Light scattering and turbidity measurements on lipid vesicles

The dynamic behaviour of model membranes in the form of sonicated liposomes in excess water was studied by means of 90 °C light scattering and turbidity measurements. Computer calculations based on the Rayleigh-Gans theory of light scattering were used to estimate the average size of lipid vesicles dispersed in water, taking into account the various structures of the vesicles. Normal reversible changes in the scattered light intensity and turbidity with temperature could be accounted for mainly by the change in the refractive index of the lipid and irreversible anomalous changes were explained on the basis of fusion of smaller aggregated vesicles.     

Phase properties of the aqueous dispersions of n-octadecylphosphocholine

Properties of the aqueous dispersions of n-octadecylphosphocholine are examined by differential scanning calorimetry, fluorescence depolarization, light scattering, ³¹P-NMR, pig pancreatic phospholipase A₂ binding, and X-ray diffraction. On heating, these dispersions exhibit a sharp lamellar to micelle transition at 20.5°C. The lamellar phase consists of frozen (gel-state) alkyl chains which do not bind phospholipase A₂. The kinetics of the transition are asymmetric: the micelle to lamellar transition is very slow and the lamellar to micelle transition is fast. It is suggested that the lamellar phase is a frozen chain bilayer in which the chains interdigitate.     

Turbidity changes of lipid vesicles near the phase transition temperature as an indication of fusion

Sonicated liposomes of dipalmitoyl phosphatidylcholine show sharp turbidity changes on heating at two distinct temperatures. A decrease in turbidity at the lower temperature (approx. 37°C) is thought to be associated with the phase transition of small vesicles and a decrease at about 44°C with larger vesicles or multilayer. An increase of turbidity between 38 and 43°C is attributed to the fusion of small vesicles. The turbidity changes were studied under various modes of vesicle preparation to confirm the interpretation of the turbidity data. Alternate interpretations are discussed.     

The influence of charge on bilayer membranes. Calorimetric investigations of phosphatidic acid bilayers.

The pH-dependence of the phase transition of dimyristoyl phosphatidic acid and dihexadecyl phosphatidic acid has been investigated using differential scanning calorimetry. Varying the pH induces different degrees of ionization of the polar head group. The changes in transition temperature with pH as observed by calorimetry are in good agreement with those obtained by measuring the changes in light scattering, whereas the transition temperatures reported by the fluorescent probe N-phenylnaphthylamine do not always coincide with those determined from calorimetry [1]. The observed maximum of the transition temperature at pH 3.5 corresponds to a minimum in the transition enthalpy vs. pH diagram. At this pH a particular stable bilayer phase is formed. Full protonation of phosphatidic acids leads to suspensions of mycrocrystals. The transition enthalpy approaches the value of the melting enthalpy of crystalline anhydrous phosphatidic acid. The decrease in the transition enthalpy at high pH values is due to a change in the hydrocarbon chain interactions induced by the doubly charged head groups. The cooperativity of the transition varies with the degree of ionization of the head group, being lower for doubly charged phosphatidic acids.     

Light scattering and turbidity measurements on lipid vesicles.

The dynamic behaviour of model membranes in the form of sonicated liposomes in excess water was studied by means of 90 degrees C light scattering and turbidity measurements. Computer calculations based on the Rayleigh-Gans theory of light scattering were used to estimate the average size of lipid vesicles dispersed in water, taking into account the various structures of the vesicles. Normal reversible changes in the scattered light intensity and turbidity with temperature could be accounted for mainly by the changes in the refractive index of the lipid and irreversible anomalous changes were explained on the basis of fusion of smaller aggregated vesicles.     

Thermotropic and structural evaluation of the interaction of natural sphingomyelins with cholesterol

