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PubMed2006

Phase separation of a binary two-dimensional core-softened fluid.

Using molecular dynamics simulations, we study the phase separation in a binary two-dimensional core-softened fluid with different size ratios and concentrations. The correlation functions for both components are analyzed to show the dependence of the configurational structure of the binary fluid on size ratio and concentration. A phase separation diagram is obtained and the structural features of the phase separation are further investigated using the direct imaging method.

Journal Article↗

PubMed2004

Coarsening mechanism of phase separation caused by a double temperature quench in an off-symmetric binary mixture.

We study phase-separation behavior of an off-symmetric fluid mixture induced by a "double temperature quench." We first quench a system into the unstable region. After a large phase-separated structure is formed, we again quench the system more deeply and follow the pattern-evolution process. The second quench makes the domains formed by the first quench unstable and leads to double phase separation; that is, small droplets are formed inside the large domains created by the first quench. The complex coarsening behavior of this hierarchic structure having two characteristic length scales is studied in detail by using the digital image analysis. We find three distinct time regimes in the time evolution of the structure factor of the system. In the first regime, small droplets coarsen with time inside large domains. There a large domain containing small droplets in it can be regarded as an isolated system. Later, however, the coarsening of small droplets stops when they start to interact via diffusion with the large domain containing them. Finally, small droplets disappear due to the Lifshitz-Slyozov mechanism. Thus the observed behavior can be explained by the crossover of the nature of a large domain from the isolated to the open system; this is a direct consequence of the existence of the two characteristic length scales.

Journal Article↗

PubMed1977

Lipid phase separation induced by a hydrophobic protein in phosphatidylserine--phosphatidylcholine vesicles.

Differential scanning calorimetry (DSC) was used to detect phase separation induced by hydrophobic myelin protein, lipophilin, in a mixture of phosphatidylserine (PS) and dipalmitoylphosphatidylcholine (DPPC). Preferential binding of PS to the boundary layer of lipophilin causes a decrease in the PS content of the remaining lamellar phase with a resultant shift in the phase-transition temperature to a higher temperature. The phase diagram for this mixture in the presence and absence of lipophilin is presented. From the phase diagram, it can be estimated that for an equimolar mixture of PS and DPPC, the boundary layer contains only PS, although for higher DPPC contents, some DPPC can also be found in the boundary layer. In the case where partial phase separation in induced in this mixture by Ca2+ alone, lipophilin increases the phase separation indicating that it also binds PS preferentially in the presence of Ca2+. Preferential binding of two other acidic lipids, phosphatidic acid and phosphatidyl-glycerol, to the boundary layer was also found, including a mixture where the acidic lipid was the higher melting component in the mixture.

Calcium↗

PubMed2026

Viral replication through phase separation: Cytosolic and nuclear condensates.

Replication of many RNA and DNA viruses occurs within specialized intracellular hubs organized as membraneless biomolecular condensates (BCs) driven by liquid-liquid phase separation. As obligate intracellular parasites, viruses depend on the host cell machinery to complete their replication cycles and therefore actively remodel the intracellular environment to favor viral genome replication, transcription, and assembly. Cytosolic and nuclear phase-separated replication compartments (RC) provide concentrated and dynamic platforms that promote efficient interactions between viral genomes and viral or host proteins essential for infection. The formation of viral replication BCs is typically facilitated by viral proteins enriched in intrinsically disordered regions and low-complexity domains, which enable multivalent interactions with viral nucleic acids and cellular factors. These interactions are mediated by diverse biophysical forces, including hydrophobic and π interactions, hydrogen bonding, molecular crowding, and osmotic effects. Throughout infection, viral BCs remain highly dynamic, allowing continuous exchange of components and functional maturation of replication hubs. Their properties and activities are further regulated by post-translational modifications of viral and host proteins, such as phosphorylation, acetylation, and methylation. In this review, we summarize current evidence supporting liquid-liquid phase separation as a central organizing principle of viral RCs. We focus on representative RNA and DNA viruses that replicate in the cytosol or nucleus, highlighting virus-specific strategies, conserved mechanisms, and the consequences of BC formation for viral replication efficiency, host antiviral responses, and therapeutic intervention.

