Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems

The partition of hemoglobin, lysozyme and glucose-6-phosphate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na(2)SO(4), pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively.     

Affinity partitioning of erythrocytic phosphofructokinase in aqueous two-phase systems containing poly(ethylene glycol)-bound cibacron blue. Influence of pH, ionic strength and substrates/effectors.

Phosphofructokinase (PFK) from rat erythrocyte haemolysates has a high affinity for Cibacron Blue F3G-A covalently bound to poly(ethylene glycol) (PEG-Cb) and thus the enzyme can be extracted into the top phase of poly(ethylene glycol)-dextran aqueous two-phase systems containing PEG-Cb. The pH, ionic strength and presence of substrates/effectors affect to different extents the affinity of the enzyme for PEG-Cb and the number of PEG-Cb molecules attached per molecule of PFK (the latter probably reflecting, at saturation, the influence on the aggregation state of the enzyme) and thus influence the yield of enzyme recovered in the top phase. Increasing the pH from 6 to 7 and then to 8 leads to a higher yield of PFK in the top phase. A change in pH from 6 to 7 and 8 results in an increased number of PEG-Cb molecules attached per molecule of enzyme while the affinity of PFK for PEG-Cb shows a minimum at pH 7. The ionic strength in the range 0.017-0.164 has less influence on the partitioning of PFK. The presence of substrates or effectors of the enzyme in general reduces the recovery of PFK in the top phase. Fructose 6-phosphate increases the number of PEG-Cb molecules attached but greatly reduces the affinity of PFK for PEG-Cb. In contrast, AMP slightly reduces the number of PEG-Cb molecules attached and the affinity of PFK for PEG-Cb. ATP and ATP-Mg2+ compete with PEG-Cb for the same binding sites in PFK.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase separation in aqueous two-phase systems containing poly(ethylene glycol) and magnesium sulphate at different temperatures

New experimental (liquid + liquid) equilibrium data have been determined for aqueous systems containing poly(ethylene glycol) of nominal molar mass 10,000 and magnesium sulphate at T = (295.15, 301.15, 305.15, and 311.15) K. The effect of temperature on the liquid compositions of coexisting phases is discussed. The experimental (liquid + liquid) equilibrium data of the systems were correlated by non-random two-liquid (NRTL) activity coefficient model. The interaction parameters of the NRTL activity coefficient model are obtained and reported. The calculated root mean square deviations (RMSD) showed that NRTL activity coefficient model can be used satisfactorily to correlate the (liquid + liquid) equilibrium data in aqueous solution of the {poly(ethylene glycol) + magnesium sulphate} system.     

Extraction mechanism of sulfamethoxazole in water samples using aqueous two-phase systems of poly(propylene glycol) and salt

Based on the poly(propylene glycol)₄₀₀ (PPG₄₀₀)–salt aqueous two-phase system (ATPS), a green, economical and effective sample pretreatment technique coupled with high performance liquid chromatography was proposed for the separation and determination of sulfamethoxazole (SMX). The extraction yield of SMX in PPG₄₀₀–salt ATPS is influenced by various factors, including the salt species, the amount of salt, pH, and the temperature. Under the optimum conditions, most of SMX was partitioning into the polymer-rich phase with the average extraction efficiency of 99.2%, which may be attributed to the hydrophobic interaction and salting-out effect. This extraction technique has been successfully applied to the analysis of SMX in real water samples with the recoveries of 96.0–100.6%, the detection limits of 0.1 μg L⁻¹, and the linear ranges of 2.5–250.0 μg L⁻¹.     

Partition of macromolecules and cell particles in aqueous two-phase systems based on hydroxypropyl starch and poly(ethylene glycol)

The partition behavior of proteins, nucleic acids, cell membranes, cell organelles and whole cells has been studied in liquid-liquid two-phase systems composed of water, poly-(ethylene glycol), and an hydroxypropyl starch. The properties of the systems are in many respects comparable with the traditional poly(ethylene glycol)-dextran systems, but the cost is reduced to around one-fifth.     

