Phase Behavior Of Phospholipids Research Articles

H-bond network, interfacial tension and chain melting temperature govern phospholipid self-assembly in ionic liquids

HypothesisThe self-assembly structures and phase behaviour of phospholipids in protic ionic liquids (ILs) depend on intermolecular forces that can be controlled through changes in the size, polarity, and H-bond capacity of the solvent. ExperimentsThe structure and temperature stability of the self-assembled phases formed by four phospholipids in three ILs was determined by a combination of small- and wide-angle X-ray scattering (SAXS and WAXS) and small-angle neutron scattering (SANS). The phospholipids have identical phosphocholine head groups but different alkyl tail lengths and saturations (DOPC, POPC, DPPC and DSPC), while the ILs’ amphiphilicity, H–bond network density and polarity are varied between propylammonium nitrate (PAN) to ethylammonium nitrate (EAN) to ethanolammonium nitrate (EtAN). FindingsThe observed structures and phase behaviour of the lipids becomes more surfactant–like with decreasing average solvent polarity, H-bond network density and surface tension. In PAN, all the investigated phospholipids behave like surfactants in water. In EAN they exhibit anomalous phase sequences and unexpected transitions as a function of temperature, while EtAN supports structures that share characteristics with water and EAN. Structures formed are also sensitive to proximity to the lipid chain melting temperature.

Phase transitions of the pulmonary surfactant film at the perfluorocarbon-water interface

Pulmonary surfactant is a lipid-protein complex that forms a thin film at the air-water surface of the lungs. This surfactant film defines the elastic recoil and respiratory mechanics of the lungs. One generally accepted rationale of using oxygenated perfluorocarbon (PFC) as a respiratory medium in liquid ventilation is to take advantage of its low surface tensions (14–18 mN/m), which was believed to make PFC an ideal replacement of the exogenous surfactant. Compared with the extensive studies of the phospholipid phase behavior of the pulmonary surfactant film at the air-water surface, its phase behavior at the PFC-water interface is essentially unknown. Here, we reported the first detailed biophysical study of phospholipid phase transitions in two animal-derived natural pulmonary surfactant films, Infasurf and Survanta, at the PFC-water interface using constrained drop surfactometry. Constrained drop surfactometry allows in situ Langmuir-Blodgett transfer from the PFC-water interface, thus permitting direct visualization of lipid polymorphism in pulmonary surfactant films using atomic force microscopy. Our data suggested that regardless of its low surface tension, the PFC cannot be used as a replacement of pulmonary surfactant in liquid ventilation where the air-water surface of the lungs is replaced with the PFC-water interface that features an intrinsically high interfacial tension. The pulmonary surfactant film at the PFC-water interface undergoes continuous phase transitions at surface pressures less than the equilibrium spreading pressure of 50 mN/m and a monolayer-to-multilayer transition above this critical pressure. These results provided not only novel biophysical insight into the phase behavior of natural pulmonary surfactant at the oil-water interface but also translational implications into the further development of liquid ventilation and liquid breathing techniques.

Effect of Phase Behavior of Phospholipids on Lipid Membrane Damage Induced by Graphene Oxide

Effect of Phase Behavior of Phospholipids on Lipid Membrane Damage Induced by Graphene Oxide

Temperature-Responsive Hydrophobic Silica Nanoparticle Ultrasound Contrast Agents Directed by Phospholipid Phase Behavior.

In this paper, we report ultrasonically active nanoscale contrast agents that behave as thermometric sensors through phase change in their stabilizing phospholipid monolayer. Phospholipid-stabilized, hydrophobic mesoporous silica nanoparticles (P@hMSNs) are known to interact with high-intensity focused ultrasound (HIFU) to promote cavitation at their surfaces, which can be used for both imaging and therapy. We show that the lateral lipid phase behavior of the phosphocholine lipid dictates the acoustic contrast of the P@hMSNs. When the lipids are in the gel phase below their melting temperature, the P@hMSNs generate detectable microbubbles when exposed to HIFU. However, if the lipids exhibit a liquid expanded phase, the P@hMSNs cease to generate bubbles in response to HIFU insonation. We verify that the heating and subsequent transition of lipid coating the hMSN are associated with the loss of acoustic response by doping laurdan dye into the lipid monolayer and imaging lipid phase through red shifts in emission spectra. Similarly, cessation of cavitation was also induced by adding a fluidizing surfactant such as Triton X, which could be reversed upon washing away the excess surfactant. Finally, by controlling for the partial fluidization caused by the adsorption of protein, P@hMSNs may be used as thermometric sensors of the bulk fluid temperature. These findings not only impact the utilization of nanoscale agents as stimulus-responsive ultrasound contrast agents but also have broader implications for how cavitation may be initiated at surfaces coated by a surfactant.

