Phase Separation Experiments Research Articles

Polymer–polymer interaction parameters for homopolymers and copolymers from light scattering and phase separation experiments in a common solvent

The thermodynamic behavior of the quaternary system DMAc/PS/PAN/SAN and of all its solvent containing subsystems [DMAc: dimethylacetamide, PS: polystyrene, PAN: polyacrylonitrile, SAN: P(S–ran–AN)] was studied in terms of second virial coefficients and phase separation in the range of moderate polymer concentrations. The most remarkable finding consists in fundamental discrepancies between the Flory–Huggins parameters obtained for the interaction between the segments of polymer X and polymer Y either straightforwardly from the light scattering experiments (ranging from −0.88 to −0.14) and that resulting from the modeling of the measured phase diagrams (+0.01 to +0.33) or from solubility parameter theory (+0.03 to +1.92). The reasons for the disagreement even in the sign of the parameters are discussed in terms of A2XY, the second osmotic virial coefficients referring to the opening of intermolecular contacts between X and Y by the insertion of solvent and in terms of W2, the normalized excess Gibbs energy of contact formation between the two types of solute in the concentration region of pair interaction according to X–X+Y–Y→2X–Y. The pair interaction coefficient W2 results negative for PS/SAN, but positive for PAN/SAN and for PAN/PS. A theoretical explanation of this unexpected result is offered.

The Use of Liquid−Liquid Extraction in the EPDM Solution Polymerization Process

The application of liquid−liquid separation as a means of polymer−solvent separation was studied. Phase separation experiments were performed with ethene−propene−diene terpolymer (EPDM) solved in hexane, using propene as an antisolvent. The results were interpreted using a thermodynamic model based on the Sako−Wu−Prausnitz (SWP) equation of state. The description of homogeneous polymers is unambiguous, but for heterogeneous polymers further development of the SWP theory is required. It was found that the model shows promising results with respect to multicomponent polymer systems. However, the description of the interaction between the components is still unsatisfactory. Therefore, an empirical model was developed to describe the experimental results. A flowsheet model was constructed to describe a novel EPDM process containing a liquid−liquid countercurrent extraction column. The effects of temperature, pressure, antisolvent concentration, and number of theoretical equilibrium stages were studied. With respect to variable costs, liquid−liquid extraction is a cost-effective option in the purification of rubber polymer solutions.

Modification of Rab5 with a photoactivatable analog of geranylgeranyl diphosphate.

A photoprobe analog of geranylgeranyl diphosphate (2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate or DATFP-FPP) inhibits mevalonate-dependent prenylation of in vitro translated Rab5 in rabbit reticulocyte lysate, suggesting that it competes for lipid binding to the Rab geranylgeranyl transferase. Modification of Rab5 with DATFP-FPP, demonstrated by gel mobility shift and Triton X-114 phase separation experiments, confirms that the enzyme uses the analog as a substrate. The sedimentation of DATFP-modified Rab5 as a larger mass complex on sucrose density gradients indicates that it binds to other factors in rabbit reticulocyte lysate. Most importantly, DATFP-Rab5 cross-links to these soluble factors upon exposure to UV light. Immunoprecipitation with antibodies raised against proteins known to interact with Rab5 reveals that the cross-linked complexes contain Rab escort protein and GDI-1. DATFP-Rab5 also associates with membranes in a guanosine-5'-O-(3-thiotriphosphate)-stimulated manner. However, although prenylated Rab5 can be cross-linked to two unknown membrane-associated factors by the chemical cross-linker disuccinimidyl suberate, these proteins fail to be UV cross-linked to membrane-bound DATFP-Rab5. These results strongly suggest that membrane-associated factors bind Rab5 through protein-protein interactions rather than protein-prenyl interactions. The modification of Rab5 with DATFP-FPP establishes a novel photoaffinity technique for the characterization of prenyl-binding sites.

