Proton-induced phase separation in phosphatidylserine/phosphatidylcholine membranes

Effects of ph and ionic strength on phosphatidylserine/phosphatidylcholine mixed membranes prepared on Millipore filter pore surfaces have been studied using spin-labeled phosphatidylcholine. Lowering pH at constant ionic strength and lowering ionic strength at constant pH caused a lateral reorganization of the membrane. The trigger was protonation of the serine carboxyl group which caused solidification of phosphatidylserine molecules in the membrane, leaving a fluid phase consisting mainly of phosphatidylcholine. The appearent pK for the proton-induced phase separation was measured in a wide range of salt concentrations. The ionic strength dependence was satisfactorily explained based on the electrostatic free energy of proton in the field of membrane surface potential. The Gouy-Chapman theory gave a good approximation for the surface potential. The surface pK of phosphatidylserine and phosphatidic acid vesicles was directly measured in various salt concentrations by 31P-NMR and the results confirmed validity of the Gouy-Chapman-type analysis. The lateral reorganization was triggered by electrostatic interaction but the bulk of the stabilization energy for the structural changes would be the gains in intermolecular van der Waals energy due to closer packing of phosphatidylserine on solidification.     

Protein partitioning in aqueous two-phase systems composed of a pH-responsive copolymer and poly(ethylene glycol)

The effect of pH and salt concentration on the partitioning behavior of bovine serum albumin (BSA) and cytochrome c in an aqueous two-phase polymer system containing a novel pH-responsive copolymer that mimics the structure of proteins and poly(ethylene glycol) (PEG) was investigated. The two-phase system has low viscosity. Depending on pH and salt concentration, the cytochrome c was found to preferentially partition into the pH-responsive copolymer-rich (bottom) phase under all conditions of pH and salt concentrations considered in the study. This was caused by the attraction between the positively charged protein and negatively charged copolymer. BSA partitioning showed a more complex behavior and partitioned either to the PEG phase or copolymer phase depending on the pH and ionic strength. Extremely high partitioning levels (partition coefficient of 0.004) and very high separation ratios of the two proteins (up to 48) were recorded in the new systems. This was attributed to strong electrostatic interactions between the proteins and the charged copolymer.     

Incorporation of sulfonylurea into N-isopropylacrylamide as an extracellular matrix for an artificial pancreas.

High-molecular-weight N-isopropylacrylamide copolymers with small amounts of sulfonylurea (SU, typically 2-4 mol% in the feed) were synthesized by free radical polymerization in benzene. SU-incorporated polymer solutions (5, 6, 8, and 10% w/v) in a culture medium (pH 7.4, 0.15 M ionic strength) with islet cells were mixed and poured into Millicells which supported gel formation. In order to increase the gelation temperature, the SU-incorporated copolymer gel, p(NiPAAm-co-SU), was blended with the p(NiPAAm-co-AAc) polymer at a ratio of 4 to 96. Interaction between the islet cells and the synthetic matrix of SU-incorporated copolymer gel resulted in effective cell viability and such cell functions as insulin secretion. To verify the specific interaction between the SU (K+ channel closer)-incorporated copolymer and islet cells, the cells were pretreated with diazoxide, an agonist of the ATP-sensitive K+ channel (K+ channel opener), before interaction between the polymer and islet cells. This treatment suppressed the action of SU on the islet cells. The results from this study provide evidence that the SU-incorporated copolymer stimulated insulin secretion by specific interaction between SU moieties in the polymer and the islet cells.     

Preparation of an immobilized two-enzyme system, beta-amylase-pullulanase, to an acrylic copolymer for the conversion of starch to copolymer for the conversion of starch to maltose. I. Preparation and stability of immobilized beta-amylase

β-Amylase (EC 3.2.1.2), obtained from barley, was chemically attached to a crosslinked copolymer of acrylamide-acrylic acid using a water-soluble carbodiimide. The derivative showed 23% β-amylase activity in relation to that of free enzyme with a coupling yield of 40% based on the amount of added β-amylase. In order to find optimal coupling conditions, the effect of pH and different carbodiimide concentrations was investigated. The enzymic activity associated with different β-amylase concentrations was further outlined. A slightly increased operational stability for the enzyme upon immobilization was observed. Markedly improved operational stability has been obtained by coupling in the presence of reduced glutathione of bovine serum albumin.     