Interaction of a Peptide Derived from the N-Heptad Repeat Region of gp41 Env Ectodomain with Model Membranes. Modulation of Phospholipid Phase Behavior

The HIV-1 gp41 envelope protein mediates the entry of the virus into the target cell by promoting membrane fusion. With a view toward possible new insights into the protein membrane alteration leading to the viral fusion mechanism, we have studied by infrared and fluorescence spectroscopies a fragment of 21 amino acids corresponding to the N-heptad repeat region of the gp41 ectodomain. Information on the structure of the peptide both in solution and in the presence of model membranes, its incorporation and location in the phospholipid bilayer, and the modulation of the phase behavior of the membrane has been gathered. Here we demonstrate that the peptide binds to and interacts with phospholipid model membranes, changing its conformation and inducing leakage of vesicle contents. These characteristics suggest that different specific regions of gp41 are capable of modifying the biophysical properties of phospholipid membranes and, therefore, might be essential for the assistance and enhancement of the viral and cell fusion process.

Thermotropic and piezotropic phase behaviour of phospholipids in propanediols and water

The investigation of the solvation process of phospholipids includes the investigation of the thermotropic and piezotropic phase behaviour in presence of non-aqueous polar solvents. 1,2-propanediol and 1,3-propanediol were chosen to investigate the influence of the solvents on the parameters of the main phase transition. The non-polar parts of the propanediols slightly reduce the main phase transition temperature in contrast to water. On the other hand, the pressure induced increase of the transition temperature hardly differs between propanediols and water. Furthermore, a coexistence of a lamellar gel phase and an isotropic liquid phase was found for a number of phospholipid/1,2-propanediol dispersions.

The effects of ring-size analogs of the antimicrobial peptide gramicidin S on phospholipid bilayer model membranes and on the growth of Acholeplasma laidlawii B.

We have examined the effects of three ring-size analogs of the cyclic beta-sheet antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior and permeability of phospholipid model membranes and on the growth of the cell wall-less Gram-positive bacteria Acholeplasma laidlawii B. These three analogs have ring sizes of 10 (GS10), 12 (GS12) or 14 (GS14) amino acids, respectively. Our high-sensitivity differential scanning calorimetric studies indicate that all three of these GS analogs perturb the gel/liquid-crystalline phase transition of zwitterionic phosphatidylcholine (PtdCho) vesicles to a greater extent than of zwitterionic phosphatidylethanolamine (PtdEtn) or of anionic phosphatidylglycerol (PtdGro) vesicles, in contrast to GS itself, which interacts more strongly with PtdGro than with PtdCho and PtdEtn bilayers. However, the relative potency of the perturbation of phospholipid phase behavior varies markedly between the three peptides, generally decreasing in the order GS14 > GS10 > GS12. Similarly, these three GS ring-size analogs also differ considerably in their ability to cause fluorescence dye leakage from phospholipid vesicles, with the potency of permeabilization also generally decreasing in the order GS14 > GS10 > GS12. Finally, these GS ring-size analogs also differentially inhibit the growth of A. laidlawii with growth inhibition also decreasing in the order GS14 > GS10 > GS12. These results indicate that the relative potencies of GS and its ring-size analogs in perturbing the organization and increasing the permeability of phospholipid bilayer model membranes, and of inhibiting the growth of A. laidlawii B cells, are at least qualitatively correlated, and provide further support for the hypothesis that the primary target of these antimicrobial peptides is the lipid bilayer of the bacterial membrane. The very high antimicrobial activity of GS14 against the cell wall-less bacteria A. laidlawii as compared to various conventional bacteria confirms our earlier suggestion that the avid binding of this peptide to the bacterial cell wall is primarily responsible for its reduced antimicrobial activity against such organisms. The relative magnitude of the effects of GS itself, and of the three ring-size GS analogs, on phospholipid bilayer organization and cell growth correlate relatively well with the effective hydrophobicities and amphiphilicities of these peptides but less well with their relative charge density, intrinsic hydrophobicities or conformational flexibilities. Nevertheless, all of these parameters, as well as others, may influence the antimicrobial potency and hemolytic activity of GS analogs.