Phase Separation↗

PubMed2003

Acyl chain unsaturation in PEs modulates phase separation from lipid raft molecules.

By one hypothesis, phospholipids containing unsaturated fatty acids may be involved in phase separation from the lipid raft molecules sphingomyelin (SM) and cholesterol (CHOL). We tested the effect of increasing the number of double bonds in the acyl chains of phosphatidylethanolamines (PEs) on phase separation from SM/CHOL. The detergent extraction method was employed on various homoacid and heteroacid PEs in mixed vesicles composed of PE/SM/CHOL (1:1:1mol). The disaturated homoacid 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (16:0-16:0PE) showed the least solubility upon detergent extraction whereas maximal solubility was observed for the polyunsaturated homoacid 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6-22:6PE). Increasing the number of double bonds in the sn-2 position of heteroacid saturated-unsaturated PEs resulted in an increase in detergent solubility, which correlated with a general decrease in the gel-to-liquid crystalline phase transition temperature of the PEs. Our findings demonstrate that increasing unsaturation in PEs results in increased phase separation from SM/CHOL membranes, which may have implications for cellular signaling.

Calorimetry, Differential Scanning↗

PubMed1997

The relationship between compositional phase separation and vesicle morphology: implications for the regulation of phospholipase A2 by membrane structure.

The action of phospholipase A2 (PLA2) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA2. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.

Light↗

PubMed2005

Phase separation of water-alcohol binary mixtures induced by the microheterogeneity.

The relationship between liquid-liquid phase separation and microheterogeneity in water-primary alcohol mixtures was examined by analysing the mass spectra of clusters generated through the fragmentation of liquid droplets. By comparing the cluster structures of water-ethanol, -1-propanol, and -1-butanol binary mixtures at various alcohol concentrations, we discovered differences in the molecular clusters that control phase separation. We also studied the role of water in alcohol self-association. Alcohol self-association is promoted in the presence of a small amount of water (ca. 10 approximately 20 wt%), in which the water-water hydrogen-bonding network is weak and does not contribute to alcohol self-association. We have demonstrated that alcohol self-association is also promoted by non-ideal mixing with other alcohols. The self-association of alcohol molecules complements the loss of stabilization energy caused by the relatively weak coexisting interactions. This complementary relationship among intermolecular interactions is an inherent property of solutions, and plays a key role in the phase separation process.

Journal Article↗

PubMed2005

Cationic surfactant-based polyfluorate salts: phase separation and analytical applications in the extraction and preconcentration of ionic species prior to liquid chromatography.

The liquid-solid phase separation originating from the formation of cationic surfactant-based polyfluorate salts (CSBPS) has been explored for extracting and preconcentrating ionic species. Two cationic surfactants were tested; one with aliphatic hydrocarbon tail [Cetyltrimethylammonium bromide (CTAB)]and the other containing a heterocyclic ring [Hexadecylpyridinium bromide (HPyBr)]. Phase separation possibility was investigated with the use of hexafluorophosphates (PF6-) and tetrafluoroborates (BF4-). The effect of added acid, base and salt on the phase separation and analyte extraction was also investigated. In all cases the obtained phase diagrams consisted of two regions: a homogeneous liquid region and a solid-liquid region. Analytes of hydrophilic and hydrophobic nature such as amines, amino acids and organic chromophores were used as test compounds in both their anionic and cationic forms. The respective recoveries ranged from over 90% for anionic species and in the proximity of 50% for cationic species, remaining below 20% for neutral species. Extracts from alkaline aqueous and plasma samples spiked with tyrosine and phenylalanine were also subjected to HPLC separation with UV detection with satisfactory results. On line application was also enabled using a flow through-solid phase extraction-HPLC hyphenated apparatus, thus adding the element of automatization and increased reproducibility.

Borates↗

PubMedDate not supplied

Morphology and kinetics of phase separating transparent xanthan-colloid mixtures.

