Dextran Two-phase Systems Research Articles

Formation and Evaluation of a Two-Phase Polymer System in Human Plasma as a Method for Extracellular Nanovesicle Isolation.

The aim of the study was to explore the polyethylene glycol–dextran two-phase polymer system formed in human plasma to isolate the exosome-enriched fraction of plasma extracellular nanovesicles (ENVs). Systematic analysis was performed to determine the optimal combination of the polymer mixture parameters (molecular mass and concentration) that resulted in phase separation. The separated phases were analyzed by nanoparticle tracking analysis and Raman spectroscopy. The isolated vesicles were characterized by atomic force microscopy and dot blotting. In conclusion, the protein and microRNA contents of the isolated ENVs were assayed by flow cytometry and by reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR), respectively. The presented results revealed the applicability of a new method for plasma ENV isolation and further analysis with a diagnostic purpose.

Technical aspects of separation using aqueous two‐phase systems in enzyme isolation processes

Technical aspects of the separation of aqueous two-phase systems in a commercial separator were studied in detail. For the Gyrotester B, the smallest available separator, a flow rate of 200 ml/min and a length of the regulating screw in the outlet port of 13.5 mm were found as optimal operation parameters for the separation of a poly(ethylene glycol) (PEG)/dextran two-phase system. In the presence of cells and cell debris the characteristics of the carrier two-phase systems are changed, most notably the phase ratio. Nevertheless good separation and high throughput can be maintained up to 30% wet cell material in the complete system. Using this method the enzyme pullulanase was extracted from 6.65 kg Klebsiella pneumoniae in 88% yield in a single step in less than 2 hr. A yield of 90% was predicted for this step based upon laboratory data, indicating that the performance of the extraction and separation can be calculated with the necessary accuracy and the further scale-up of the process should be accomplished quite easily. The hydrophilic polymers Constituting the phase system will often stabilize the enzymes, So that the separation can be carried out at room temperature without extensive cooling. The method of enzyme solubilization or cell disruption is not decisive for the successful extraction of the enzymes, the only limitation being the necessity to find a suitable two-phase system where the desired product and the cells or cell debris will partition in opposite phases. This is shown for α-glucosidase from Saccharomyces carlsbergensis and three aminoacyl-tRNA-synthetases from Escherichia coli. The results obtained demonstrate that aqueous two-phase systems can be separated in commercially available separators with high capacity and efficiency. It can be expected that the advanced separation technology available from chemical engineering studies can also be used for the development of large-scale isolation processes for enzymes involving liquid–liquid partitions.

Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin–dextran

A new concept for affinity two-phase partitioning was tested. The partitioning was based on the interaction of target membranes with a primary antibody which, in turn, interacted with a biotinylated secondary antibody and NeutrAvidin–dextran in a poly(ethylene glycol)/dextran two-phase system. Caveolae selectively redistributed from the top phase to the NeutrAvidin–dextran-containing bottom phase by employing anti-caveolin as the primary antibody. This immunoaffinity approach was more selective than the established sucrose gradient centrifugation method and resulted in highly purified caveolae from Triton X-100-treated liver and lung plasma membranes. The same approach, employing other selective primary antibodies, should facilitate the purification also of other membrane fractions.

Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin–dextran

A new concept for affinity two-phase partitioning was tested. The partitioning was based on the interaction of target membranes with a primary antibody which, in turn, interacted with a biotinylated secondary antibody and NeutrAvidin–dextran in a poly(ethylene glycol)/dextran two-phase system. Caveolae selectively redistributed from the top phase to the NeutrAvidin–dextran-containing bottom phase by employing anti-caveolin as the primary antibody. This immunoaffinity approach was more selective than the established sucrose gradient centrifugation method and resulted in highly purified caveolae from Triton X-100-treated liver and lung plasma membranes. The same approach, employing other selective primary antibodies, should facilitate the purification also of other membrane fractions.