Phase behavior, photopolymerization, and morphology development in mixtures of eutectic nematic liquid crystal and photocurable monomer

Phase behavior and photopolymerization-induced morphology development in mixtures of eutectic nematic liquid crystals (LC) (designated E44) and photocurative monomers (designated NOA65) have been investigated by means of optical microscopy and light scattering. The observed phase diagram of the E44/NOA65 system prior to photopolymerization is of an upper critical solution temperature (UCST) type overlapping with the nematic–isotropic transition of E44. It displayed isotropic liquid, isotropic liquid+isotropic liquid, isotropic liquid+nematic, and pure nematic coexistence regions that were verified experimentally. Using the phase diagram as a guide, photopolymerization-induced phase separation experiments were performed in the isotropic region in order to examine the morphology development in multicomponent LC (E44)-based system in comparison with that of a single component LC (K21)-based system. Of particular interest is that the emerging LC droplets turned out to be unevenly distributed in the E44/NOA65 system as opposed to the K21/NOA65 system where the droplet size was uniform. The non-uniform distribution of the droplets in the E44/NOA65 system may be attributed to compositional fractionation associated with different affinities of the constituent LC in E44 to the NOA65 networks.

Differential glycosylation and proteolytical processing of LeechCAM in central and peripheral leech neurons

LeechCAM is a recently described member of the Ig-superfamily which has five Ig-domains, two FNIII-domains, a transmembrane domain, and a cytoplasmic domain. Phylogenetic analysis indicated that LeechCAM is the leech homolog of apCAM, FasII, and vertebrate NCAM. Using a leechCAM-specific monoclonal antibody we show by immunoblot analysis and by Triton X-114 phase separation experiments that in addition to existing in a transmembrane version LeechCAM is likely to be proteolytically cleaved into a secreted form without the transmembrane domain and the intracellular tail. Furthermore, by immunoprecipitation we demonstrate that LeechCAM is glycosylated with the Laz2-369 glycoepitope, an epitope that has been specifically implicated in regulation of axonal outgrowth and synapse formation.

Functional reconstitution of β-glucan elicitor-binding activity upon incorporation into lipid vesicles

In temperature-induced Triton X-114 phase separation experiments the β-glucan elicitor-binding site from soybean ( Glycine max L.) root membranes was identified as (a) hydrophobic membrane protein(s). The Zwittergent 3-12-solubilized β-glucan-binding proteins were incorporated into lipid vesicles by the detergent-dilution procedure. Reconstituted binding proteins were functional in that binding of the hepta-β-glucoside ligand was saturable, reversible and of high affinity ( K d=6–7 nM). Competition studies using β-glucans with different degrees of polymerization (DP 7–15; DP 15–25) showed effective displacement of the radioligand from the binding site whereas β-glucan fragments with DP <7 were ineffective. The total amount of reconstituted binding activity was dependent on the acyl chain length of the phospholipids used for the reconstitution with a preference for decanoic (C10) and dodecanoic (C12) chains. Restored ligand binding was maximally 37% as compared to the former detergent-solubilized binding activity. The presence of a lipid environment stabilized the purified β-glucan-binding proteins.

Plasma membrane association and preliminary characterization of Drosophila sperm surface glycosidases.

Previous studies have identified beta-N-acetylglucosaminidase (GlcNAc'ase) and (alpha-mannosidase activities on the Drosophila melanogaster sperm surface which may have a role in fertilization. The aim of this study was to investigate their linkage to the sperm plasma membrane. We verified that glycosidases are not peripherally adsorbed to the cell surface by evaluating their resistance to release by KI, by buffered salt solutions of high ionic strength or alkaline buffers. Glycosidases were released from the sperm surface by detergents and, only to a minor extent, by mild proteolysis. Differential detergent solubilization pointed out that Triton X-114 was the most effective releasing agent for GlcNAc'ase and CHAPS for mannosidase. No activity was released from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). The released forms were quite hydrophilic in phase separation experiments with Triton X-114. This finding indicates the presence of a hydrophobic domain limited to a single transmembrane helix or/and the presence of an extensive glycosylation. The use of a Con-A binding assay demonstrated that both the enzymes are glycosylated. The molecular weight of the released glycosidases estimated by gel filtration was 158 kDa for GlcNAc'ase and 317 kDa for mannosidase. These results suggest that Drosophila melanogaster GlcNAc'ase and mannosidase are mannosylated integral membrane proteins that would function as exoenzymes with their active sites accessible in the extracellular space.

