MLVs extruded through the 50 nm pore size filter.     

Antioxidant activity of probucol and its effects on phase transitions in phosphatidylcholine liposomes

The effect of probucol on the phase behavior of dimyristoylphosphatidylcholine (DMPC) was examined by fluorescence polarization and differential scanning calorimetry (DSC). Probucol broadens and shifts the temperature of the main phase transition of DMPC liposomes as measured by fluorescence polarization with diphenylhexatriene and trimethyl-ammonium-diphenylhexatrine at concentrations as low as 5 mole%. As measured by DSC, probucol reduces the transition temperature of the gel----liquid-crystalline phase transition of DMPC by approx. 2 C degrees at all concentrations above about 5 mole% probucol and eliminates the pretransition at less than 1 mole%. In addition, the phase transition of DMPC is broadened and the enthalpy of the transition reduced by approx. 50%. Even at high concentrations of probucol, the gel----liquid-crystalline phase transition of DMPC is not eliminated. Similar effects are observed with dipalmitoylphosphatidylcholine liposomes. Based on these DSC measurements, measurements of the melting of probucol in dry mixtures with DMPC and observations of probucol mixtures with DMPC under polarizing optics, the maximum solubility of probucol in DMPC is approx. 10 mole%. This concentration exceeds that required (approx. 0.5 mole%) to prevent peroxidation of 10 mole% arachidonic acid in DMPC liposomes for 30 min in the presence of 0.05 mM Fe(NH4)(SO4)2 at 4 degrees C. Thus, probucol has a limited solubility in saturated phosphatidylcholine bilayers, but is an effective antioxidant at concentrations lower than its maximum solubility.     

Incorporation of highly purified melittin into phosphatidylcholine bilayer vesicles

Melittin free of phospholipase A2 was prepared. In the absence of salt this highly pure protein starts to aggregate in solution at a protein concentration of Cp greater than 10(-3) M. In high salt solution (2 M) aggregation starts at Cp greater than 10(-6) M. This was determined from the blue shift of the intrinsic fluorescence of the protein. Reinvestigation of the quenching behaviour clearly shows that self-aggregation cannot be deduced from quenching experiments using nitrate or 2,2,6,6-tetramethylpiperidine-1-oxyl as quencher. The incorporation of melittin into phosphatidylcholine bilayer vesicles was studied by fluorescence quenching and by energy-transfer experiments using 2- and 6-anthroyloxypalmitic acid as acceptor and peptide tryptophan as donor. Incorporation of melittin into small unilamellar vesicles was found to be reduced below the lipid phase transition temperature, Tt, whereas it incorporates and distributes more randomly above Tt. Cooling the temperature below Tt after incubation at T greater than Tt leads to a deeper incorporation of the peptide into the lipid bilayer due to electrostatic interaction between the lipid phosphate groups and the positively charged amino acids. This stabilizing effect is lost above Tt and melittin is extruded to the polar phase. Quenching experiments support this finding. EPR measurements clearly demonstrate that even in the presence of high amounts of melittin up to 10 mol% with respect to the lipid broadening of the phase transition curves was only observed with fatty acid spin labels, where the doxyl group is localized near the bilayer surface. The order degree of the inner part of the bilayer remains almost unchanged even in the presence of high melittin content.     

Effects of vitrified and nonvitrified sugars on phosphatidylcholine fluid-to-gel phase transitions

DSC was used to study the ability of glass-forming sugars to affect the gel-to-fluid phase transition temperature, T(m), of several phosphatidylcholines during dehydration. In the absence of sugars, T(m) increased as the lipid dried. Sugars diminished this increase, an effect we explain using the osmotic and volumetric properties of sugars. Sugars vitrifying around fluid phase lipids lowered T(m) below the transition temperature of the fully hydrated lipid, T(o). The extent to which T(m) was lowered below T(o) ranged from 12 degrees to 57 degrees, depending on the lipids' acyl chain composition. Sugars vitrifying around gel phase lipids raised T(m) during the first heating scan in the calorimeter, then lowered it below T(o) in subsequent scans of the sample. Ultrasound measurements of the mechanical properties of a typical sugar-glass indicate that it is sufficiently rigid to hinder the lipid gel-to-fluid transition. The effects of vitrification on T(m) are explained using the two-dimensional Clausius-Clapeyron equation to model the mechanical stress in the lipid bilayer imposed by the glassy matrix. Dextran and polyvinylpyrrolidone (PVP) also vitrified but did not depress T(m) during drying. Hydration data suggest that the large molecular volumes of these polymers caused their exclusion from the interbilayer space during drying.     