The structural transitions in aqueous dispersions of egg-sphingomyelin and bovine brain-sphingomyelin and sphingomyelin co-dispersed with different proportions of cholesterol were compared during temperature scans between 20° and 50 °C using small-angle and wide-angle X-ray scattering techniques. The Bragg reflections observed in the small-angle scattering region from pure phospholipids and codispersions of sphingomyelin:cholesterol in molar ratios 80:20 and 50:50 could all be deconvolved using peak fitting methods into two coexisting lamellar structures. Electron density profiles through the unit cell normal to the bilayer plane were calculated to derive bilayer and water layer thicknesses of coexisting structures at 20° and 50 °C. Codispersions of sphingomyelin:cholesterol in a molar ratio 60:40 consisted of an apparently homogeneous bilayer structure designated as liquid-ordered phase. Curve fitting analysis of the wide-angle scattering bands were applied to correlate changes in packing arrangements of hydrocarbon in the hydrophobic domain of the bilayer with changes in enthalpy recorded by differential scanning calorimetry. At 20 °C the wide-angle scattering bands of both pure sphingomyelins and codispersions of sphingomyelin and cholesterol could be deconvolved into two symmetric components. A sharp component located at a d-spacing of 0.42 nm was assigned to a gel phase in which the hydrocarbon chains are oriented perpendicular to the bilayer plane. A broader symmetric band centered at d-spacings in the region of 0.44 nm was assigned as disordered hydrocarbon in dispersions of pure sphingomyelin and as liquid-ordered phase in codispersions of sphingomyelin and cholesterol. It is concluded from the peak fitting analysis that cholesterol is excluded from gel phases of egg and brain sphingomyelins at 20 °C. The gel phases coexist with liquid-ordered phase comprised of egg-sphingomyelin and 27 mol% cholesterol and brain-sphingomyelin and 33 mol% cholesterol, respectively. Correlation of the disappearance of gel phase during heating scans and the enthalpy change recorded by calorimetry in codispersions of sphingomyelin and cholesterol leads to the conclusion that a major contribution to the broadened phase transition endotherm originates from dilution of the cholesterol-rich liquid-ordered phase by mobilization of sphingomyelin from the melting gel phase.     

The bilayer melting transition in lung surfactant bilayers: the role of cholesterol

Aqueous dispersions of a porcine lung surfactant (PLS) extract with and without cholesterol supplementation were analyzed by X-ray scattering. Lamellar liquid-crystalline and gel-type bilayer phases are formed, as in pure phosphatidylcholine (PC)-cholesterol systems. This PLS extract, developed for clinical applications, has a cholesterol content of less than 1% (w/w). Above the limit of swelling, the bilayer structure shows a melting (main) transition during heating at about 34 degrees C. When 13 mol% cholesterol was added to PLS, so that the cholesterol content of natural lung surfactant was reached, the X-ray scattering pattern showed pronounced changes. The main transition temperature was reduced to the range 20-25 degrees C, whereas according to earlier studies of disaturated PC-cholesterol bilayers in water the main transition remains almost constant when the amount of solubilized cholesterol is increased. Furthermore, the changes in scattering pattern at passing this transition in PLS-cholesterol samples were much smaller than at the same transition in PLS samples. These effects of cholesterol solubilization can be related to phase segregation within the bilayers, known from pure PC-cholesterol systems. One phase, solubilizing about 8 mol% cholesterol, exhibits a melting transition, whereas the other bilayer phase, with a liquid-crystalline disordered conformation, has a cholesterol content in the range 20-30 mol% and this phase shows no thermal transition. The relative amount of bilayer lipids that is transformed at the main transition in the PLS-cholesterol sample is therefore only half compared to that in PLS samples. The reduction in transition temperature in the segregated bilayer of lung surfactant lipids is probably an effect of enrichment of disaturated PC species in the phase, which is poor in cholesterol. This work indicates that cholesterol in lung surfactant regulates the crystallization behavior.     

Change in volume magnetic susceptibility at the phase transition of dipalmitoylphosphatidylcholine.

We have observed a change at 41 degrees C in the relative volume magnetic susceptibility of an aqueous dispersion containing 13 wt% multilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles. The magnitude of the change is consistent with the known density change of the phospholipid bilayer and the assumption that the mass susceptibility of the system is constant through the transition. The superconducting susceptometer used in this study of the sharp transition of DPPC will be able to detect 1% changes in bilayer density for 10 wt% dispersions even when the transition temperature and transition width of phospholipid vesicle under various experimental conditions.     

Author index

Using dynamic light scattering we have been able to determine precisely the hydrodynamic radius of l-α-dimyristoylphosphatidylcholine (DMPC) vesicles as a function of temperature. We have detected a sharp, thermally reversible change in the vesicle radius at a phase transition temperature 24°C, corresponding to an approximate 11% increase in surface are. In the range 10–20°C, the change in radius is less than 1%.     