The influence of structure and redox state of prenylquinones on thermotropic phase behaviour of phospholipids in model membranes

Our study was aimed to investigate the significance of the isoprenoid side chain size as well as redox state of the quinone ring for interaction of two main classes of prenylquinones: plastoquinones (PQ) and ubiquinones (UQ) with lipid bilayers. By use of differential scanning calorimetry (DSC) we have followed the thermotropic behaviour of multilamellar vesicles prepared from dipalmitoylphosphatidylcholine (DPPC) upon incorporation of increasing amount (1.3–12 mol%) of quinone (quinol) molecules. Our studies reveal that as the side chain is shorter (from 9 to 2 isoprenoid units) the height of the calorimetric profiles is reduced and the temperature of the main transition of DPPC ( T m) decreases ( T m=39.4 °C for a sample with 12 mol% of PQ-2), and then increases up to 39.8 °C for PQ-1. For the samples containing quinols the effect is more pronounced even at lower concentration. The greater influence of the added prenylquinones on the pretransition demonstrates a stronger distortion of the DPPC packing in the gel state . It seems that this is the isoprenoid side chain length rather than the redox state of prenylquinones that determines their effectiveness in perturbation of thermotropic properties of lipid bilayer.

Structural Analysis of the Protein/Lipid Complexes Associated with Pore Formation by the Bacterial Toxin Pneumolysin

Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.

The influence of alkali fatty acids on the properties and the stability of parenteral O/W emulsions modified with Solutol HS 15 ®

Arachis oil based parenteral O/W emulsions were prepared using soya bean phosphatidylcholine (SPC) and different combinations of co-emulsifiers containing polyethylene glycol fatty acid esters (Solutol HS 15 ®) and alkali fatty acids (sodium laurate, sodium stearate). The parameters measured were droplet size (both by photon correlation spectroscopy and laser diffractometry), pH and zeta potential. All emulsions were subjected to autoclaving. The addition of polyethylene glycol 12-hydroxy stearate (Solutol HS 15 ®) led to a significant decrease of mean oil droplet size. For long-term stability the amount added turned out to be the most important factor. With increased amounts of Solutol HS 15 ® the packing density of the emulsifier layer and the zeta potential decreased leading to instability. The optimum load of Solutol HS 15 ® was found to be 15 μmol/ml. Alkali fatty acids markedly improved the physical stability of the emulsions. Improved stability properties conferred to emulsions by alkali fatty acids could be attributed to the zeta potential increase even in the presence of Solutol HS 15 ®. Consequently a mixed emulsifier film was established in which the ionized fatty acids determined the interface charge. In addition to this a strengthening of the molecular interactions occurring between phospholipid and Solutol HS 15 ® emulsifier in the presence of ionized fatty acids at the O/W interface can be assumed (L. Rydhag, The importance of the phase behaviour of phospholipids for emulsion stability, Fette Seifen Anstrichm. 81 (1979) 168–173). Different co-emulsifier mixtures were shown to have a pronounced impact on the plasma protein adsorption onto emulsion droplets.