Fraction of cholesterol undergoing spontaneous exchange between small unilamellar phosphatidylcholine vesicles

The kinetics of the spontaneous exchange of [3H]cholesterol between small unilamellar vesicles of phosphatidylcholine has been reexamined. Although first-order exchange kinetics were observed (k = 0.0117 min-1), in good agreement with previous studies, about 20% of the total cholesterol was found to be nonexchangeable in the 8-h time frame of the experiments. The size of this nonexchangeable pool was found to depend on the type of phospholipid and the temperature. It seems probable that the two pools of cholesterol defined in these experiments reflect the complex phase structure of the cholesterol-phosphatidylcholine vesicles.     

Steady-state fluorescence anisotropy and multifrequency phase fluorometry on oxidized phosphatidylcholine vesicles

Multilamellar liposomes, from mixtures of unoxidized (control) and singlet oxygen oxidized phosphatidylcholine, were studied by steady-state fluorescence anisotropy and multifrequency phase fluorometry using 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probe. Lifetime fluorescence decay of the DPH-labeled liposomes was analyzed either by a model of discrete exponential components and a model that assumes a continuous distribution of lifetime values. Increasing the oxidized phosphatidylcholine content in the liposomes, an increase of the membrane interior polarity and a decrease of membrane fluidity occurs which can be related to the hydroperoxide-lipids and double bonds conjugation, respectively.     

Stabilizing effect of cholesterol on phosphatidylcholine vesicles observed by ultrasonic velocity measurement.

The temperature dependence of the ultrasonic velocity was measured in sonicated vesicles of dipalmitoylphosphatidylcholine by varying the content of cholesterol. When cholesterol is incorporated, an anomalous dip of the ultrasonic velocity gradually smeared out. At the same time, the ultrasonic velocity of the membrane increased remarkably above 30 degrees C due to the increase of the bulk modulus by about 15%. On the other hand, the ultrasonic velocity and the bulk modulus decreased below 30 degrees C. Comparing the cholesterol-incorporated membrane with vesicles of bovine brain sphingomyelin and human erythrocyte membrane, we discuss the role of cholesterol in biological membranes in terms of the stability of the membrane as a barrier.     

Incorporation of amphotericin B into large unilamellar vesicles composed of phosphatidylcholine and phosphatidylglycerol

The spontaneous incorporation of the polyene antibiotic amphotericin B from a micellar solution into phospholipid vesicles was examined as a function of the lipid composition of the vesicles and their physical state. Virtually no insertion of the antibiotic into egg phosphatidylcholine vesicles was observed even when cholesterol was also present in the bilayer. In contrast, rapid incorporation occurred into systems containing an anionic phospholipid such as phosphatidylglycerol or phosphatidylserine with the fastest rates observed for lipids containing the saturated dimyristoyl fatty acyl species. Insertion of amphotericin B into vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (7:3 mole ratio) was rapid either above, below or within the gel-to-liquid-crystalline phase transition temperature (23 degrees C). The ability of amphotericin B to intercalate into lipid vesicles is discussed in relation to their relative bilayer stabilities.     

Effect of cholesterol on the ethanol-induced interdigitated gel phase in phosphatidylcholine: use of fluorophore pyrene-labeled phosphatidylcholine

It is now recognized that many amphiphilic molecules such as ethanol can induce the formation of the fully interdigitated gel phase (L beta I) in phosphatidylcholines (PC's). In the present study, we have developed a simple detection method for the L beta I phase using pyrene-labeled PC (PyrPC), which is a PC analogue with covalently coupled pyrene moiety at the end of one of its acyl chains. The intensity ratio of its fluorescence vibrational bands is a reflection of the polarity of the environment of the fluorophore. We have tested this fluorophore in several established interdigitated lipid systems, including 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC) in the presence of high concentrations of ethanol and 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (DHPC) and 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC) in the absence of any additives. We have found in each of these systems that the ratio of the intensities of band III (387.5 nm) to band I (376.5 nm) is sensitive to the lipid phase change from the noninterdigitated L beta' phase to the interdigitated L beta I phase. By comparison of the III/I ratios for PyrPC in the lipid systems with the III/I ratios for methylpyrene in organic solvents, it was shown that the polarity of the PyrPC environment in the L beta I phase is similar to that of pentanol or ethanol. Using this method, we investigated the effect of cholesterol on the ethanol induction of the interdigitated gel phase in 1,2-DPPC. We found that the ethanol induction of the interdigitated gel phase is prevented by the presence of 20 mol % cholesterol.     