Effects of polypeptide-phospholipid interactions on bilayer reorganizations Raman spectroscopic study of the binding of polymyxin B to dimyristoylphosphatidic acid and dimyristoylphosphatidylcholine dispersions

The interactions of the antibiotic polymixin B, a polycationic cyclic polypeptide containing a branched acyl side chain, with dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidic acid (DMPA) bilayers were investigated by Raman spectroscopy for a wide range of lipid/polypeptide mole fractions. Temperature profiles, constructed from peak height intensity ratios derived from the lipid methylene C-H stretching and acyl chain C-C stretching mode regions, reflected changes originating from lateral chain packing effects and intrachain trans / gauche rotamer formation, respectively. For DMPC/polymyxin B bilayers the temperature dependent curves indicate a broadening of the gel-liquid crystalline phase transition accompanied by an approx. 3 C deg. increase in the phase transition temperature from 22.8°C for the pure bilayer to 26°C for the polypeptide complex. For a 10:1 lipid/polypeptide mole ratio the temperature profile derived from the C-C mode spectral parameters displays a second order/disorder transition, at approx. 35.5°C, associated with the melting behavior of approximately three bilayer lipids immobilized by the antibiotic's charged cyclic headgroup and hydrophobic side chain. For the 10:1 mole ratio DMPA/polypeptide liposomes, the temperature profiles indicate three order/disorder transitions at 46, 36 and 24°C. Pure DMPA bilayers display a sharp lamellar-micellar phase transition at 51°C.     

The influence of charge on phosphatidic acid bilayer membranes.

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, 1,2-dihexadecyl-sn-glycerol-3-phosphoric acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of the phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; 1,2-dimyristoyl-sn-glycerol-3-phosphoric acid, 1,3-dimyristoylglycerol-2-phosphoric acid and 1,2-dipalmitoyl-sn-glycerol-3-phosphoric acid. The phase transition temperatures (Tt) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90 degrees light scattering. The Tt values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states. The Tt vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 (pK1). The regions between the first and the second pK of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H+ concentration results in an abrupt change of the transition temperature. The slope of the Tt vs. pH diagram beyond pK2 becomes very steep. This is the result of reduced hydrocarbon interaction energy, which was demonstrated by differential scanning calorimetry (Blume, A. and Eibl, H., unpublished data).     

A low-temperature structural phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the gel phase

A new thermotropic phase transition, at ?30°C and atmospheric pressure, was found to occur in the gel phase of aqueous DPPC dispersions. The Raman spectral changes at this phase transition are similar to those observed in the gel phase of DMPC dispersions at ?60°C. The thermotropic phase transition at ?30°C is equivalent to the barotropic GII to GIII phase transition observed in DPPC at 1.7 kbar and 30°C. It is shown that the rate of the large angle interchain reorientational fluctuations decreases gradually with decreasing temperature, and that the orientationally disordered acyl chain structure of the GII phase is extended into the GIII phase of DPPC. The interchain interaction, arising from the damping of the reorientational fluctuations, increases with decreasing temperature in the GII gel phase as well as in the GIII gel phase.     

Laser raman studies of lipid disordering by the B-protein of fd phage

Complexes of the B-protein of fd phage with the model lipid dipalmitoyl phosphatidylcholine (DPPC) were made by sonication of the fd phage in the presence of dipalmitoyl phosphatidylcholine. Both laser Raman spectra and circular dichroism show the protein in the membrane to be almost entirely in the β-sheet conformation. This β-sheet conformation is found to be independent of the temperature between 10° C and 50° C. On the other hand, the protein has a very dramatic effect on the organization of the lipid bilayer. An aqueous dispersion of 1 : 1 lipid/protein mixture gives a broad conformational transition of DPPC which occurs between 10° C and 30° C. This contrasts markedly with simple aqueous DPPC dispersions which show a sharp transition at 41°C. This appears to be the first reported example of the lowering of the conformational transition of a membrane bilayer by an intrinsic membrane protein.     

Cooperativity of the phase transition in single- and multibilayer lipid vesicles

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersions of dimyristoyl phosphatidylcholine, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, $\text{2,2,6,6-}\text{tetramethylpiperdine}\text{-N-}\text{oxyl}$, phosphatidylcholine and stearic acid, and also 90° light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526–535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/?σ, is a measure of the mean number of molecules, per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.     