Incorporation of Sulfonylurea into N-Isopropylacrylamide as an Extracellular Matrix for an Artificial Pancreas

High-molecular-weight N-isopropylacrylamide copolymers with small amounts of sulfonylurea (SU, typically 2-4 mol% in the feed) were synthesized by free radical polymerization in benzene. SU-incorporated polymer solutions (5, 6, 8, and 10% w/v) in a culture medium (pH 7.4, 0.15 M ionic strength) with islet cells were mixed and poured into Millicells which supported gel formation. In order to increase the gelation temperature, the SU-incorporated copolymer gel, p(NiPAAm-co-SU), was blended with the p(NiPAAm-co-AAc) polymer at a ratio of 4 to 96. Interaction between the islet cells and the synthetic matrix of SU-incorporated copolymer gel resulted in effective cell viability and such cell functions as insulin secretion. To verify the specific interaction between the SU (K⁺ channel closer)-incorporated copolymer and islet cells, the cells were pretreated with diazoxide, an agonist of the ATP-sensitive K⁺ channel (K⁺ channel opener), before interaction between the polymer and islet cells. This treatment suppressed the action of SU on the islet cells. The results from this study provide evidence that the SU-incorporated copolymer stimulated insulin secretion by specific interaction between SU moieties in the polymer and the islet cells.     

Conjugation of Arg-Gly-Asp sequence into thermo-reversible gel as an extracellular matrix for 3T3-L1 fibroblast cell culture

High molecular weight N-isopropylacrylamide copolymers with small amounts of acrylic acid (typically 2–5 mol% in feed) were synthesized by free radical polymerization in benzene and then conjugated with adhesion molecules of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides. Aqueous polymer solutions (5, 6, 8 and 10% w/v) in culture medium (pH 7.4, ionic strength; 0.15 M) with 3T3-L1 fibroblast cells were mixed and poured in Millicells, which supported the gel formation without a significant gel induction time at 36 °C (gelation temperature). The initially formed gel was translucent and became more opaque as the temperature increased. The interaction between fibroblast cells and an artificial matrix of GRGDS containing p(NiPAAm-co-AAc) copolymer gel resulted in effective cell attachment, proliferation and growth. This study supported that specific attachment is the result of the interaction between the integrin families on the fibroblast and the RGD sequence on the p(NiPAAm-co-AAc) copolymer gel.     

Synthesis and electron microscopic investigation of model polyacryloynucleosides

Using the interaction of polyacrylic anhydride with uridine or N-acetyl derivatives of adenosine, cytidine, and guanosine, the water-soluble copolymers, polyacryloylnucleosides, were obtained. The acryloylnucleoside units to acrylic acid units ratio in the copolymer was usually about 1:20. The attaching of nucleosides to the polymer occurs mostly through the 5′-hydroxyl group of the sugar. The prominent feature of all polyacryloylnucleosides obtained is their fibrous structure at an ionic strength 0.2 and neutral pH. At concentrations <200 μg/ml the separate strands with a length of 0.2–1.0 μ and diameter of 30–40 Å are distinguishable. Evidently they are formed by side association of two molecules of copolymer or by folding of one molecule on itself. At higher concentrations branched multistranded structures are formed. In the same conditions polyacrylic acid alone does not form the fibrous structures. Heating of polyacryloylnucleoside solutions at 100°C and fast cooling in ice water, or raising of the pH to 12 turned the stranded structures to coils. After annealing or neutralization the stranded structures reformed. These transformations are similar to those which occur with nucleic acids. The results show that the fibrous structure of the copolymers depends on the hydrogen bonds formed by purine and/or pyrimidine bases.     

Adsorption of alcohol from water by poly(ionic liquid)s

Bioethanol is used widely as a solvent and is considered a potential liquid fuel. Ethanol can be produced from biomass by fermentation, which results in low concentrations of alcohol in water. Conventional distillation is normally used to separate ethanol from water, but it required high energy consumption. Therefore, alternative approaches to this separation are being pursued. This study examined the potential use of poly(ionic liquid)s (PILs) for the extraction and separation of alcohols from the aqueous phase. Hydrophobic PILs were developed and evaluated by the adsorption of ethanol from ethanol/water solutions. All the necessary parameters, such as cations and anions of the ionic liquid, morphology of the polymer and processing conditions, were evaluated.     