Chain fluidity and phase behaviour of phospholipids as revealed by FTIR and sum-frequency spectroscopy

The functioning of biological membranes seems to require a certain degree of fluidity of their components. The fluidity of the lipids forming the matrix of such membranes is related to the order/disorder state of their hydrocarbon chains. In this study, vibrational spectroscopy is applied to probe the chain conformation (as determining the order) of a number of phospholipids with varying intrinsic fluidities as a function of water activity (hydration). Using conventional Fourier-transform infrared (FTIR) spectroscopy and sum-frequency spectroscopy (SFS) enables one to characterize and, thus, to compare physical properties of the molecules in the bulk and in the superficial layer of a specimen, respectively. The results demonstrate the ability of FTIR spectroscopy not only to classify the lipids with respect to chain ordering, but also to detect lyotropic (hydration-driven) phase transitions. It could be shown that the main transition of mixed-chain oleoyl/palmitoyl phosphatidylcholines (POPC, OPPC) occurs at room temperature and a defined water activity of the films investigated, as also confirmed by small-angle X-ray scattering (SAXS). Equivalent effects were found for POPC in appropriately designed SFS experiments thus evidencing lipid phase transitions by this method for the first time. This opens up a new avenue to elucidate basic aspects of lipid phase behaviour using single bilayer membranes as models of the in vivo state.

The effect of storage temperature on interactions between dehydrated sugars and phosphatidylcholine

We studied the effects of storage temperature on the stability of dehydrated POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine) mixed with sucrose, trehalose, or a sucrose/raffinose mixture. We used DSC to measure the gel-to-fluid phase transition temperature (T m) of POPC after incubation either below or near the glass transition temperature (T g) of the sugars in the mixture. Glass formation by the sugars around fluid-phase POPC led to the lowering ofT m below that of the fully hydrated lipid. Phospholipid phase behavior did not change during storage belowT g. In some samples stored aboveT g, trehalose crystallized completely; in these samples, theT g of POPC increased to that of the partially dehydrated phospholipid. Melting the crystalline sugar re-established its ability to lower POPC'sT m. We conclude that prevention of complete sugar crystallization was important for stability in the dry state, and that storage belowT g conferred long-term stability to the dehydrated sugar-lipid mixtures.

Molecular order and hydration property of amine group in phosphatidylethanolamine and its N-methyl derivatives at subzero temperatures

The molecular order and hydration properties of the amine group in phosphatidylethanolamine and its N-methyl derivatives were studied by 2H-NMR at subzero temperatures. Three coexisting signals with 2H-NMR quadrupolar splittings of 146, 106, and 28.8 KHz were detected from the fully hydrated phosphatidylethanolamine/D2O at the lowest studied temperature of -120 degrees C by using short recycle time in the applied NMR pulse sequence. These signals have been assigned to originate from frozen D2O in the interbilayer space and the deuterated amine group, i.e., -ND, with and without threefold symmetric motions. Comparative 2H-NMR studies of phosphatidylethanolamine/D2O with different degrees of methylation over a temperature range between -40 and -120 degrees C lead to the following conclusions. First, the bond angle of -D attached to the nitrogen atom of the amine group may be determined by the 2H-NMR quadrupolar splittings, i.e., 106 and 28.8 KHz, of the two coexisting signals of the deuterated amine group and found to be 112.9 for the gel-state phosphatidylethanolamine. Second, assuming the applicability of the empirical equation for the hydrogen bond distance of N+D--O with deuteron quadrupole coupling constants and using the intermolecular hydrogen bond distance of the amine group determined in single crystals of phosphatidylethanolamine bilayers, the largest measured quadrupolar splitting (delta nu Q) of N-D in this study, i.e., 106 KHz, is close to the static value. This interpretation is also consistent with the fact that the delta nu Q value determined remains constant in the temperature range between -70 and -120 degrees C. Third, the molecular order parameter of the amine group, as calculated from the ratio of the libration-averaged and static delta nu Q value for the lipid with different degrees of methylation, suggests that the perturbation of the headgroup interaction is most significant for the final methylation step. Finally, measurement of the spectral intensity of isotropic unfrozen D2O signals in D2O/phospholipid dispersions at temperatures below the homogeneous nucleation temperature of ice formation for D2O, i.e., below -34 degrees C, suggests that the first methylation step perturbs the neighboring water most significantly. Assuming that the molecular order of the amine group and the amount of unfrozen water detected under the present experimental condition can be taken as a measure of the hydrogen-bonding ability and the extent of perturbation caused by the methyl group, respectively, the gradual methylation of the amine group perturbs the interactions of the N-methylated headgroups in a nonlinear fashion. The results provide a molecular explanation for the phase behavior of phospholipids with different degrees of methylation.