We present a study on the morphology and kinetics of depletion-induced phase separation in aqueous xanthan-colloid mixtures with light microscopy and small angle light scattering (SALS), using fluorinated colloids with a refractive index close to that of water to prevent complications of multiple scattering. Microscopy with the direction of observation perpendicular to gravity enabled us to observe the development of the microstructure during the entire phase separation process including the formation of a macroscopic interface. Bicontinuous structures typical of a spinodal decomposition mechanism were observed at early times. These structures coarsened in time until hydrodynamic flow resulted in lane formation. Close to the binodal, a nucleation-and-growth mechanism was observed with formation of droplets. The coarsening kinetics were studied in more detail with SALS and turbidity measurements. Above polysaccharide concentrations at which entanglements become dominant, a slower coarsening and macroscopic phase separation were found because of the high continuous phase viscosity.

Carbohydrate Conformation↗

PubMed2001

Lipid phase separation in phospholipid bilayers and monolayers modeling the plasma membrane.

It is postulated that biological membrane lipids are heterogeneously distributed into lipid microdomains. Recent evidence indicates that docosahexaenoic acid-containing phospholipids may be involved in biologically important lipid phase separations. Here we investigate the elastic and thermal properties of a model plasma membrane composed of egg sphingomyelin (SM), cholesterol and 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE). Two techniques are employed, pressure-area isotherms on monolayers to examine condensation and interfacial elasticity behavior, and differential scanning calorimetry (DSC) on bilayers to evaluate phase separations. Significant levels of condensation are observed for mixtures of SM and cholesterol. Surface elasticity measurements indicate that cholesterol decreases and SDPE increases the in-plane elasticity of SM monolayers. At X(SDPE)> or =0.15 in SM, a more horizontal region emerges in the pressure-area isotherms indicating 'squeeze out' of SDPE from the monolayers. Addition of cholesterol to equimolar amounts of SM and SDPE further increases the amount of 'squeeze out', supporting the concept of phase separation into a cholesterol- and SM-rich liquid ordered phase and a SDPE-rich liquid disordered phase. This conclusion is corroborated by DSC studies where as little as X(Chol)=0.0025 induces a phase separation between the two lipids.

Calorimetry, Differential Scanning↗

PubMed1993

Phase separated anionic domains in ternary mixed lipid monolayers at the air-water interface.

A series of ternary mixed monolayers containing varying amounts of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and equimolar additions of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LYSO-PC) and palmitic acid (PA) were studied at the air-water interface. These mixed monolayers were used to model phospholipid biomembrane interfaces resulting from phospholipase A2 (PLA2) hydrolysis. Recent work [D.W. Grainger A. Reichert, H. Ringsdorf and C. Salesse (1989) Biochim. Biophys. Acta. 1023, 365-379] has shown that PLA2 hydrolysis of pure phospholipid monolayers results in formation of large PLA2 domains at the air-water interface. These domains are proposed to result from PLA2 adsorption to phase separated regions in the hydrolyzed monolayer. To elucidate the phase behaviour in these monolayer systems, surface pressure-area isotherms were measured for the ternary mixtures on pure water and buffered subphases. Fluorescence microscopy at the air-water interface was used to image fluorescent probe-doped monolayer mixtures during isothermal compressions. A water-soluble cationic carbocyanine dye was used to probe the interfacial properties of the mixed monolayers. Isotherm data do not provide unambiguous evidence for either phase separation or ideal mixing of monolayer components. Fluorescence microscopy is more revealing, showing that lateral phase separation of microstructures containing palmitic acid occurred only when monolayer subphases contained Ca2+ ions at alkaline pH. At either low pH or on Ca(2+)-free subphases, phase separation was not observed.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

Phase Separation and Dewetting of Weakly Incompatible Polymer Blend Films.

The phase separation and dewetting of thin films of blends of deuterated polystyrene (dPS) and poly(p-methylstyrene) (PpMS) were investigated during annealing. The surface morphology, obtained from atomic force microscopy and phase measurement interference microscopy, the density profile, determined by X-ray reflectivity in the region of total external reflection, and the surface composition obtained from static secondary ion mass spectroscopy, are reported. This system is only weakly incompatible. The interaction of the components with substrate and air during phase separation leads to a bilayer formation with a broad polymer-polymer interface. PpMS segregates to the air interface. The bilayer structure is unstable and defines the starting point for the dewetting of PpMS on top of the dPS layer. In the final dewetting state a homogeneous layer of dPS on top of the substrate is covered with an ultrathin layer of PpMS as well as with quite thick mesoscopic drops of PpMS.