Poly(N-isopropylacrylamide-co-propylacrylic acid) copolymers that respond sharply to temperature and pH

Temperature- and pH-sensitive random copolymers of N-isopropylacrylamide (NIPAAm) and propylacrylic acid (PAA) were prepared using the reversible addition fragmentation chain transfer (RAFT) polymerization method. The lower critical solution temperatures (LCSTs) (or phase separation temperatures) of the NIPAAm-co-PAA copolymer solutions were measured by the cloud-point method. At slightly acidic conditions, the LCST decreased with increase in PAA content, which suggests that the hydrophobic propyl group of PAA has a greater influence on the LCST than the polar carboxylic acid group at those conditions. An increase of pH led to a significant increase in LCST of the copolymers due to the ionization of the -COOH group. The LCSTs were studied as a function of copolymer composition over the pH range from 5.0 to 7.0. Because the pK(a) of the polymers can be tuned to fall close to neutral pH, these polymer compositions can be designed to have phase transitions triggered near physiological pH or at slightly acidic pH values that fall within acidic gradients found in biology. The NIPAAm-co-PAA copolymers thus display tunable properties that could make them useful in a variety of molecular switching and drug delivery applications where responses to small pH changes are relevant.     

Protein salting-out: phase equilibria in two-protein systems

The phase behavior of two aqueous binary protein mixtures, lysozyme-chymotrypsin and lysozyme-ovalbumin, was determined in ammonium sulfate solutions. Protein concentrations were determined in both phases as a function of pH and ionic strength. For lysozyme-chymotrypsin mixtures, the observed phase behavior was similar to that for each individual protein; the presence of the second protein had little influence. The phase behavior of lysozyme-ovalbumin mixtures, however, was different from that of the respective single-protein systems. Lysozyme and ovalbumin are found together in egg whites; their association is both pH and ionic-strength dependent. The association of proteins is a key determinant of protein solubility in salt solutions. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 567-574, 1997.     

Ionic strength dependence of the hysteresis in the polyriboadenylate-polyribouridylate system.

The hysteresis observed in cyclic acid-base titrations of the three-standed polyribonucleotide helix poly (A)-2 POLY (U) strongly depends on ionic strength. For NaCl and at 25 degrees C, hysteresis occurs in the limited concentration range between 0.03 M and 1.0 M(NaCl). The transition points associated with the cyclic conversions between the triple helix and the poly (A)-poly (A) double helix and (free) poly (U) constitute a (pH ionic strength) phase diagram covering the ranges of stability and metastability of the hysteresis system. Variations with NaCl concentration of some hysteresis parameters can be quantitatively described in terms of polyelectrolyte theories based on the cylinder-cell model for rodlike polyions. The results of this analysis suggest that the metastability is predominantly due to dlectrostatic energy barriers preventing the equilibrium transition of the partially protonated triple helix above a critical pH value. Ultraviolet absorbance and potentiometric titration data of poly (A)in the acidic pH range can be analyzed in terms of two types of double-helical structures. Spectrophotometric titrations reveal isosbestic wavelengths for structural transitions of poly (A). "Time effects" commonly observed in poly (A) titrations are suggested to reflect helix transitions between the two acidic structures.     

Effects of polyols, saccharides, and glycoproteins on thermoprecipitation of phenylboronate-containing copolymers

The copolymer of 3-(acrylamido)phenylboronic acid and N-isopropylacrylamide (82:18, Mn = 47000 g/mol) was prepared by free radical polymerization. The copolymer showed typical thermoprecipitation behavior in aqueous solutions; its phase transition temperature (TP) was 26.5 +/- 0.2 degrees C in 0.1 M glycine-NaOH buffer containing 0.1 M NaCl, pH 9.2. Due to specific complex formation of the pendant boronates with sugars, TP was strongly affected by the type of sugar and its concentration at pH 9.2. Fructose, lactulose, and glucose caused the largest increase in TP (up to 4 degrees C) at 0.56 mM concentration, attributed to the high binding affinity of the sugars to borate and phenylboronate. Among the sugars typical of nonreducing ends of oligosaccharides, N-acetylneuraminic acid had the strongest effect on TP (ca. 2 degrees C at 0.56 mM concentration and pH 9.2), while the effects of other sugars are well expressed at the higher concentrations (16 and 80 mM) and decreased in the order xylose approximately galactose >or= N-acetyllactosamine >or= mannose approximately fucose > N-acetylglucosamine. The effect exerted on the phase transition by glycoproteins was the strongest with mucin from porcine stomach and decreased in the series mucin > horseradish peroxidase > human gamma-globulin at pH 9.2. As a first approximation, the weight percentage and/or the number of oligosaccharides in glycoproteins determined the character of their interaction with the pendant phenylboronates and, therefore, the effect on the copolymer phase transition.     