Light-Scattering and Phase-Separation Studies on Cyclohexane Solutions of Four-Arm Star Polystyrene

Light-scattering and phase-separation experiments were performed on cyclohexane solutions of four narrow-distribution samples of four-arm star polystyrene with molecular weights of 8.5 × 10 4 to 1.4 × 10 6 , and the results were compared with literature data for linear polystyrene in cyclohexane. The upper critical solution temperatures T c for the star polymer were systematically lower than those for the linear polymer, showing a considerable effect of chain branching on phase separation. The apparent second virial coefficients J of light scattering for the two polymers, plotted against o/P 0.1 at a fixed temperature below Θ, happened to form a composite curve at high polymer concentrations, regardless of the relative degree of polymerization P (o denotes the polymer volume fraction), whereas they appreciably differed at low concentrations, reflecting the finding that the (true) second virial coefficient for the star polymer is significantly larger than that for the linear polymer below Θ. The chemical potentials of the solvent and solute components derived from the J data were shown to explain the phase diagrams of the two systems fairly well. Thus it was concluded that the difference in T c between four-arm star and linear polystyrenes arises primarily from the difference in J in dilute solution.

Inhibition and Kinetic Properties of Membrane-Bound Carbonic Anhydrases in Rabbit Skeletal Muscles

It was the aim of this study to investigate whether the carbonic anhydrases associated with the sarcoplasmic reticulum (SR) and sarcolemmal membranes differ in their kinetic and inhibitory properties. To this end, sarcolemmal and SR membrane vesicle fractions were prepared from rabbit white and red skeletal muscles, the white muscle sarcolemmal fraction (WSL), the red muscle sarcolemmal fraction (RSL), the white muscle SR fraction (WSR), and the red muscle SR fraction (RSR). WSL displayed a specific carbonic anhydrase activity of 22.1 U · ml/mg and RSL of 7.5 U · ml/mg, whereas the SR fractions showed a much lower activity of 0.5 U · ml/mg for WSR and of 2.4 U · ml/mg for RSR. In both SR fractions phase separation experiments with Triton X-114 demonstrated that the carbonic anhydrase activity is due to a membrane-bound enzyme and not due to a cytosolic isozyme. The kinetic properties of carbonic anhydrase from the four distinct membane fractions were evaluated by determination of the Michaelis constant,Km, and of the catalytic centre activitykcat.Kmappears to be somewhat lower for SR than for SL. Inhibition constants of SR and SL carbonic anhydrases were determined applying six carbonic anhydrase inhibitors: chlorzolamide, ethoxzolamide, methazolamide, benzolamide, and acetazolamide, and also cyanate. The inhibition constants of the SR fractions were significantly different from those of the corresponding sarcolemmal fractions, indicating that the carbonic anhydrase measured in the SR fractions does not originate from contaminating sarcolemmal membrane vesicles, but appears to represent a distinct carbonic anhydrase associated with the SR membrane.

Liquid crystal formation in DNA fragment solutions

The critical volume fractions pertaining to the formation of DNA liquid crystals were obtained from polarization microscopy, 31P-nmr, and phase separation experiments. The DNA length (approximately one to two times the persistence length 50 nm), ionic strength, and counterion variety dependencies are reported. The cholesteric–isotropic transition is interpreted in terms of the coexistence equations, which are derived from the solution free energy including orientational entropy and excluded volume effects. With the wormlike chain as reference system, the electrostatic contribution to the free energy is evaluated as a thermodynamic perturbation in the second virial approximation with a Debye–Hückel potential of mean force. The hard core contribution has been evaluated with scaled particle theory and/or a simple generalization of the Carnahan–Starling equation of state for hard spheres. For sufficiently high ionic strengths, the agreement is almost quantitative. At lower amounts of added salt deviations are observed, which are tentatively attributed to counterion screening effects. The contour length dependence agrees with a DNA persistence length 50 nm. © 1998 John Wiley & Sons, Inc. Biopoly 46: 31–37, 1998

Liquid crystal formation in DNA fragment solutions.