Structural changes in lecithin-water systems thermal-turbidimetric studies

Turbidity measurements were made of dilute aqueous dispersions of 1,2-dipalmitoyl-l-lecithin as the temperature was varied. In the range from 24 to 33° a decrease in turbidity is associated with the penetration of water between the layers of lipid in the crystalline structure. At the transition to the liquid crystalline form (39–41°) a sharp decrease in turbidity occurs. In solutions of 0.06 M LiCl, 1 mM CaCl₂, MgCl₂ or 0.05 M PO₄³ an increase in turbidity resulted at the transition temperature, whereas in 0.1 M NaCl, or quarternary ammonium salts the turbidity decreased. The presence of small amounts of dicetylphosphoric acid mixed with the lecithin decreased the turbidity, and at elevated pH levels there was no change at the transition temperature.     

Studies on the Critical Micellar Concentration and Phase Transitions of Stearoylcarnitine

The critical micellar concentration (CMC) of stearoylcarnitine was determined at different pH values at room temperature by fluorescence spectroscopy, monitoring the spectral changes of 8-anilinonaphthalene-1-sulfonate (ANS). The CMC was found to vary with pH, increasing from about 10 μM at pH 3.0 to ca. 25 μM at pH 7.0, but decreasing slightly with further increase in pH to approximately 19 μM at pH 10.0. Differential scanning calorimetry (DSC) shows that stearoylcarnitine dispersed in water at low concentration undergoes a broad thermotropic phase transition at 44.5°C, with a transition enthalpy of 15.0 kcal/mol. The transition temperature (T _t) shifts to ca. 50.5°C in the presence of 1 mM EDTA or when the concentration is increased significantly. The turbidity of aqueous dispersions of stearoylcarnitine was found to be considerably high at low temperatures, which decreases quite abruptly over a short temperature range, indicating that a transition occurs from a phase of large aggregates to one of much smaller aggregates, most likely micelles. The phase transition temperature was determined as 29.1°C at pH 3.0, which increased with increasing pH up to a value of 55.3°C at pH 8.6 and remains nearly constant thereafter up to pH 11.2. The pH dependence of CMC and T _t suggest that the pK _a of the carboxyl group of long chain acylcarnitines shifts to higher temperatures upon aggregation (micelles or bilayer membranes).     

An X-ray diffraction study of the effect of alpha-tocopherol on the structure and phase behaviour of bilayers of dimyristoylphosphatidylethanolamine.

The effect of alpha-tocopherol on the thermotropic phase transition behaviour of aqueous dispersions of dimyristoylphosphatidylethanolamine was examined using synchrotron X-ray diffraction methods. The temperature of gel to liquid-crystalline (Lbeta-->Lalpha) phase transition decreases from 49.5 to 44.5 degrees C and temperature range where gel and liquid-crystalline phases coexist increases from 4 to 8 degrees C with increasing concentration of alpha-tocopherol up to 20 mol%. Codispersion of dimyristoylphosphatidylethanolamine containing 2.5 mol% alpha-tocopherol gives similar lamellar diffraction patterns as those of the pure phospholipid both in heating and cooling scans. With 5 mol% alpha-tocopherol in the phospholipid, however, an inverted hexagonal phase is induced which coexists with the lamellar gel phase at temperatures just before transition to liquid-crystalline lamellar phase. The presence of 10 mol% alpha-tocopherol shows a more pronounced inverted hexagonal phase in the lamellar gel phase but, in addition, another non-lamellar phase appears with the lamellar liquid-crystalline phase at higher temperature. This non-lamellar phase coexists with the lamellar liquid-crystalline phase of the pure phospholipid and can be indexed by six diffraction orders to a cubic phase of Pn3m or Pn3 space groups and with a lattice constant of 12.52+/-0.01 nm at 84 degrees C. In mixed aqueous dispersions containing 20 mol% alpha-tocopherol, only inverted hexagonal phase and lamellar phase were observed. The only change seen in the wide-angle scattering region was a transition from sharp symmetrical diffraction peak at 0.43 nm, typical of gel phases, to broad peaks centred at 0.47 nm signifying disordered hydrocarbon chains in all the mixtures examined. Electron density calculations through the lamellar repeat of the gel phase using six orders of reflection indicated no difference in bilayer thickness due to the presence of 10 mol% alpha-tocopherol. The results were interpreted to indicate that alpha-tocopherol is not randomly distributed throughout the phospholipid molecules oriented in bilayer configuration, but it exists either as domains coexisting with gel phase bilayers of pure phospholipid at temperatures lower than Tm or, at higher temperatures, as inverted hexagonal phase consisting of a defined stoichiometry of phospholipid and alpha-tocopherol molecules.     