AQUEOUS POLYMER TWO-PHASE SYSTEMS AND THEIR USE IN FRAGMENTATION AND SEPARATION OF BIOLOGICAL MEMBRANES FOR THE PURPOSE OF MAPPING THE MEMBRANE STRUCTURE

When solutions of two different polymers are mixed, phase separation often occurs even at low concentrations of polymers. One polymer usually collects in one phase and the other polymer in the other phase. When water is used as solvent, two aqueous, immiscible, phases are obtained. The same holds for aqueous mixtures of a salt and a polymer. Such aqueous two-phase systems (ATPS) are very useful for separation of high-molecular-weight biomolecules such as proteins and nucleic acids and also for cells, cell organelles, and membrane vesicles. The phase systems can be made highly selective and they are also mild toward biomolecules and cell particles. In this review we describe how ATPS can be used for fragmentation and separation analyses of biological membranes and how this can be used for mapping of the photosynthetic membrane, the thylakoid, of green leaves.     

Standard thermodynamic formation properties for the adenosine 5'-triphosphate series.

The criterion for chemical equilibrium at specified temperature, pressure, pH, concentration of free magnesium ion, and ionic strength is the transformed Gibbs energy, which can be calculated from the Gibbs energy. The apparent equilibrium constant (written in terms of the total concentrations of reactants like adenosine 5'-triphosphate, rather than in terms of species) yields the standard transformed Gibbs energy of reaction, and the effect of temperature on the apparent equilibrium constant at specified pressure, pH, concentration of free magnesium ion, and ionic strength yields the standard transformed enthalpy of reaction. From the apparent equilibrium constants and standard transformed enthalpies of reaction that have been measured in the adenosine 5'-triphosphate series and the dissociation constants of the weak acids and magnesium complexes involved, it is possible to calculate standard Gibbs energies of formation and standard enthalpies of formation of the species involved at zero ionic strength. This requires the convention that the standard Gibbs energy of formation and standard enthalpy of formation for adenosine in dilute aqueous solutions be set equal to zero. On the basis of this convention, standard transformed Gibbs energies of formation and standard transformed enthalpies of formation of adenosine 5'-trisphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate, and adenosine at 298.15 K, 1 bar, pH = 7, a concentration of free magnesium ions of 10(-3) M, and an ionic strength of 0.25 M have been calculated.     

Water-soluble complexes through coulombic interactions between bovine serum albumin and anionic polyelectrolytes grafted with hydrophilic nonionic side chains

The interaction between bovine serum albumin (BSA) and the anionic graft copolymers poly(sodium acrylate-co-sodium 2-acrylamido-2-methyl-1-propanesulfonate)-graft-poly(N,N-dimethylacrylamide) (P(NaA-co-NaAMPS)-g-PDMAMx) was investigated within the acid pH region, 2 < or = pH < or = 7. The weight percentage, x, of the poly(N,N-dimethylacrylamide) (PDMAM) side chains varied from 0 up to 75% (w:w). When BSA and P(NaA-co-NaAMPS)-g-PDMAMx are oppositely charged, i.e., when pH is lower than the isoelectric point of BSA, the two macromolecules associate through Coulombic attractions. When the anionic graft copolymer is rich enough to the nonionic PDMAM side chains, x > or = 50% w:w, the associative phase separation is practically prevented, as revealed by the turbidimetric study of the BSA/P(NaA-co-NaAMPS)-g-PDMAMx mixtures in aqueous solution vs pH. In addition, the viscosity measurements support the formation through a charge neutralization process of a rather compact protein-polyelectrolyte complex stabilized by the hydrophilic PDMAM side chains grafted onto the anionic copolymer backbone.     

pK-matched running buffers for gel electrophoresis.

Electrophoresis through agarose and polyacrylamide-type gels is the standard method to separate, identify, and purify nucleic acids. Properties of electrophoresis buffers such as pH, ionic strength, and composition affect performance. The buffers in use contain a weak acid or weak base buffered by a compound with a dissimilar pK. Herein, three pK-matched buffers were developed, each containing two effective buffering components: one weak base and one weak acid which have similar pKa at 25 degrees C (within 0.3 pK units): (i) Ethanolamine/Capso, pH 9.6; (ii) triethanolamine/Tricine, pH 7.9; and (iii) Bis-Tris/Aces, pH 6.7. On agarose gels, the buffers in various concentrations were tested for separation of double-stranded DNA fragments with various DNA markers, agarose gel concentrations, and field strengths. Mobility was inversely proportional to the logarithm of molecular weight. The buffers provided high resolution without smearing at more dilute concentration than is possible with standard TAE (Tris/acetate, pH 8.0) or TBE (Tris/borate, pH 8.3) buffers. The buffers were also tested in 7 M urea denaturing LongRanger sequencing gels and in nondenaturing polyacrylamide SSCP gels. The pK-matched buffers provide good separation and high resolution, at a broad range of potential pH values. In comparison to TAE and TBE, pK-matched buffers provide higher voltage and current stability, lower working concentration, more concentrated stock solutions (up to 200x), and lower current per unit voltage, resulting in less heat generation.     