The critical volume fractions pertaining to the formation of DNA liquid crystals were obtained from polarization microscopy, 31P-nmr, and phase separation experiments. The DNA length (approximately one to two times the persistence length 50 nm), ionic strength, and counterion variety dependencies are reported. The cholesteric-isotropic transition is interpreted in terms of the coexistence equations, which are derived from the solution free energy including orientational entropy and excluded volume effects. With the wormlike chain as reference system, the electrostatic contribution to the free energy is evaluated as a thermodynamic perturbation in the second virial approximation with a Debye-Hückel potential of mean force. The hard core contribution has been evaluated with scaled particle theory and/or a simple generalization of the Carnahan-Starling equation of state for hard spheres. For sufficiently high ionic strengths, the agreement is almost quantitative. At lower amounts of added salt deviations are observed, which are tentatively attributed to counterion screening effects. The contour length dependence agrees with a DNA persistence length 50 nm.

Characterization of high affinity progesterone-binding membrane proteins by an anti-peptide antiserum

A chemically synthesized 15-mer oligopeptide derived from the N terminus of high affinity progesterone-binding membrane site(s) from porcine liver was used to generate site-specific antibodies. Western blotting experiments confirmed the specificity of the anti-peptide serum obtained. In further investigations this antiserum was used for the identification of the native progesterone-binding membrane protein complex that represents an oligomer with an apparent molecular mass of about 200 kDa. In temperature-induced Triton X-114 phase separation experiments combined with Western-blotting, the progesterone-binding site was identified as an hydrophobic (integral) membrane protein. In addition, in Western blotting analyses the antiserum reacted with the progesterone-binding or related proteins in membrane fractions from a wide array of different tissues in various species.

Localization of alanyl aminopeptidase and leucyl aminopeptidase in cells of Pseudomonas aeruginosa by application of different methods for periplasm release.

Various methods for the isolation of periplasm were examined and compared with regard to the complete release of known periplasmic marker enzymes and the contamination of the periplasm by cytosol for Pseudomonas aeruginosa PAO1 as a significant Gram-negative test strain. The aim of the investigations was to clarify the exact localization of alanyl aminopeptidase (AAP) and leucyl aminopeptidase (LAP) of this microorganism and to evaluate these methods. The osmotic shock of NOSSAL and HEPPEL (1996) was the most effective method with the lowest contamination by the cytosolic marker enzyme malic enzyme, but some proteins, which are located near the inner side of the cytoplasmic membrane, can be released additionally into the periplasm. All other procedures like chloroform or polymyxin treatment, the magnesium chloride washing of intact bacteria and spheroblasting by lysozyme in the presence of EDTA or magnesium chloride resulted only in a partial, sometimes only very low release of periplasm. The periplasmic enzymes are bound either more by hydrophobic or more by ionic interactions to the cell envelope and show a different behaviour with the different releasing agents. These methods are useful for a further differentiation between really periplasmic protein, and those proteins, which were false positive found in periplasm as a result of the osmotic shock. Our results show that AAP from Pseudomonas aeruginosa is a periplasmic enzyme with hydrophobic interactions to the cytoplasmic membrane, corresponding to the early results of LAZDUNSKI and MURGIER for Escherichia coli (LAZDUNSKI et al. 1975a and b, MURGIER et al. 1977), and LAP is cytosolic, but located near the cytoplasmic membrane. The AAP is not a real amphipatic membrane protein, as could be demonstrated by phase separation experiments with Triton X-114.

Pressure-induced reentrant phase behavior in the poly(N-vinyl-2-pyrrolidone)-water system.