Mesoscopic structure in the chain-melting regime of anionic phospholipid vesicles: DMPG

In a range of low ionic strength, aqueous dispersions of the anionic phospholipid DMPG (dimyristoylphosphatidylglycerol) have a transparent intermediate phase (IP, between T(m)(on) congruent with 20 degrees C and T(m)(off) congruent with 30 degrees C) between the turbid gel and fluid membrane phases, evidenced in turbidity data. Small angle x-ray scattering results on DMPG dispersions show that, besides the bilayer peak present in all phases, a peak corresponding to a mesoscopic structure at approximately 400 A is detected only in IP. The dependence of this peak position on DMPG concentration suggests a correlation in the bilayer plane, consistent with the stability of vesicles in IP. Moreover, observation of giant DMPG vesicles with phase contrast light microscopy show that vesicles "disappear" upon cooling below T(m)(off) and "reappear" after reheating. This further proves that although vesicles cannot be visualized in IP, their overall structure is maintained. We propose that the IP in the melting regime corresponds to unilamellar vesicles with perforations, a model which is consistent with all described experimental observations. Furthermore, the opening of pores across the membrane tuned by ionic strength, temperature, and lipid composition is likely to have biological relevance and could be used in applications for controlled release from nanocompartments.     

Thermal transitions of DMPG bilayers in aqueous solution: SAXS structural studies

Dimyristoylphosphatidylglycerol (DMPG) has been extensively studied as a model for biological membranes, since phosphatidylglycerol is the most abundant anionic phospholipid in prokaryotic cells. At low ionic strengths, this lipid presents a peculiar thermal behavior, with two sharp changes in the light scattering profile, at temperatures named here T(on)(m) and T(off)(m). Structural changes involved in the DMPG thermal transitions are here investigated by small angle X-ray scattering (SAXS), and compared to the results yielded by differential scanning calorimetry (DSC) and electron spin resonance (ESR). The SAXS results show a broad peak, indicating that DMPG is organized in single bilayers, for the range of temperature studied (10-45 degrees C). SAXS intensity shows an unusual effect, starting to decrease at T(on)(m), and presenting a sharp increase at T(off)(m). The bilayer electron density profiles, obtained from modeling the SAXS curves, show a gradual decrease in electron density contrast (attributed to separation between charged head groups) and in bilayer thickness between T(on)(m) and T(off)(m). Results yielded by SAXS, DSC and ESR indicate that a chain melting process starts at T(on)(m), but a complete fluid phase exists only for temperatures above T(off)(m), with structural changes occurring at the bilayer level in the intermediate region.     

Lecithin bilayers. Density measurement and molecular interactions.

Density measurement are reported for bilayer dispersions of a series of saturated lecithins. For chain lengths with, respectively, 14, 15, 16, 17, and 18 carbons per chain, the values for the volume changes at the main transition are 0.027, 0.031, 0.037, 0.040 and 0.045 ml/g. The main transition temperature extrapolates with increasing chain length to the melting temperature of polyethylene. Volume changes at the lower transition are an order of magnitude smaller than the main transition. Single phase thermal expansion coefficients are also reported. The combination of X-ray data and density data indicated that the volume changes are predominantly due to the hydrocarbon chains, thus enabling the volume vCH2 of the methylene groups to be computed as a function of temperature. From this and knowledge of intermolecular interactions in hydrocarbon chains, the change in the interchain van der Waals energy, delta UvdW, at the main transition is computed for the lecithins and also for the alkanes and polyethylene at the melting transition. Using the experimental enthalpies of transition and delta UvdW, the energy equation is consistently balanced for all three systems. This yields estimates of the change in the number of gauche rotamers in the lecithins at the main transition. The consistency of these calculations supports the conclusion that the most important molecular energies for the main transition in lecithin bilayers are the hydrocarbon chain interactions and the rotational isomeric energies, and the conclusion that the main phase transition is analogous to the melting transition in the alkanes from the hexagonal phase to the liquid phase, but with some modifications.