Influence of cholesterol on gramicidin-induced HII phase formation in phosphatidylcholine model membranes

The influence of cholesterol incorporation on gramicidin-induced hexagonal HII phase formation in different phosphatidylcholine model systems was investigated by 31P- and 2H-NMR, small-angle X-ray diffraction and differential scanning calorimetry. In liquid-crystalline distearoylphosphatidylcholine systems cholesterol inhibits gramicidin-induced HII phase formation. In dioleoylphosphatidylcholine the opposite effect is observed. Cholesterol appears to preferentially interact with gramicidin under liquid-crystalline conditions in both systems. Two phenomena that had been reported for gramicidin-treated erythrocyte membranes and derived liposomes (Tournois, H., Leunissen-Bijvelt, J., Haest, C.W.M., De Gier, J. and De Kruijff, B. (1987) Biochemistry, 26, 6613-6621) could also be observed in more simple dioleoylphosphatidylcholine-gramicidin-cholesterol systems. These are (i) an increase in tube diameter in the gramicidin-induced HII phase with increasing temperature, which is ascribed to the presence of cholesterol in this phase, and (ii) the loss of the hexagonal HII phase related 31P-NMR line shape at lower temperatures despite the presence of this phase as demonstrated with X-ray diffraction. This latter phenomenon appears to be due to restrictions in the rate of lateral diffusion of the phospholipids around the HII tubes due to the presence of gramicidin.     

Spontaneous fusion of phosphatidylcholine small unilamellar vesicles in the fluid phase

Using a high-sensitivity differential scanning microcalorimeter capable of performing cooling scans, we have examined the phase behavior of small unilamellar vesicles (SUV) as a function of time of storage above their order-disorder phase transition. Vesicles composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were examined. Cooling scans on fresh (5-7-h postsonication) samples revealed broad, relatively simple heat capacity peaks (peak temperatures: 19.9 degrees C for DMPC, 37.8 degrees C for DPPC) free of high-temperature spikes or shoulders. Subsequent heating scans displayed a sharp peak characteristic of previously described fusion products formed below the phase transition. SUV samples stored for 1 or more days above their phase transition displayed a moderately broad, high-temperature shoulder (23.8 degrees C for DMPC and 40.2 degrees C for DPPC) in the cooling profile. For DMPC, the enthalpy associated with this peak increased in a first-order fashion with time. Hydrolysis products were not detected until 12-20 days of storage. Both the rate and extent of shoulder appearance increased with temperature (k = 0.0017 h-1, fraction of total enthalpy = 0.1 at 36 degrees C; k = 0.0037 h-1, fraction = 0.2 at 42 degrees C). Freeze-fracture electron micrographs confirmed that an intermediate-sized vesicle population (diameters 400-500 A) appeared in SUV samples stored above their phase transition. Also, the trapped volume of DMPC SUV increased from 0.26 microL/mumol after 17 h of storage to 0.54 microL/mumol after storage for 16 days at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulse NMR study of phase transitions in dipalmitoyl phosphatidylcholine multilayer systems

Pretransition and main transition of aqueous dipalmitoyl phosphatidylcholine (DPPC) dispersions were investigated by pulse NMR. The second moment M2 inter of the proton absorption line shows significant changes at 42 degrees C and about 35 degree C. Over the whole investigated temperature range between 25 and 50 degree C a superposition of at least two distinct second moments assigned to different molecular regions was observed.     

Lipid phase transitions in model biomembranes: The effect of ions on phosphatidylcholine bilayers

Differential scanning calorimetry has been used to study the endothermic phase behaviour of some model biomembranes (i.e. phosphatidylcholine-water systems) in the presence of a wide range of alkaline, alkaline earth and heavy metal salts. Studies and comparisons were made of both cation and anion effects. Shifts occur in the temperatures of both the pre-transition and main transition endotherms. The observed shifts are smaller than those which have been reported for charged lipids, and no evidence has been found for the formation of specific complexes. Electron microscopic studies on freeze-fractured dispersions of phosphatidylcholine-water-salt systems show that with some salts the typical rippled surface observed with l-α-dimyristoyl phosphatidylcholine, when in the gel state, is replaced by a smooth surface.     