Phase separation and dynamic light scattering experiments were performed on aqueous solutions of poly(N-vinyl-2-pyrrolidone) under high pressure conditions to help elucidate the role that hydrogen bonding plays in the solution behavior of water-soluble polymers. From the pressure-induced phase separation experiments we observe reentrant phase behavior in the pressure-temperature plane at fixed composition. Variation of the phase separation pressure with both composition and molecular weight is also investigated. The concentration dependence on the phase separation pressure is weak and the molecular weight dependence exhibits an approximately ${\mathit{M}}_{\mathit{w}}^{\mathrm{\ensuremath{-}}1/2}$ scaling. All this behavior can be explained through a modified Flory-Huggins theory that includes the effect of pressure and temperature on hydrogen bonds and the hydrophobic interactions. We use our data to derive fit parameters for this model and the results suggest that not only does the hydrogen bond weaken at high pressure but hydrophobic interactions increase. Dynamic light scattering (DLS) experiments on solutions within the one-phase region of the phase diagram quantify the influence of changing solvent quality on the polymer's conformation. For dilute solutions, which at ambient temperature and pressure exhibit classic good-solvent behavior, a pressure-induced crossover from good to poor solvent behavior is found. Such measurements are shown to give a measure of ${\mathit{A}}_{2}$, an otherwise difficult to measure property at elevated pressure. Above the overlap concentration, this polymer system exhibits aggregate formation. The autocorrelation spectrum contains two relaxations: a fast relaxation mode associated with the usual cooperative diffusion and a slow relaxation mode associated with internal dynamics of the aggregates. Through DLS experiments in the semidilute regime we show that these two modes merge upon increasing pressure, an effect that is completely reversible. The dispersal of the aggregates under pressure suggests that the aggregates depend strongly on water-mediated hydrogen bonds between monomers. \textcopyright{} 1996 The American Physical Society.

Phase Separation of Mixtures of Colloidal Boehmite Rods and Flexible Polymer

Phase separation experiments have been performed on mixtures of colloidal polyisobutene grafted boehmite rods and flexible polymers. Two systems of rods have been studied, one with an average length and diameter of 71 nm and 11.1 nm, and the other with an average length and diameter of 253 nm and 9.4 nm. The polymers used have molecular weights varying from 35 to 186 kg mol-1, leading to radii of gyration of 5.9 to 20 nm. Bi- and triphasic equilibria involving a dilute isotropic phase, a concentrated isotropic phase, and a nematic phase are observed. The phase behavior is compared to a recent theory, which is an extension of the treatment for sphere/polymer mixtures (H. N. W. Lekkerkerker et al., Europhys. Lett. 20, 559 (1992)). The main features of the phase behavior can be understood on the basis of this simple theory. There remain significant discrepancies between theory and experiment which are attributed to the limitations of the theory on the one hand and to the polydispersity of the colloidal rods and the formation of metastable and nonequilibrium states such as colloidal gels and glasses on the other hand.

Behaviour of differently produced methylalumoxanes in the phase separation with diethyl ether and molecular weight estimations

AbstractCryoscopic molecular weight estimations and phase separation experiments of differently produced methylalumoxanes showed diverse behaviour. The cryoscopic molecular weight estimations showed higher molecular weights caused by increasing methyl conversion. The average molecular weight in benzene lay between 1000 to 2000 while the solvent 1, 4‐dioxane delivered smaller molecular weights (factor 2 to 4). Molecular weights in trimethylaluminium were similar to those in benzene. In phase separation experiments we obtained an increasing volume portion of lower phase with increasing methyl conversion during production. There was a decisive influence of circulation circumstances at the ice surface and the trimethylaluminium concentration at production according to the behaviour in phase separation experiments.

Proposals for structure and effect of methylalumoxane based on mass balances and phase separation experiments

AbstractMethylaluminoxane prepared from trimethylaluminium on a surface of ice mainly can be described as [Al4O3(CH3)6]4. This is shown through analysis, phase separation experiments with diethyl ether and molecular weight determinations in benzene, 1, 4‐dioxane, tetrahydrofuran and trimethylaluminium. It is discussed that the reason for forming this ball‐like structure is the saturation of four coordinated Al4O3(CH3)6. This molecule has a cavern which contains a solvent molecule or a molecule of trimethylaluminium. The consequences for the formation of the catalytically active structure together with metallocene are discussed.