Determination of glass transition temperature of poly(ethylene glycol) by spin probe technique

The rotational relaxation data for free nitroxyl radicals in poly(ethylene glycol) samples of mol. wt 3000-22,000 gave for the glass-transition temperature (T_p) a value of ?60°, independent of molecular weight. The rotational activation energy was ～ 2 kJ/mole below T_g and ～ 10 kJ/mole above T_g. This indicated that the mechanism of motion below T_g differs from the above T_g. Evidently the rotating units of polymer are much smaller below T_g than above it. The rotational relaxation time (T) was found to be dependent on the molar volume of PEG (V) and to follow the empirical equations t = A exp (?kV₁) and t = B exp (?kV₁) where V_a and V₁ are the molar volumes (cm³/mol) when T < T_g and T > T_g respectively, and k ～ 1. Therefore the defects in which radicals are located are perhaps the dominant factor determining the dynamic state of probe radicals in polymers at low temperatures.     

A structural transition in duplex DNA induced by ethylene glycol

The twist energy parameter ( E T) that governs the supercoiling free energy, and the linking difference (Delta l) are measured for p30delta DNA in solutions containing 0-40 w/v % ethylene glycol (EG). A plot of E T vs -ln a w, where a w is the water activity, displays the full (reverse) sigmoidal profile of a discrete structural transition. A general theory for the effect of added osmolyte on a cooperative structural transition between two duplex states, 1 right arrow over left arrow 2, is formulated in terms of parameters applicable to individual base-pair subunits. The resulting fraction of base pairs in the 2-state ( f 2 (0)) is incorporated into expressions for the effective torsion and bending elastic constants, the effective twist energy parameter ( E T (eff)), and the change in intrinsic twist (delta l 0). Fitting the expression for E T (eff) to the measured E T values yields reasonably unambiguous estimates of E T 1 and E T 2 , the midpoint value (ln a w) 1/2, and the midpoint slope ( partial differential E T/ partial differential ln a w) 1/2, but does not yield unambiguous estimates of the equilibrium constant ( K 0), the difference in DNA-water preferential interaction coefficient (DeltaGamma), or the inverse cooperativity parameter ( J). Fitting a noncooperative model (assumed J = 1.0) to the data yields K 0 = 0.067 and DeltaGamma = -30.0 per base pair (bp). Essentially equivalent fits are provided by models with a wide range of correlated J, DeltaGamma, and K 0 values. Other results favor DeltaGamma in the range -1.0 to 0, which then requires K 0 > or = 0.914, and a cooperativity parameter, 1/ J > or = 30.0 bp. The measured delta l 0 and circular dichroism (CD) at 272 nm are found to be compatible with curves predicted using the same f 2 (0) values that best-fit the E T data. At least 7-10% of the base pairs are inferred to exist in the 2-state in 0.1 M NaCl in the complete absence of added osmolyte. Compared with the 1-state, the 2-state has a approximately 2.0- to 2.1-fold greater torsion elastic constant, a approximately 0.70-fold smaller bending elastic constant, a approximately 0.91-fold smaller E T value, a approximately 0.2% lower intrinsic twist, a somewhat lower CD near both 272 and 245 nm, and less water and/or more EG in its neighborhood. However, the relative change in preferential interaction coefficient associated with the transition is likely rather slight.     

Metal cation induced cubic phase in poly(ethylene glycol)-functionalized dioleoylphosphatidylethanolamine aqueous dispersions

Metal cations (Mn(2+) or Ca(2+)) in aqueous dispersions of mixtures of dioleoylphosphatidylethanolamine (DOPE) and poly(ethylene glycol)-functionalized DOPE (DOPE-PEG(350)) induce, above a certain amount of the PEG lipid component, a phase transition from the inverted hexagonal phase H(II) to the bicontinuous inverted cubic phase Q(224) with space group Pn3m. The process is driven by the decrease of free elastic energy due to the Gaussian curvature of the cubic phase. The structural characterization of the phase behavior over the whole explored range of DOPE-PEG/DOPE weight ratio (3-25%) is reported, focusing on the role of the metal cation in the formation of the 3D cubic lattice. This result may represent a significant progress toward a design-based approach to drug delivery.     

