Aqueous Polymer Two-phase Systems Research Articles

One-step purification of proteins from chicken egg white using counter-current chromatography

Proteins present in chicken egg white are separated by counter-current chromatography (CCC) in one step using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% (w/w) poly(ethylene glycol) 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 1.0 ml/min. From about 20 g of the crude egg white solution, lysozyme, ovalbumin, and ovotransferrin were resolved within 5.5 h. Each component was identified by 12% SDS gel electrophoresis with Coomassie brilliant blue staining.

Separation of Listeria monocytogenes and Salmonella berta from a complex food matrix by aqueous polymer two-phase partitioning.

In the present study, the use of aqueous polymer two-phase systems for separation of pathogenic bacteria from a complex food sample was investigated. Three different two-phase systems, a polyethylene glycol 3350/dextran T 500, a methoxy polyethylene glycol 5000/dextran T 500 and a polyethylene glycol 3350/hydroxypropyl starch system, were compared at pH 3 and pH 6 for their capacity to separate the pathogenic bacteria Listeria monocytogenes and Salmonella berta from a Cumberland sausage. In all three phase systems, the food particles partitioned to the lower phase. Best performance was obtained by the polymer combinations, polyethylene glycol 3350/dextran T 500 and polyethylene glycol 3350/hydroxypropyl starch. In these systems, Salmonella berta partitioned to the hydrophobic upper phase both at pH 3 and pH 6 with an average partitioning ratio of 80% and a recovery of 56%. Listeria monocytogenes partitioned to the upper phase at pH 3 only with an average partitioning ratio of 72% and a recovery of 45%. This method may become a valuable tool for separation of bacteria from complex food matrices.

Partitioning of Polymer and Inorganic Colloids in Two-Phase Aqueous Polymer Systems

The well-known phenomenon of partitioning of bioparticles in two-phase aqueous polymeric systems is extended to a variety of polymer (acrylic latex) and inorganic (TiO2) colloidal particles. It is demonstrated that particle partitioning into a particular phase is controlled in rational ways by particle surface chemistry, viz., hydrophobicity, functional groups such as hydroxyl and carboxyl, and pH effects, in relation to the chemical nature of the water soluble polymers (WSPs) that comprise the phase-separated system. WSPs include poly(ethylene oxide), poly(N-vinylpyrrolidone), dextran, and a hydrophobe-terminated water soluble polyurethane based on polyethylene oxide. Mixtures of colloidal particles can be separated by selective partitioning.

Analytical partitioning of poly(ethylene glycol)-modified proteins.

Covalently grafting proteins with varying numbers (n) of poly(ethylene glycol) molecules (PEGs) often enhances their biomedical and industrial usefulness. Partition between the phases in aqueous polymer two-phase systems can be used to rapidly characterize polymer-protein conjugates in a manner related to various enhancements. The logarithm of the partition coefficient (K) approximates linearity over the range O<n<x. However, x varies with the nature of the conjugate (e.g., protein molecular mass) and such data analysis does not facilitate the comparison of varied conjugates. The known behavior of surface localized PEGs suggests a better correlation should exist between log K and the weight fraction of polymer in PEG-protein conjugates. Data from four independent studies involving three proteins (granulocyte-macrophage colony stimulation factor, bovine serum albumin and immunoglobulin G) has been found to support this hypothesis. Although somewhat simplistic, 'weight fraction' based analysis of partition data appears robust enough to accommodate laboratory to laboratory variation in protein, polymer and phase system type. It also facilitates comparisons between partition data involving disparate polymer-protein conjugates.

Interaction between tryptophan residues and hydrophobically modified dextran: Effect on partitioning of peptides and proteins in aqueous two-phase systems