Phase separation of methylalumoxane with diethyl ether

AbstractAfter the addition of diethylether to a solution of methylalumoxane in toluene, phase separation can be observed. The investigation of several phase separation experiments led to an idea of the possible composition of methylalumoxane that could be consisting of a basic element and differently associated trimethylaluminium.Investigations of the system methylalumoxane–diethylether–trimethylaluminium show that diethylether is stronger complexed at methylalumoxane than with trimethylaluminium. A second model of the structure of methylalumoxane allows an attempt to explain the phenomenum of phase separation.

Effects of Taurine and Taurine Analogues on the Phosphorylation of a 44 kDa Protein Present in a Mitochondrial Subfraction of the Rat Heart: Partial Characterization of the 44 kDa Phosphoprotein

Recent studies suggest that taurine (2-aminoethanesulfonic acid) is involved in the regulation of protein phosphorylation in excitable tissues such as the retina, brain and heart. In order to determine the structural requirements for the effect of taurine on the phosphorylation of a 44 kDa protein(s), a series of taurine analogues were tested in an in vitro assay using a subcellular mitochondrial fraction of rat heart. Inhibitors of the phosphorylation of the 44 kDa protein include taurine and close structural analogues of taurine such as aminoethylhydrogen sulfate and α-sulfo-β-alanine. Secondary amines with the taurine structure partially locked into a saturated 5-membered ring such as (±) piperidine-3-sulfonic acid and 1,2,3,4-tetrahydroquinoline-8-sulfonic acid also possess inhibitory activity. Sulfone analogues of taurine such as 2-aminoethylmethylsulfone, a non-restricted taurine analogue with maximal conformational flexibility about its amino and sulfone moieties, and (±) 3-aminotetrahydrothiopyran-1.1-dioxide, an analogue containing the sulfone moiety in a six-membered ring structure, were found to be more potent inhibitors of phosphorylation than taurine despite the fact that the sulfone moiety is neither an isosteric nor isoelectronic substitution for the sulfonic acid moiety. The results of this study indicate that the inhibition of the phosphorylation of the 44 kDa protein in a rat heart mitochondrial fraction is relatively specific for the taurine structure. Two analogues of taurine with unsaturated rings containing a primary sulfonic acid and a secondary amine, pyridine-3-sulfonic acid and quinoline-8-sulfonic acid, were observed to be stimulators of the phosphorylation of the 44 kDa protein. In addition, 2-aminobenzenesulfonic acid also stimulated phosphorylation. Phase separation experiments with Triton X-114 suggest that the 44 kDa phosphoprotein is a soluble protein and not an integral membrane protein of the mitochondria. Phosphate incorporation into specific amino acids was determined by two-dimensional electrophoresis on celluloses plates and was found exclusively in the serine residues.

Lysyl hydroxylase, a collagen processing enzyme, exemplifies a novel class of luminally-oriented peripheral membrane proteins in the endoplasmic reticulum.

Lysyl hydroxylase (LH), an enzyme required early during collagen biosynthesis, appears to be exceptional among proteins that are thought to be residents of the endoplasmic reticulum (ER). It is a homodimer and does not contain either of the two previously characterized ER-specific retention motifs (KDEL or the double lysine motif) in its primary structure. We now show that LH, nevertheless, resides in the lumen of the ER. In immunofluorescence experiments, LH co-localizes with a KDEL-containing protein, protein disulfide isomerase (PDI), and also co-sediments with it after fractionation of subcellular organelles by sucrose density gradient centrifugation. In addition, LH seems to be stress-inducible. In one respect, however, LH differs from PDI and other known luminal proteins in the organelle. It is found in situ only in association with the ER membranes. Our cell fractionation and Triton X-114 phase separation experiments suggest that it binds to the membranes via weak electrostatic interactions. LH can thus be regarded as a first luminally-oriented "peripheral membrane" protein which has been characterized in the ER. The results suggest a novel possibility by which ER lumen can acquire its specific protein components from the bulk flow.