Journal Article↗

PubMed1987

Effect of aspirin and vitamin E on phase separation in calf lens homogenate.

Vitamin E (d-alpha-tocopherol) and aspirin were evaluated for their effects on the phase separation temperature, Tc, of calf lens cytoplasm. Vitamin E and aspirin were added to nuclear homogenate and the change in Tc was measured. Aspirin had no measurable effect on Tc. Vitamin E lowered Tc at a rate of -22 degrees C M-1 l-1. The results suggest that the effects of aspirin on cataract are unrelated to phase separation and that vitamin E may act on the phase separation in precataractous lens cells.

Animals↗

PubMed2004

Associative phase separation of beta-lactoglobulin/pectin solutions: a kinetic study by small angle static light scattering.

Interpolymer complexation between beta-lactoglobulin (beta-lg) and pectin led to phase separation. Small angle static light scattering and phase contrast microscopy were used to monitor the phase separation of beta-lg/low-methoxyl or high-methoxyl-pectin (LM- or HM-pectin) dispersions as they were slowly acidified from pH 7 to 4 with glucono-delta-lactone (GDL). The monotonic decrease in scattered light intensity with the wave vector was associated with a nucleation and growth phase separation mechanism. Microscopic observations and turbidity measurements showed the increase of complex amounts with lower pH and at higher beta-lg/pectin ratios. The formation of intrapolymer complexes was initiated at pH 6.4 with the LM-pectin and at pH 5.0 with the HM-pectin. Local ordering with increasing amounts of small complexes was observed as scattered light intensity increased at intermediate q values. The beta-lg/LM-pectin complexes at the 5:1 and the 2:1 weight ratios and the beta-lg/HM-pectin complexes at 5:1 weight ratio have fractal structures. The formation of large amounts of small assemblies and sedimentation would be responsible for the decrease in the number and volume mean diameters and fractal dimension of beta-lg/LM-pectin complexes over time.

Kinetics↗

PubMedDate not supplied

Analytical evaluation of methods for serum creatine kinase-MB. Electrophoresis, immunoinhibition and solid phase separation.

The purpose of this study was to evaluate immunoassay methods for the measurement of serum cardiac creatine kinase isoenzyme (CK-MB) with respect to sensitivity and specificity. The CK-MB electrophoretic assay (Helena Laboratories) was used as the reference. Two principles of immunoassay were included in the evaluation,--immunoinhibition and solid phase separation. The direct immunoinhibition techniques were from Beckman Instruments (CKMB reagent) and DuPont Medical Products (CKMB). Three solid phase separation techniques were from Abbott Laboratories (IMx CKMB), DuPont (acaPlus MCKMB), and Tosoh Medics Inc. (AIA-Pack CKMB). The electrophoretic method for separation of the CK isoenzymes has good specificity but lacks sensitivity for CK-MB in low concentrations. The immunoinhibition methods lack specificity and correlate poorly with the electrophoresis method and with the solid phase methods. The solid phase separation techniques are highly sensitive and show an excellent correlation with electrophoresis when based on specificity. The solid phase separation methods correlate well with each other.

Creatine Kinase↗

PubMed2004

Oleic and docosahexaenoic acid differentially phase separate from lipid raft molecules: a comparative NMR, DSC, AFM, and detergent extraction study.