Hydrophobically modified dextrans, benzoyl dextran and valeryl dextran, have been used to study the interactions between tryptophan residues and benzoyl or valeryl groups by partitioning of tryptophan, tryptophan–tryptophan, (tryptophan) 3, poly(lysine, tryptophan), β-galactosidase and lysozyme in polymer aqueous two-phase systems. The two-phase systems used were polyethylene glycol (PEG)–benzoyl dextran, PEG–valeryl dextran, dextran–benzoyl dextran and dextran–valeryl dextran. Interaction between tryptophan residues and benzoyl or valeryl groups was observed by partitioning of tryptophan containing compounds to the phase containing hydrophobically modified dextran. At a certain phase composition the interactions were increased with increasing number of tryptophan per molecule. In a PEG–dextran system the partitioning of tryptophan peptides to the PEG phase was increased with increased number of tryptophan. In a PEG–benzoyl dextran system the opposite effect was obtained. At similar conditions benzoyl groups showed stronger interactions with tryptophans compared to valeryl groups. The partition coefficient of salts (sodium phosphate, NaCl, NaI and NaClO 4) was determined in PEG–benzoyl dextran and PEG–valeryl dextran aqueous two-phase systems. The effect of addition of these salts on partitioning of poly(lysine, tryptophan), β-galactosidase and lysozyme was studied. Salt effects on partitioning could be explained by the relative affinities of the ions for the polymers in the system. Charged molecules containing tryptophan were to an increasing degree partitioned to the phase for which the counterions had highest affinity. Strong effects on the partitioning of positively charged poly(lysine, tryptophan) and lysozyme were obtained with the ions I − and ClO 4 −.

Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR

A sample preparation method based on a two-phase aqueous polymer system, composed of 8% (w/w) polyethylene glycol 4000 and 11% (w/w) dextran 40, was employed to remove PCR-inhibitory substances from human faeces prior to PCR. The majority of the PCR-inhibitory substances, including bile salts, were shown to be distributed in the polyethylene glycol-rich top phase, whereas target bacteria were detected by PCR in the dextran-rich bottom phase. The efficiency of the aqueous two-phase system in removing PCR-inhibitory substances from faeces was evaluated with a standardised PCR assay containing different concentrations of pure Helicobacter pylori DNA. The detection level for a pure water solution of DNA was in the range 10 −12 to 10 −13 g DNA per reaction tube. Untreated faecal homogenate totally inhibited PCR even after 1000 times dilution in water. A positive PCR result was obtained when 10 −6 g H. pylori DNA was present in a 50 μl reaction mixture containing 10 μl homogenised faeces, which had been briefly centrifuged, boiled and diluted 100 times in water. After extraction of an undiluted heat-treated homogenate in the aqueous two-phase system, the detection level was lowered to the range 10 −10 to 10 −11 g DNA. When the aqueous two-phase system was evaluated on five faecal samples the detection level was decreased by three to five orders of magnitude. Finally, the aqueous two-phase system was applied to faecal samples inoculated with H. pylori cells. The sensitivity of the PCR assay after extraction was found to be 40 times above the detection level of H. pylori in a pure water solution. The target bacteria were detected directly by PCR in the dextran-rich bottom phase. Furthermore, most of the H. pylori cells were shown to be present in the dextran-rich bottom phase.

Enzyme-catalyzed peptide syntheses in aqueous polymer two-phase systems

Peptide synthesis in the aqueous polymer two-phase system was examined. Water soluble polymers adopted were dextran and polyethyleneglycol (PEG). By the modification of a proteolytic enzyme, trypsin, with dextran carrying a very small number of carboxyl groups, a separation of the enzyme from its peptide product, N-benzoyl-L-arginine glycinamide, which is relatively more soluble in a PEG phase could be realized. This separation drastically reduced a so-called "mass-law" effect, and increased the yield of the reaction product. The aqueous polymer two-phase system would be useful to enhance the usefulness of biocatalysts in organic syntheses.

Regulated insulin release from biodegradable dextran hydrogels containing poly(ethylene glycol)

Biodegradable dextran hydrogels containing poly(ethylene glycol) (PEG) were prepared as a model of heterogeneous-structured devices composed of drug reservoirs and degradable matrices, and insulin release during their enzymatic degradation was investigated. Specific distribution of bioactive peptides has been well known in a combination of PEG and dextran as an aqueous polymer two-phase system. Insulin was preferentially loaded into PEG phase when methacrylic dextran was crosslinked in the presence of PEG and insulin. Microscopic observation, thermal analysis and small-angle light scattering measurements revealed that PEG-Dex hydrogels exhibited a variation of microdomain structures, the extent of which was dependent upon the molecular weight of PEG. The degradation of both cross-linked dextran hydrogels containing PEG (PEG-Dex hydrogels) and cross-linked dextran hydrogel (Dex hydrogel) by dextranase were found to proceed from the surface front. However, release profiles of insulin from PEG-Dex hydrogels were different from that of Dex hydrogel: insulin release from PEG-Dex hydrogels was regulated by the surface degradation of PEG-Dex microdomain-structured matrix, whereas diffusion was the dominant factor in insulin release from Dex hydrogel.