Partitioning of l-methionine in aqueous two-phase systems containing poly(propylene glycol) and sodium phosphate salts

The partitioning behavior of l-methionine has been studied in aqueous two-phase systems of (poly(propylene glycol) + sodium phosphate salts + H₂O) at different temperatures. The salts used were sodium di-hydrogen phosphate (NaH₂PO₄), di-sodium hydrogen phosphate (Na₂HPO₄) and tri-sodium phosphate (Na₃PO₄). The effects of tie line length, salt type, and temperature on the partition coefficient of this amino acid have been studied. In addition, thermodynamic parameters (ΔH°, ΔS° and ΔG°) as a function of temperature were calculated. The results showed that increasing tie line length led to decreasing of the partition coefficient. We also showed that the partition coefficients of the amino acid in the systems containing Na₃PO₄ are greater than the other two salts. Moreover, it is verified that increasing temperature led to decreasing the partition coefficient. The experimental partition coefficient data are correlated using a modified virial-type model.     

Subcellular distribution of rat pituitary homogenates by poly(ethylene glycol)-dextran countercurrent partitioning

Rat pituitary homogenates were subjected to two phase countercurrent partition in a poly(ethylene glycol)-dextran mixture using a simple apparatus with enhanced gravity to facilitate the phase separations. Assay of the fractions for organelle marker enzymes and prolactin after 17 transfers showed similar distributions for endoplasmic reticulum, lysosomes, prolactin granules and plasma membrane at the lowest dextran concentrations. Increasing the dextran concentrations had a differential effect on the various organelles. Excellent resolution of endoplasmic reticulum from the other organelles was obtained and marked organelle heterogeneity was demonstrated. Two-phase countercurrent partition thus offers an alternative approach to the subcellular fractionation of pituitary homogenates and should prove useful in separating endoplasmic reticulum from plasma membrane and other cell components.     

Solubility of amino acids in aqueous poly (ethylene glycol) solutions

The solubilities of amino acids have been measured in water and aqueous poly(ethylene glycol) (PEG) solutions as a function of temperature and PEG concentration. The free energies of transfer from water to aqueous PEG solutions forl-alanine,l-valine,l-isoleucine andl-leucine were positive, while those forl-phenylalanine andl-tryptophan were negative. The corresponding enthalpies of transfer were almost zero for all amino acids. The equilibrium constants of the binding of amino acids to PEG chain were estimated from the solubility data. Amino acids with larger hydrophobicity are bound more strongly to the PEG chain due to the hydrophobic interaction between the methylene groups of PEG and the side chain of amino acid. The equilibrium constants showed a correlation with the dynamic hydration number (n _DHN) which expresses the hydration properties of amino acids in aqueous solution.     

Study of the self‐diffusion of poly(ethylene glycol)s in poly(vinyl alcohol) aqueous systems

We have measured the self‐diffusion coefficients of a series of oligo‐ and poly(ethylene glycol)s with molecular weights ranging from 150 to 10,000, in aqueous solutions and gels of poly(vinyl alcohol) (PVA), using the pulsed‐gradient spin‐echo NMR techniques. The PVA concentrations varied from 0 to 0.38 g/mL which ranged from dilute solutions to polymer gels. Effects of the diffusant size and polymer concentration on the self‐diffusion coefficients have been investigated. The temperature dependence of the self‐diffusion coefficients has also been studied for poly(ethylene glycol)s with molecular weights of 600 and 2,000. Several theoretical models based on different physical concepts are used to fit the experimental data. The suitability of these models in the interpretation of the self‐diffusion data is discussed. © 1999 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 37: 2396–2403, 1999     

Extraction of humic acids and their fractions in poly(ethylene glycol)-based aqueous biphasic systems

The possibility of extraction and fractionation of humic acid (HA) in the aqueous biphasic systems (ABS) was shown for the first time. The 10% PEG-10% (NH₄)₂SO₄-H₂O and 5% PEG-7.5% dextran (or dextran sulphate, sodium salt)-H₂O systems were used. HA originated from peat, soddy-podzolic soil and chernozem were studied. The distribution coefficients were measured for HA of different origin, size of the molecules, molecular weights (MW) of the polymers and pH of the system. The PEG-(NH₄)₂SO₄-H₂O system was found to be better for preconcentration and isolation of HA, whereas the PEG-dextran-H₂O system is preferable for HA fractionation. The extractability of HA in ABS increases with increasing the MW of HA molecules. Peat HA are extracted in ABS with higher distribution coefficients compared with chernozem and soddy-podzolic soil HA. It is consistent with higher hydrophobicity of peat HA revealed by hydrophobic interaction chromatography. ABS are promising for HA separation into fractions that differ in their hydrophobic/hydrophilic properties as well as for comparing HA of different origin by their relative hydrophobicity.     