Isolation of a caveolae-enriched fraction from rat lung by affinity partitioning and sucrose gradient centrifugation

Caveolae were isolated from rat lungs by a combination of affinity partitioning and sucrose gradient centrifugation. After homogenization of the lungs directly in a polyethylene glycol–dextran two-phase system and conventional phase partitioning, the polyethylene glycol-rich top phase was affinity partitioned with fresh bottom phase containing dextran-linked wheat-germ agglutinin. The lectin selectively attracted plasma membranes to the bottom phase. The isolated plasma membrane fraction was treated with Triton X-100 or, alternatively, sonicated before centrifugation in a stepwise sucrose gradient. Caveolin-enriched material collected at the 5/24% sucrose boundary. This material also contained 5 ′-nucleotidase activity and actin. Electron microscopy showed the material to consist of a homogeneous population of 50- to 100-nm vesicles. This purification protocol should allow the facile purification of caveolae also from other tissues, facilitating structural and functional studies.

Bioconversion in an aqueous two-phase system using a smart biocatalyst: casein hydrolysis by a α-chymotrypsin derivative

Casein hydrolysis was carried out in PEG–dextran two-phase system by using α-chymotrypsin immobilized on a water soluble polymer Eudragit S-100. This biocatalyst along with the substrate casein was predominantly found to partition into the PEG phase. Under these optimized conditions, the product was found to partition (84%) in the dextran phase. Removal of dextran phase at an appropriate time interval and replacing it with fresh dextran phase led to considerable enhancement of casein hydrolysis. The biocatalyst could be separated from the PEG phase by lowering the pH to 3.8 and again dissolved in PEG phase by increasing the pH to 7.6. Thus, this smart biocatalyst could be reused for casein hydrolysis in PEG–dextran two-phase aqueous system.

Affinity partitioning for membrane purification exploiting the biotin–NeutrAvidin interaction: Model study of mixed liposomes and membranes

Biotinylated negatively charged liposomes as well as membranes were affinity partitioned in an aqueous poly(ethylene glycol)–dextran two-phase system using NeutrAvidin conjugated to dextran as affinity ligand. Both liposomes and membranes redistributed from top to bottom phase upon addition of NeutrAvidin–dextran. The presence of 35–60 m M Li 2SO 4 was necessary both to force the components into the top phase without ligand and for ligand-dependent redistribution into the bottom phase. Attaching biotin via a hexanamidohexanoyl spacer and an increased density of biotin or NeutrAvidin enhanced the affinity separation. The separation conditions in these model experiments provide a basis for affinity partitioning of membranes using other affinity ligands.

Partitioning of Ganglioside-Reconstituted Liposomes in Aqueous Two-Phase Systems

The partitioning of ganglioside (GM3,GD1a, GD1b or GT1b)-reconstituted liposomes was investigated in an aqueous poly(ethylene oxide)/dextran two-phase system to evaluate the carbohydrate–carbohydrate interactions in water. The partitioning of the ganglioside-reconstituted liposome was strongly affected by the buffer employed. As the concentration of sodium phosphate decreased, the ganglioside-reconstituted liposomes were partitioned to the bottom, dextran-rich, phase. In 10 mM sodium phosphate containing 150 mM sodium chloride, the conventional liposome without ganglioside were located mostly at the interface between the two phases. On the other hand, the ganglioside-reconstituted liposomes were significantly partitioned to the bottom dextran-rich phase, which was related to the ganglioside density on the liposomal surface. This partitioningto the dextran-rich phase also depended on the chemical structure of the ganglioside on the liposomal surface. The affinity of liposomal ganglioside being on the liposomal surface to dextran was the following sequence; GT1b>GD1a>GD1b>GM3. Partitioning of liposomes to the top poly(ethylene oxide)-rich phase was negligible irrespective of the characteristics of the liposomal surface.