Effect of Cd on plasma membrane ATPase from plant roots differing in tolerance to Cd

Abstract The effect of Cd on a H+/K+ antiport in bean was determined and the effect of Cd on the activity of the plasma membrane ATPase partially purified from the roots of plants differing in tolerance to Cd2+ using an aqueous polymer two-phase system was compared. Cadmium inhibited the H+/K+ antiport in vivo and inhibited the plasma membrane ATPase in vitro. The order of Cd tolerance of the plasma membrane ATPase partially purified from the roots was as follows: rape > pumpkin > pea > rice > kidney bean in the treatment at 10-50 μM Cd. Cadmium tolerance of ATPase appeared to be positively correlated with Cd tolerance in intact plants, except in rice. The results suggested that Cd may be associated with many components in the plasma membrane and may affect ion transport and that the Cd tolerance of ATPase may be one of the mechanisms of Cd tolerance in intact plants.

Purification of pea nodule symbiosomes using an aqueous polymer two-phase system

Symbiosomes were obtained from mature pea ( Pisum sativum cv. Argona) root nodules infected with a Rhizobium leguminosarum strain (biov. viciae 3841) and purified using an aqueous polymer two-phase system (APS). The APS consists of a mixture of polymers, usually dextran T500 and poly(ethylene glycol) 3350, prepared as aqueous solutions on a weight per weight basis, where each fraction distributes according to their surface characteristics. Results of ATPase activity, cytochrome c oxidase activity, glucan synthase II activity, NAD(P)H-cytochrome c reductase activity, NO 3 −-sensitive ATPase activity, transport of [ 14C]malate vs. [ 14C]glutamate and MAC 57 antigen analysis showed that the APS method provided intact symbiosomes with low bacteroid, plasma membrane, endoplasmic reticulum and/or mitochondria contamination. No complicated equipment is needed and the method was simple and fast, compared with other purification techniques.

A single partitioning step in aqueous polymer two-phase systems reduces hypotonized rat erythrocyte heterogeneity

Rat carrier erythrocytes prepared by hypotonic dialysis (80 mOsm/kg) are a heterogeneous cell population that can be fractionated into two-well-defined cell subpopulations by a single partition step, in charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. One subpopulation (65% of total cells) has a decreased cell surface charge and is partitioned at the interface in a single step and then fractionated by counter-current distribution as a low- G subpopulation. The other subpopulation (35% of total cells) has charge surface properties more like those of the untreated control rat erythrocytes. These last cells are partitioned in the top phase in a single step and then fractionated by counter-current distribution as a high- G subpopulation. Partitioning is more effective in reducing cell heterogeneity in hypotonized rat erythrocyte populations than is density separation in Ficoll-paque which only separates a small less dense cell subpopulation (5% of total cells), with the most fragile cells, from a larger and more dense cell subpopulation (95% of total cells), with a mixture of fragile and normal cells. This simple cell separation procedure quickly reduces carrier erythrocyte heterogeneity in a single partitioning step so it can be used to prepare cells for in vivo studies.

Effect of ionic strength and ionic species on partitioning behavior of hydrophobic and hydrophilic polystyrene latex beads in aqueous two-phase polymer systems