Erythrocyte partitioning in dextran-poly(ethylene glycol) aqueous phase systems. Events in phase and cell separation.

Early events in the partitioning process which involve characteristic kinetics of cell- and phase-specific interactions and phase separation have been described previously. This paper reports on red cell-phase droplet interactions pertaining at the time of usual phase sampling (i.e., the time at which a clear bulk interface is first apparent) and beyond in cell partitioning and countercurrent distribution experiments. In non-charge-sensitive phase systems close to the critical point, cells can be free or attached to phase droplets. Cells that are free are virtually completely in the top phase, whereas different cell populations that show essentially complete binding to droplets can nevertheless have different partition ratios and be separated, thus reflecting the effects of the difference in the cells' avidity for the phase droplets during the early, elapsed events in partitioning. At higher polymer concentrations (i.e., higher interfacial tensions), the cell populations, completely bound to phase droplets, partition completely to the interface, and consequently cannot be separated. When such systems are made charge-sensitive by the generation of a Donnan potential between the phases or made into affinity systems by the incorporation of PEG ligands (e.g., PEG-palmitate), there is a decrease in the avidity of the cells for phase droplets. The resulting increase in the ratio of free to droplet-bound red cells in the top phase at the time of sampling correlates with an increase in the partition ratio, P, observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isotherms of poly(ethylene glycol) adsorption from aqueous solutions by carbon adsorbents

Isotherms of adsorption of poly(ethylene glycols) PEG300, PEG6000, and PEG15 000 from aqueous solutions on graphitized carbon black and active carbon were measured at 25°C. Isotherms of excess adsorption of PEG6000 and PEG15 000 from aqueous solutions on both adsorbents were characterized by a distinct maximum. Comparison of adsorption isotherms for PEG6000 and PEG15 000 on a mesoporous AU-87 carbon testified to the existence of a sieve effect upon the adsorption of large PEG15 000 macromolecules in the accessible pores of this adsorbent. The analysis of the dependence of maximal adsorption values on the PEG molecular mass indicated a possible unfolding of macromolecular coils in the field of adsorption forces. As a result, all oxygen and carbon atoms of PEG macromolecules in a monolayer tend to approach active sites on the surface of a carbon adsorbent. The calculated values of thickness of dense adsorption monolayers of PEG300, PEG6000, and PEG15 000 lie within 0.36–0.63 nm for both adsorbents. PEG300 adsorption monolayers also contain, in addition to macromolecules, molecules of a solvent (water).Translated from Kolloidnyi Zhurnal, Vol. 66, No. 6, 2004, pp. 856–859.Original Russian Text Copyright © 2004 by Eltekov, Eltekova, Roldughin.     

Differential scanning calorimetric study of phase changes in poly(ethylene glycol) dodecyl ether-water systems

Phase changes in binary systems of poly(ethylene glycol) dodecyl ethers, C₁₂H₂₅(OC₂H₄)_xOH (x=5, 6 and 8) with water have been studied between –40 to 100 C by differential scanning calorimetry. A number of transitions, including the liquidliquid phase separation, were seen and the transition temperatures and enthalpy changes were determined. The observed temperatures were generally in good agreement with reported phase diagrams. In the C₁₂E₅ system, three of the four three-phase lines were seen and a more complete phase diagram is suggested for the water-rich part of the system. Most of the phase changes seen above 0 C are accompanied by small endothermic enthalpy changes of 0.7 to 0.9 kJ (mol C₁₂E_x)^–1, independent of system studied, type of transition and transition temperature. Water-rich solutions and liquid crystalline phases separate upon freezing into ice and crystals of hydrated amphiphile. Eutectics are developed at the following temperatures and compositions: C₁₂E₅ –3.0 C and 54 wt % C₁₂E₅; Q₂E₆ –4.5 C and 54 wt % C₁₂E₆, C₁₂E₈ –3.8 C and 49 wt % C₁₂E₈.     