Affinity partitioning of biotinylated mixed liposomes: effect of charge on biotin–NeutrAvidin interaction

The partitioning behaviour of biotinylated mixed liposomes in aqueous poly(ethylene glycol)/dextran two-phase systems containing NeutrAvidin–dextran suggests that the biotin–NeutrAvidin affinity interaction is charge dependent. Biotinylated phosphatidylcholine liposomes with a low negative surface charge distributed in the NeutrAvidin-containing bottom phase at neutral pH, but the introduction of additional negative charges by including phosphatidylserine or the surfactant sodium dodecylsulfate in the liposomes caused them to distribute in the poly(ethylene glycol)-rich top phase instead. By gradually lowering the pH of the affinity two-phase system below the isoelectric point (6.3) of NeutrAvidin, negatively charged phosphatidylserine/phosphatidylcholine liposomes increasingly were attracted by NeutrAvidin to the bottom phase. It is suggested that acidic amino acids present at the rim of the biotin-binding pocket of NeutrAvidin may interact electrostatically with charged residues of the closely apposed liposome surface affecting the affinity interaction.

Aqueous two-phase affinity partitioning of biotinylated liposomes using neutral avidin as affinity ligand

Biotinylated small unilamellar liposomes were affinity partitioned in an aqueous poly(ethylene glycol)–dextran two-phase system using avidin coupled to dextran as affinity ligand. In the absence of affinity ligand more than 90% of the liposomes partitioned in the poly(ethylene glycol)-rich top phase, whereas in its presence more than 95% partitioned in the dextran-rich bottom phase. For this redistribution to occur 10 m M and above of lithium sulphate, or other appropriate salts, had to be added to the two-phase system. Without added salt the liposomes with complexed avidin–dextran instead partitioned in the top phase. An extended mixing time for the system was required for maximum redistribution. Less than two biotin residues per liposome, coupled via a C 6 spacer arm, was required to redistribute the liposomes to the bottom phase.

Poly(ethylene glycol) Amphiphiles: Surface Behavior of Biotechnical Significance

The interfacial behavior of surface localized, poly(ethylene glycol) (PEG) esterified fatty acid amphiphiles was comparatively studied at microparticle surfaces via phase partition and at flat surfaces via in situ null ellipsometry. Ellipsometry was performed on methylsilane (MeSi), phosphatidic acid (PA), and phosphatidylcholine (PC) coated silica slides, while human erythrocyte and PC liposomes were subjected to partition in aqueous PEG, dextran two-phase systems. Analogous results from both methods suggest that PEG−amphiphile adsorption can be relatively independent of the underlying surface. Ellipsometry indicated that members of a series of PEG-fatty acid esters of the type Ci:j−EO151 (16 ≤ i ≤ 18, 0 ≤ j ≤ 2) adsorb similarly at MeSi-, PC-, and PA-coated surfaces, reaching a plateau (≈0.1 PEG chains·nm-2) independent of micelle formation. When normalized for acyl tail hydrophobicity, PEG−amphiphile adsorption is relatively noncooperative and independent of the acyl tail; saturation is largely determi...

A new approach to examine conformational changes occurring upon binding of ligand by biomolecules

Liquid-liquid partition chromatography in an aqueous poly(ethylene glycol)/dextran two-phase system (LLPC) is shown to be a quick and sensitive method for detecting conformational changes occurring upon binding of ligands by biospecific molecules. Two groups of well-characterized proteins, enzymes and monoclonal antibodies, were employed. As an example, LLPC demonstrated that isoforms of lactate dehydrogenase as well as of hexokinase existed in a ligand-dependent equilibrium between two forms and that conformational changes occurred when monoclonal antibodies bound haptens. We also demonstrate that the method could be used to detect and separate subfractions in preparations of unliganded proteins that appeared to be homogeneous when analysed by other techniques.