Sol-gel transition temperature of PLGA-g-PEG aqueous solutions

Aqueous solutions of poly(DL-lactic acid-co-glycolic acid)-g-poly(ethylene glycol) copolymers exhibited sol-to-gel transition with increasing temperature. Further increase in temperature makes the system flow and form a sol phase again. Subcutaneous injection of a copolymer aqueous solution (0.5 mL) resulted in a formation of a hydrogel depot by temperature-sensitive sol-to-gel transition in a rat model. The reliable determination and control of sol-to-gel transition temperatures are the most important issues for this kind of sol-gel reversible hydrogel. The sol-to-gel transition temperature determined by the test tube inverting method, falling ball method, and dynamic mechanical analysis coincided within 1-2 degrees C. Fine tuning of the sol-to-gel transition temperature was achieved by varying the ionic strength of the polymer solutions and by mixing two polymer aqueous solutions with different sol-to-gel transition temperatures. The sol-to-gel transition temperature of polymer mixture aqueous solutions was well described by an empirical equation of miscible blends, indicating miscibility of the two polymer systems in water on the molecular level.     

Physical gelation of chitosan in the presence of beta-glycerophosphate: the effect of temperature

When adding beta-glycerophosphate (beta-GP), a weak base, to chitosan aqueous solutions, the polymer remains in solution at neutral pH and room temperature, while homogeneous gelation of this system can be triggered upon heating. It is therefore one of the rare true physical chitosan hydrogels. In this study, physicochemical and rheological properties of chitosan solutions in the presence of acetic acid and beta-GP were investigated as a function of temperature in order to gain a better understanding of the gelation mechanisms. The gel structure formed at high temperature was only partially thermoreversible upon cooling to 5 degrees C because of the existence of remaining associations, confirmed by the spontaneous recovery of the gel after breakup at low temperature. Increasing temperature had no effect on the pH values of this system, while conductivity (and calculated ionic strength) increased. Values from the pH measurements were used to estimate the degree of protonation of each species as a function of temperature. The decreasing ratio of -NH3+ in chitosan and -OPO(O-)2 in beta-GP suggested reduced chitosan solubility along with a diminution of ionic interactions such as ionic bridging with increasing temperature. On the other hand, the increased ionic strength as a function of temperature, in the presence of beta-GP, enhanced screening of electrostatic repulsion and increased hydrophobic effect, resulting in favorable conditions for gel formation. Therefore, our study suggests that hydrophobic interactions and reduced solubility are the main driving force for chitosan gelation at high temperature in the presence of beta-GP.     

Tuning supramolecular structuring at the nanoscale level: nonstoichiometric soluble complexes in dilute mixed solutions of alginate and lactose-modified chitosan (chitlac)

Two oppositely charged polysaccharides, alginate and a lactose-modified chitosan (chitlac), have been used to prepare dilute binary polymer mixtures at physiological pH (7.4). Because of the negative charge on the former polysaccharide and the positive charge on the latter, polyanion-polycation complex formation occurred. A complete miscibility between the two polysaccharides was attained in the presence of both high (0.15 M) and low (0.015 M) concentrations of simple 1:1 supporting salt (NaCl), as confirmed by turbidity measurements; phase separation occurred for intermediate values of the ionic strength (I). The binary solutions were further characterized by means of light scattering, specific viscosity, and fluorescence quenching measurements. All of these techniques pointed out the fundamental role of the electrostatic interactions between the two oppositely charged polysaccharides in the formation of nonstoichiometric polyelectrolyte soluble complexes in dilute solution. Fluorescence depolarization (P) experiments showed that the alginate chain rotational mobility was impaired by the presence of the cationic polysaccharide when 0.015 M NaCl was used. Moreover, upon addition of calcium, the P values of the binary polymer mixture in 0.015 M NaCl increased more rapidly than that of an alginate solution without chitlac, suggesting an efficient crowding of the negatively charged alginate chains caused by the polycation.     

两价盐对C_8－卵磷脂微团溶液液－液相分离的影响

采用激光散射等方法测定了在添加不同的两价无机盐情况下,Ｃ８－卵磷脂微团溶液的液－液相分离曲线,及其相变临界温度随盐类型和盐离子强度的变化。并从理论上分析了两价盐对Ｃ８－卵磷脂微团溶液吉布斯自由能的影响,推导出－关于盐对该微团溶液相交临界温度影响的半经验半理论公式,可满意地描述该微团溶液的液－液相分离受益调控的规律。