Thermogelling hydrogels of poly(ε-caprolactone-co-D,L-lactide)–poly(ethylene glycol)–poly(ε-caprolactone-co-D,L-lactide) and poly(ε-caprolactone-co-L-lactide)–poly(ethylene glycol)–poly(ε-caprolactone-co-L-lactide) aqueous solutions

Thermogelling poly(ε-caprolactone-co-D,L -lactide)–poly(ethylene glycol)–poly(ε-caprolactone-co-D,L -lactide) and poly(ε-caprolactone-co-L -lactide)–poly(ethylene glycol)–poly(ε-caprolactone-co-L -lactide) triblock copolymers were synthesized through the ring-opening polymerization of ε-caprolactone and D,L -lactide or L -lactide in the presence of poly(ethylene glycol). The polymerization reaction was carried out in 1,3,5-trimethylbenzene with Sn(Oct)₂ as the catalyst at various temperatures, and the yields were about 96%. The molecular weights and polydispersities (M_w/M_n) by gel permeation chromatography were in the ranges of 5140–6750 and 1.35–1.45, respectively. The differential scanning calorimetry results showed that the melting temperatures of the poly(ε-caprolactone) components were between 30 and 40 °C. By the subtle tuning of the chemical compositions and microstructures of these triblock copolymers, the aqueous solutions underwent sol–gel transitions as the temperature increased, with the suitable lower critical solution temperature in the range of 17–28 °C at different concentrations. Transesterification in the polymerization process generated the redistribution of sequences, which remarkably affected the sol–gel transition temperature. The amphiphilic copolymers formed micelles in aqueous solutions with a diameter of 62 nm and a critical micelle concentration of about 0.032 wt % at 20 °C. Micelles aggregated as the temperature increased, leading to gel formation. The sol–gel transition was studied, with a focus on the structure–property relationship. It is expected to have potential applications in drug delivery and tissue engineering. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4091–4099, 2007     

Relationship between the coil-globule transition of an aqueous poly(N-isopropylacrylamide) solution and structural changes in local conformations of the polymer

Structural changes in the local conformation of poly(N-isopropylacrylamide) (PNiPA) during the thermally and solvent-induced coil-globule transitions in an aqueous solution were studied by using attenuated total reflection / infrared (ATR/IR) spectroscopy combined with density functional theory (DFT) calculation. DFT calculation makes it possible to connect the spectral changes observed during the transitions with the structural changes of the local conformation of polymer chains. The results suggest that some of the intramolecular C=O···H-N hydrogen bonds of amide groups are broken, and the changes in local conformations occur during the coil-globule transitions of PNiPA. In this paper, an overview of our recent studies on the coil-globule transitions of PNiPA is given for introducing a new idea that may explain the stimulus sensitivities of PNiPA in solutions; the solubility of segments concerning with the local conformation of repeating monomer units is changed by an external perturbation, and then the polymer system shows the coil-globule transition.     

Precipitation temperature of poly(ethylene glycol) in some electrolyte solutions

The effect of sodium fluoride, sodium bromide, tetramethyl ammonium chloride, tetramethyl ammonium bromide and sodium formate on both precipitation and θ-temperatures of poly(ethylene glycol) (M_w = 4000) in aqueous solution have been determined. Curves have been drawn to represent the effect of such ions on both the θ and the precipitation temperatures of the polymer. The effectiveness increases in the order (CH₃)₄ NBr, NaBr, (CH₃)₄NCl, NaF and HCOONa. The M_w-temperatures obtained at infinite dilution are 445 K for NaBr, NaF, (CH₃)₄ NBr and (CH₃)₄NCl and 415 K for HCOONa.     

Effect of temperature on the intrinsic viscosity of poly(ethylene glycol)/poly(vinyl pyrrolidone) blends in aqueous solutions

In this work the intrinsic viscosity of poly(ethylene glycol)/poly(vinyl pyrrolidone) blends in aqueous solutions were measured at 283.1–313.1 K. The expansion factor of polymer chain was calculated by use of the intrinsic viscosities data. The thermodynamic parameters of polymer solution (the entropy of dilution parameter, the heat of dilution parameter, theta temperature, polymer–solvent interaction parameter and second osmotic virial coefficient) were evaluated by temperature dependence of polymer chain expansion factor. The obtained thermodynamic parameters indicate that quality of water was decreased for solutions of poly(ethylene oxide), poly(vinyl pyrrolidone) and poly(ethylene oxide)/poly(vinyl pyrrolidone) blends by increasing temperature. Compatibility of poly(ethylene oxide)/poly(vinyl pyrrolidone) blends were explained in terms of difference between experimental and ideal intrinsic viscosity and solvent–polymer interaction parameter. The results indicate that the poly(ethylene glycol)/poly(vinyl pyrrolidone) blends were incompatible.