The dissimilar interactions of the calcium antagonist flunarizine with different phospholipid classes and molecular species: a differential scanning calorimetry study

The influence of the class IV calcium antagonist flunarizine on the phase behaviour of different species of the major phospholipid classes of mammalian plasma membranes has been examined using differential scanning calorimetry. We show that it has the ability to substantially influence the phase behaviour of phospholipids. Flunarizine significantly influences the gel to liquid-crystalline transition temperature of phosphatidylserines whilst having little effect of those of the phosphatidylethanolamines tested. The liquid-crystalline to inverted hexagonal phase transition of phosphatidylethanolamines is however, strongly induced by the presence of flunarizine. Examination of the effect of flunarizine on the phase behaviour of different phosphatidylcholine species revealed an acyl-chain dependent influence. Dissimilar results with phosphatidylcholines, phosphatidylethanolamines and phosphatidylserines reveal different locations and ionization states for the drug in the different phospholipid bilayers. These results not only indicate an essential role for the ionization state of the drug in determining drug-phospholipid interactions but also the role of the phospholipid in determining the ionization state of the drug and have important implications for drug-membrane interactions demonstrating that drug interaction with one phospholipid may bear no relation whatsoever to its interaction with another.

The effects of flunarizine on the phase behaviour of phospholipids

The effects of flunarizine on the phase behaviour of phospholipids

DSC-studies of phospholipids containing iso-branched and ω-cyclohexane fatty acids

Differential Scanning Calorimetry (DSC) and Differential Scanning Densitometry (DSD) was employed to study the phase behavior of phospholipids with iso-branched and ω-cyclohexyl fatty acids. Phosphatidylcholines (PC) and phosphatidylethanolamines (PE) with iso-branched fatty acids show complicated phase behavior with several gel-gel transitions with odd-even effects. These odd-even effects are even more pronounced for phosphatidylcholines with ω-cyclohexyl fatty acids (cyPCs). Odd-numbered cyPCs show a large main transition with strong hysteresis while the even-numbered cyPCs have several transitions but show no hysteresis. These differences in thermotropic behavior are also reflected in their molecular volumes. The odd-numbered cyPCs display much tighter packing in the gel phase than the even-numbered analogues. Binary mixtures of cyPCs with either straight chain PCs or iso-branched PCs show eutectic behavior. This is also observed when cyPCs of different chain length are mixed.

Influence of sterol structure on phospholipid phase behavior as detected by parinaric acid fluorescence spectroscopy.

Phospholipid-sterol interactions were investigated using parinaric acid fluorescence spectroscopy. Cholesterol and cholesterol analogues which were modified in the sterol nucleus or side chain were added at 50 mol % to multilamellar vesicles of model phospholipids selected to be representative of major components in an LM cell plasma membrane. These included sphingomyelins and saturated and monounsaturated phosphatidylcholines and phosphatidylethanolamines. Based on the changes in cis-parinaric acid steady-state fluorescence polarization observed with addition of sterol, 50 mol % cholesterol abolished the phase transition of all the model phospholipids. Dihydrocholesterol and trans-22-dehydrocholesterol behaved like cholesterol in the two systems studied. 24-Methylcholesterols interacted well with all phospholipids except phosphatidylethanolamine which contained an unsaturated fatty acid. 24-Alkyl,trans-22-dehydrocholesterols abolished the phase transition in only two systems: sphingomyelins and phosphatidylcholines possessing relatively short saturated acyl chains. Since steady-state anisotropy is a function of fluorescence lifetime, rotational diffusion rates, and limiting anisotropy, we determined these parameters for two of the phospholipid systems. The results show that steady-state anisotropy values for phospholipid-sterol interactions correlate closely with limiting anisotropy and to a lesser extent with rotational relaxation time. The behavior of the sterols in the model phospholipids are used to interpret 1) fluorescence polarization measurements made with phospholipids extracted from LM cell plasma membranes, and 2) changes in membrane lipid composition which accompany growth of LM cells on various sterols.