The effects of ion type, pH, ionic strength and colloidal surface properties on the partitioning of three different types of polystyrene latex beads with different surface properties in the two-phase polyethylene glycol (PEG)/dextran (DEX) system have been measured. Certain inorganic monovalent ion types alter the partitioning of hydrophilic polystyrene beads from the top PEG-rich phase to the bottom DEX-rich phase, while hydrophobic polystyrene beads always partition into the top PEG-rich phase, regardless of the type and concentration of salt. The partitioning of the hydrophilic beads depends on the ionic strength as well as the ion type, and is not due to changes in pH. Strong water-structure-making salts, e.g. ammonium sulfate, are least likely to induce partitioning to the bottom phase, while sodium azide, a pseudo halide, has the strongest effect on partitioning of the beads. The Hofmeister series does not predict the relative effectiveness of the different ions that do alter the partitioning of the hydrophilic beads. The polyacrylic acid (PAA) coating on the hydrophilic beads is known to strongly interact with PEG and polyoxyethylene (PEO) polymers, via hydrogen bonding, especially at lower pH values. This strong polymer-polymer interaction may be disrupted by many salts, e.g. sodium chloride, at higher ionic strengths. In general, the influence of ions on the conformation, solvation and free energy of PEG in aqueous solution, and its interaction with PAA, is a complex phenomenon and is not predicted by existing theories. Depletion flocculation may also play a role in the salt-dependent bead partitioning behavior. A general consequence of the acid-PEG interaction is that relatively hydrophilic solutes with many surface carboxylic acid groups may partition to the top PEG-rich phase, even though it is the less polar of the two phases in the PEG/DEX system. This phenomenon may explain the partitioning behavior as a function of salt observed for certain species of bacteria.

Heterogeneity of hypotonically loaded rat erythrocyte populations as detected by counter-current distribution in aqueous polymer two-phase systems

Carrier rat erythrocytes loaded with exogenous substances ([ 125I]carbonic anhydrase) by hypotonic-isotonic dialysis become heterogeneous cell populations that can be fractionated using the counter-current distribution (CCD) technique. Two well-defined low- and high-partition ratio, G, subpopulations are obtained in charge-sensitive dextran-polyethylene glycol two-phase systems. Thelow- G subpopulation, which contains the most fragile and surface-altered cells, as deduced from their osmotic fragility curves and partition behaviour, respectively, presents a high amount of exogenous substance incorporated (134.6 cpm/10 6 cells). Thehigh- G subpopulation, that contains cells similar to the control or isotonically dialyzed cells presents a lower amount of exogenous substance incorporated (69.8 cpm/10 6 cells). Cells in thishigh- G subpopulation seem to be fractionated, like the controls, according to ageing as suggested by the decline of the pyruvate kinase specific activity from the left- to the right-hand side of the CCD profile.

The purification of membranes by affinity partitioning

We describe affinity partitioning as a preparative method for membranes and membraneous structures such as organelles, cells, and viruses. Biospecific affinity partitioning is carried out in aqueous polymer two-phase systems, commonly with polyethylene glycol and dextran as phase polymers, in an environment compatible with membrane structures. Ideally, two-phase conditions are chosen to partition the bulk of membrane material into one phase, while the affinity ligand, conjugated to the second phase polymer, will selectively pull the membranes to be isolated into this phase. Suitable ligands include lectins, antibodies, and receptor-specific agents. Because the method has so far been used successfully in rather few instances, all using high ligand receptor densities in target membranes, the discussion focuses on factors to be considered when developing affinity partitioning conditions using new ligands.

Aqueous two-phase polymer systems as tools for the study of a recombinant surface-expressed Escherichia coli hemagglutinin

The surface expression of an integral membrane hemagglutinin, HRA1, cloned from Escherichia coli O9: H10:K99 in heterologous E. coli strains was studied by utilizing a variety of polyethylene glycol-dextran and dextran-Ficoll aqueous two-phase polymer systems. Bacteria containing plasmids that encoded the hemagglutinin were found to partition differently from both the host bacteria lacking the plasmid and the original hemagglutinating strain in several of these systems. By using molecular biological techniques, the origin of the partition difference was unambiguously correlated to the expression of HRA1, providing evidence independent of the agglutination phenotype that the protein was accessible to the surrounding milieu. It was demonstrated by using bacterial partition in charge-sensitive systems that the agglutination event was not likely to be due to the presence of a nonspecific positively charged surface protein, as HRA1-expressing clones showed no less affinity for the relatively positive polyethylene glycol-rich upper phase than did control bacteria. This work demonstrates the utility of aqueous polymer two-phase systems for the study of surface-expressed recombinant proteins, due to the sensitivity of the systems and the presence of excellent controls (the host bacteria before plasmid introduction). In cloning and expression studies of surface-associated proteins, two-phase aqueous polymer systems could be used as an alternative to antibody production for the monitoring of surface expression, and these systems may give valuable information on the surface exposure of the protein.