Inhibition of the binding of cytochrome b5 to phosphatidylcholine vesicles by cholesterol

The binding of cytochrome b₅ to single-walled liposomes of egg phosphatidylcholine was inhibited by the presence of cholesterol in the lipid bilayer under conditions where a limited amount of liposomes was incubated with the cytochrome. Since similar conditions seem to apply for the binding of cytochrome b₅ to erythrocyte ghosts, this observation supports the conclusion of Enomoto and Sato (Enomoto, K. and Sato, R. (1977) Biochim. Biophys. Acta 466, 136–147) that the localization of cholesterol on the outer surface of the ghost membrane prevents the binding of cytochrome b₅ to this surface. The finding reported by Roseman et al. (Roseman, M.A., Holloway, P.W. and Calabro, M.A. (1978) Biochim. Biophys. Acta 507, 552–556) that cholesterol did not prevent the cytochrome binding to phosphatidylcholine liposomes in the presence of a large excess of liposomes could be confirmed in the present study, but this does not contradict the abovementioned conclusion.     

Effects of cholesterol on the structural transitions induced by diacylglycerol in phosphatidylcholine and phosphatidylethanolamine bilayer systems

The transient membrane lipid diacylglycerol (DG) is known to modify and destabilize phospholipid bilayers and can lead to the formation of nonbilayer structures. Since cholesterol forms a major fraction of many plasma membranes, we have investigated how it modifies the structural effects of DG on bilayers of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE). We view these systems as modelling the behaviour of local, DG-containing sites in membranes. Using X-ray diffraction, we have characterized the lamellar (L alpha) and inverse hexagonal (HII) structures that these ternary lipid mixtures form in excess aqueous solution. As the DG level increases, the lipid progresses from a single L alpha structure to a mixture of L alpha and HII, and then to a pure HII structure. This allows determination of the DG levels at which the HII transition begins, which we interpret as those levels that destabilize bilayers. In both PC and PE bilayers, the presence of 30 mol% cholesterol reduces the amounts of DG required to destabilize the bilayer structure. The destabilization can be translated into the number of neighbouring lipid molecules that a DG molecule perturbs, and of bilayer areas that it affects. The data show that the presence of cholesterol greatly enhances the perturbing effects of DG. We examine the possible role of DG in enzyme activation and membrane fusion.     

Effect of the phase transition on the transbilayer movement of dimyristoyl phosphatidylcholine in unilamellar vesicles

Dimyristoyl phosphatidylcholine rapidly exchanges between vesicles at 37°C without vesicle fusion.The rate of the transbilayer movement of dimyristoyl phosphatidylcholine in sonicated vesicles has been measured employing ¹³C NMR using N-¹³CH_3? labeled lipids which are introduced into the outer monolayer of non-labeled vesicles by a phosphatidylcholine exchange protein. The rate of transbilayer movement of dimyristoyl phosphatidylcholine shows a distinct maximum (halftime 4 h) in the temperature range at which the hydrocarbon phase transition occurs.The activation energy of the flip-flop rate above the phase transition is 23.7 ± 2.0 kcal/mol.     

Influence of dodecyltrimethylammonium halides on thermotropic phase behaviour of phosphatidylcholine/cholesterol bilayers

Effects of dodecyltrimethylammonium chloride (DTAC), dodecyltrimethylammonium bromide (DTAB) and dodecyltrimethylammonium iodide (DTAI) on thermotropic phase behaviour of phosphatidylcholine bilayers containing cholesterol as well as on 1H NMR spectra were studied. Two series of experiments were performed. In the first one the surfactants were added to the water phase while in the other directly to the lipid phase (a mixed film from cholesterol, surfactant and phosphatidylcholine was formed). The effects of particular surfactants on the main phase transition temperature, Tm, were more pronounced when added to the lipid phase (2nd method) than to the water phase (1st method); the opposite happened when cholesterol was absent (Rózycka-Roszak and Pruchnik 2000, Z. Naturforsch. 55c, 240-244). Furthermore, in the case of the first method the transitions were asymmetrical while in the second method nearly symmetrical. It is suggested that surfactant poor and surfactant rich domains are formed when surfactants are added to the water phase.