LM cell growth and membrane lipid adaptation to sterol structure.

Using a sterol auxotroph of the LM cell mouse fibroblast, we demonstrate that relatively few cholesterol analogues can substitute for cholesterol as a growth factor. The auxotroph grows normally on desmosterol and trans-22-dehydrocholesterol and at reduced rates on dihydrocholesterol, campesterol, and 22,23-dihydrobrassicasterol. It does not grow with beta-sitosterol, stigmasterol, ergosterol, or cis-22-dehydrocholesterol when the sterol is present as sole supplement but does grow at normal rates when the analogue is supplied with suboptimal amounts of cholesterol. Two contrasting types of membrane lipid changes are observed in cells grown on cholesterol analogues. In cells grown with dihydrocholesterol, a marked increase in desaturation and elongation of fatty acids is noted. Conversely, when cells are grown with cis-22-dehydrocholesterol, desaturation and elongation of fatty acids are severely curtailed. Cells grown on alkyl sterols respond like cells grown on cis-22-dehydrocholesterol but in a less pronounced fashion. The effects of sterol substitution in mammalian cells versus in lower eukaryotes are compared, and an explanation for the secondary changes in fatty acid composition in terms of phospholipid phase behavior is suggested.

An alternative view of phospholipid phase behavior at the air-water interface. Microscope and film balance studies

Pure-lipid films at the water interface have surface-pressures vs. area isotherms that are often interpreted as involving first-order phase transitions from a condensed region to a liquid-expanded region. Two phases are presumed to coexist in the intermediate part of the isotherm. We constructed a film balance that could be placed on the stage of an epifluorescence microscope. A dipalmitoyl phosphatidylcholine film containing a low concentration of a fluorescent lipid probe showed an inhomogeneous fluorescence distribution in the so-called liquid-expanded region of the isotherm. Only the intermediate and condensed regions could be prepared so as to be optically homogeneous below 25 degrees C. We investigated membrane flow and lateral lipid diffusion in the membrane on the trough. The isotherms and isochores were measured. The results require, at least, a modified description of the monolayer structure in various regions of the isotherms. The solid-condensed region corresponds to a gel phase of the lipids where there is no flow in the membrane, lateral diffusion is low, the compressibility is low, and the membrane is optically homogeneous. The "liquid-condensed/liquid-expanded" region appears to be a homogeneous membrane where lateral diffusion and membrane flow are both rapid. This is a region of high compressibility. The "liquid-expanded" region is not homogeneous as seen under the microscope, and the flow of the surface layer can be very fast.

Zeaxanthin ([3R,3'R]-beta, beta-carotene-3–3'diol) as a resonance Raman and visible absorption probe of membrane structure

When zeaxanthin ([3R,3R']-beta, beta-carotene-3,3'diol) is inserted into phospholipid dispersions and the latter heated through their gel-liquid crystal phase transitions, large changes are noted in the resonance Raman and absorption spectra of the carotenoid molecule. By analogy with the data of Carey and co-workers (J. Raman Spectrosc. 6:282) who studied the aggregation of zeaxanthin in acetone-water solutions, it is suggested that the carotenoid aggregates in the phospholipid gel state while forming a monomer in liquid crystal phases. The alterations in both the visible absorption and resonance Raman data have been used to monitor phospholipid phase behavior in dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine, (DSPC) one-component systems and binary mixtures. The phase diagram obtained for the binary system, as constructed from visible absorption and resonance Raman data, is compared with that of Shimshick and McConnell (Biochemistry. 12:2351) obtained from electron spin resonance (ESR) studies. Although the agreement between absorption and ESR data is generally satisfactory, onset temperatures for phase separation at low DSPC mole fractions deduced from resonance Raman measurements are several degrees lower than those from the other methods. Nevertheless, the use of zeaxanthin as a resonance Raman and visible absorption probe behavior will be useful in some situations where ordinary Raman spectroscopic data cannot be obtained easily. The advantage of the resonance Raman approach is illustrated in a study of the phase behavior of a phospholipid extract of a cel- mutant of Neurospora crassa. A phase separation region is observed with onset and completion temperatures of -19 and -6 degrees C, respectively.