Analysis of the electrostatic potential difference in aqueous polymer two-phase systems

Experimental partition coefficients of two ionic dyes in two-phase systems water + poly(ethylene glycol) 3000 + dextran 500000 containing various salts are presented. Based on these data unambiguous information on the electrostatic potential differences induced by the salts is obtained. To achieve this goal the model proposed by Albertsson (1986) is applied to systems containing mixed salts to account for an ionic impurity present in the polymer samples. With both dyes the value of Δϕ Na2SO4 − Δϕ NaCl = 4.7 mV at salt concentrations up to 50 mmol kg −1 is obtained. Also the partition coefficients of the salts are predicted in agreement with the experiment. These results indicate that up to a salt concentration of approximately 50 mmol kg −1 the Albertsson model can be applied. Above this concentration limit, the basic two-phase system is altered and the model predictions are severely in error.

A rapid, simple method for the isolation and characterization of the photoreceptor of Dictyostelium discoideum amoebae

A membrane-bound 45.5 kDa protein has been isolated from Dictyostelium discoideum amoebae. It shows an absorption spectrum, which closely resembles the action spectrum for amoebal phototaxis, leading to the conclusion that this protein might play an important role in the photoreception of Dictyostelium amoebae. For further characterization we employed phase partition in an aqueous polymer two-phase system, which was developed by Widell and Larsson for the separation of plasma membrane proteins of higher plants. This method clearly shows that the 45.5 kDa protein is a plasma membrane protein and not an intracellular protein. Furthermore, by using phase systems with increasing polymer concentrations, this simple and rapid purification of plasma membrane proteins allowed us to isolate the putative photoreceptor in one single step. Compared to standard biochemical methods phase partition provides an enormous facilitation of the isolation of D. discoideum membrane proteins.

Surface properties of crosslinked erythrocytes as studied by counter-current distribution in aqueous polymer two-phase systems.

The bifunctional imidoester dimethyl suberimidate hydrochloride can stabilize rat red blood cells (RBCs) by membrane protein crosslinking, and in that way they can be used as carrier systems for exogenous substances. Counter-current distribution fractionation in charge-sensitive dextran-polyethyleneglycol two-phase systems has been used to detect slight changes in surface charge in stabilized cells. A decrease in the surface charge of crosslinked RBCs and an apparent masking of the age-related cell surface properties have been found to result from the protein crosslinking. Digitonin treatment used to permeabilize crosslinked RBCs produces a significant decrease of the cell surface charge while the age-related surface properties do not seem to be modified by the treatment.

Cultivation of Plant Cell suspensions from Nicotiana Tabacum 1507 and Lavandula Vera mm in Aqueous Two-Phase Polymer Systems

ABSTRACTThe growth experiments in which plant cell suspensions of N. tabacum 1507 and L. vera MM were cultivated in two-phase systems were carried out in order to select the best phase formulation ensuring culture growth. The highest growth observed for N. tabacum 1507 occurred in 5% PEG 6000/10% dextran 70 000. The overproduction of extracell phenolic compounds from both cell cultures in the formulated two-phase systems was investigated. The best two-phase system ensuring higher production of phenolic compounds from both cell cultures is 4% PEG 20 000/7.5% dextran 70 000. The plant cell suspensions of N. tabacum 1507 produced 0.171 g dm−3 total phenolic compounds in this system (against 0.028 g dm−3 from control fermentation). In the same two-phase system, the plant cell suspension from L. vera MM produced 0.387 g dm−3 total phenolic compounds (against 0.058 g dm−3 from control fermentation). In both cases the phenolic partitioned mainly in the top phase.

Partitioning of high-density lipoproteins in charge-sensitive two-phase systems

Aqueous polymer two-phase systems characterized by a difference in the electrical potential between the upper and the lower phase (charged systems) are useful tools for the detection of changes in the surface charge and hydrophobicity of high-density lipoproteins (HDL). While the large particle size of low-density lipoproteins (LDL) leads to accumulation at the interface, the smaller diameter and the higher surface charge density of the native HDL particles allows partitioning without aggregation at the interface. Charged two-phase systems can be used to check the native state of HDL samples. Moreover, these systems would be suitable for investigating the hydrophobicity and surface charge of modified HDL.