We have previously suggested that the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) may in part function by enhancing membrane lipid phase separation into lipid rafts. Here we further tested for differences in the molecular interactions of an oleic (OA) versus DHA-containing phospholipid with sphingomyelin (SM) and cholesterol (CHOL) utilizing (2)H NMR spectroscopy, differential scanning calorimetry, atomic force microscopy, and detergent extractions in model bilayer membranes. (2)H NMR and DSC (differential scanning calorimetry) established the phase behavior of the OA-containing 1-[(2)H(31)]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE-d(31))/SM (1:1) and the DHA-containing 1-[(2)H(31)]palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE-d(31))/SM (1:1) in the absence and presence of equimolar CHOL. CHOL was observed to affect the OA-containing phosphatidylethanolamine (PE) more than the DHA-containing PE, as exemplified by >2 x greater increase in order measured for the perdeuterated palmitic chain in 16:0-18:1PE-d(31)/SM (1:1) compared to 16:0-22:6PE-d(31)/SM (1:1) bilayers in the liquid crystalline phase. Atomic force microscopy (AFM) experiments showed less lateral phase separation between 16:0-18:1PE-rich and SM/CHOL-rich raft domains in 16:0-18:1PE/SM/CHOL (1:1:1) bilayers than was observed when 16:0-22:6PE replaced 16:0-18:1PE. Differences in the molecular interaction of 16:0-18:1PE and 16:0-22:6PE with SM/CHOL were also found using biochemical detergent extractions. In the presence of equimolar SM/CHOL, 16:0-18:1PE showed decreased solubilization in comparison to 16:0-22:6PE, indicating greater phase separation with the DHA-PE. Detergent experiments were also conducted with cardiomyocytes fed radiolabeled OA or DHA. Although both OA and DHA were found to be largely detergent solubilized, the amount of OA that was found to be associated with raft-rich detergent-resistant membranes exceeded DHA by almost a factor of 2. We conclude that the OA-PE phase separates from rafts far less than DHA-PE, which may have implications for cellular signaling.

Animals↗

PubMed2005

Genetically engineered elastin-protein A fusion as a universal platform for homogeneous, phase-separation immunoassay.

A simple and universal platform for competitive phase-separation immunoassay is reported based on a fusion protein composed of a temperature-responsive elastin-like polypeptide (ELP) and the antibody-binding staphylococcal protein A (SpA). The basic principle is to take advantage of the ability of SpA to bind a variety of antibodies with high affinity, allowing simple separation of antigen-antibody complex by thermal precipitation. The resulting ELP-SpA fusion was shown to preserve the ability to reversibly precipitate as well as its high affinity toward different IgGs and IgMs. As a model system, a competitive phase-separation immunoassay based on the ELP-SpA format was established for paclitaxel (taxol) with IC(50) (20.18 nM) and the lower detection limit (2.94 nM) very similar to those reported for the ELISA format. Unlike the heterogeneous interaction in ELISA, which decreases the antibody-binding activity, the reported homogeneous immunoassay not only alleviates this problem but also enables the potential for high-throughput automation. We believe that the reported ELP-SpA fusion will find applications not only as a powerful diagnostic tool for diverse analytes but also a potential useful tool for purification and immobilization of antibody.

Elastin↗

PubMed1996

Effects of sugars and polymers on crystallization of poly(ethylene glycol) in frozen solutions: phase separation between incompatible polymers.

PURPOSE: This study examined the effect of third components (low-molecular-weight saccharides and polymers) on the crystallization of poly(ethylene) glycol (PEG) in frozen solutions, focusing on the relationship between their crystallization-inhibiting ability and molecular compatibility. METHODS: Effects of sugars and polymers on the crystallization of PEG 3000 in frozen solution were monitored by differential scanning calorimetry (DSC). Pulsed-NMR was employed to monitor the molecular mobility of water and solutes in the frozen solutions. Miscibility between PEG and third components in aqueous solution was estimated from the lowering of cloud point of PEG 20,000. Thermal analysis of frozen solutions containing some non-crystallizing solutes was used to examine the possibility of phase separation in frozen solutions. RESULTS: Some sugars and polymers inhibited the crystallization of PEG and formed practically stable amorphous phases among ice crystals. The mobility of solute molecules in the amorphous phase increased above the softening temperature of maximally concentrated solutions (Ts), whereas that of water molecules appeared at a lower temperature. Mono- and disaccharides that are relatively less miscible with PEG in solution inhibit PEG crystallization to a lesser degree. Two Ts regions were observed in frozen solutions containing both polyvinylpyrrolidone (PVP) and dextran, at much lower concentrations than those causing aqueous two-phase separation at ambient temperatures. CONCLUSIONS: Ice crystallization raises the concentration of solutes in the remaining solution, which can lead to phase separation in the amorphous phase. Molecular compatibility between components is an important factor determining their propensity to phase separate and crystallize.

Calorimetry, Differential Scanning↗