Estimation of the effective hydrophobicity of protiens: Re-evaluation of methods

Partition coefficients of distribution of proteins were measured in two systems: i) 3-phenoxy-2-hydroxypropyl derivatives of bead cellulose (PHPC)/water solution — coefficients P; and ii) Aqueous polyethylene glycol (PEG)/dextran (DXT) two-phase system — coefficient K. Following proteins were used for the measurements: lysozyme, trypsin, chymotrypsin, ovalbumin, bovine serum albumin and immunoglobulin G. The obtained P and K values were correlated with previous data about hydrophobicity of the above proteins available in the literature. The literary data concerned: i) the efficacy of energy transfer from tryptophan residues of proteins to cis-parinaric acid applied as hydrophobic probe, estimated by fluorescent spectroscopy; ii) the hydrophobic ratio indicating the ratio between the hydrophobic and hydrophilic parts (in volumes) of protein molecules deduced from their primary structure; and iii) the interfacial tension at 0.2% protein-water solution/corn oil interface. Significant corrlations were obtained for P and efficacy of energy transfer (r=0.964; p<0.01) and for K and interfacial tension (r=0.936; p<0.05). When P and K were fitted as exponential function of three independent variables (i.e., efficacy of energy transfer, hydrophobic ratio and interfacial tension) good agreement between the measured and computed data was obtained. The increases in efficacy of energy transfer, hydrophobic ratio and decrease in interfacial tension were found to be accompanied by increase in P. In contrary, K behaved always similarly as efficacy of energy transfer, hydrophobic ratio and interfacial tension.

Immobilized metal-ion affinity partitioning of NAD +-dependent dehydrogenases in poly(ethylene glycol)-dextran two-phase systems

Affinity partitioning of yeast alcohol dehydrogenase (YADH), lactate dehydrogenase from rabbit muscle (MLDH) and lactate and malate dehydrogenases from pig heart (HLDH and HMDH, respectively) were studied in aqueous two-phase systems containing metal ions (Cu 2+, Ni 2+, Zn 2+ and Cd 2+) chelated by iminodiacetate-poly(ethylene glycol) (IDA-PEG). The partitioning behaviour of the enzymes in the presence of Cu(II)-IDA-PEG was studied as a function of the concentration of NaCl, the pH of the medium and the concentration of added selected agents. It was demonstrated that the partition effect (Δ log K) of dehydrogenases in the presence of Cu(II)-IDA-PEG and the affinity of enzymes for immobilized Cu 2+ ions increases in the order MLDH > YADH > HMDH ⩾ HLDH. It was shown that the determined variations in the enzyme affinities for Cu(II)-IDA-PEG might be related to the differences in the content of histidine residues accessible to the solvent.

Partitioning of chemically modified low-density lipoprotein in aqueous polymer two-phase systems

Aqueous polyethylene glycol (PEG)-dextran two-phase systems containing 10 m M Tris·HCl (pH 7.4) were used for the partitioning of chemically modified low-density lipoprotein (LDL). Anionic modification connected with an increase in the negative surface charge of lipoproteins favours the accumulation of modified LDL in the top phase. The partition coefficient increases depending on the extent of modification. Cationic modification yields lower values for the partition coefficient. Positively charged LDL favours a bottom-phase accumulation. With weakly charged and nearly neutral particles, the Van der Waals interaction between polymer and particle preponderates over electrostatic interactions, leading to a favoured accumulation of LDL in the PEG-rich top phase. Results of measurements of the relative electrophoretic mobility and the determination of free amino groups are in agreement with the calculated values of the partition coefficient. Because the partitioning of LDL is accompanied by aggregation at the interface, experimental techniques have to be carefully standardized. Subtle differences in the surface properties of modified LDL can be detected.

Thermodynamics of aqueous poly(ethylene glycol)-dextran two-phase systems using the consistent osmotic virial equation

Abstract In this work we present a thermodynamic model for the prediction of the liquid-liquid phase behavior of aqueous poly(ethylene glycol) (PEG) - dextran two-phase systems. The model is based on the McMillan-Mayer solution theory (1945) and results in thermodynamically consistent expressions for the chemical potentials of the solutes derived from the osmotic virial equation (COVE). Applying the COVE, we have examined the predictability using a complete and reliable data- base of liquid-liquid equilibrium (LLE) and vapor-liquid equilibrium (VLE) data. As a result of this examination, we were able to demonstrate the essential influence of the molecular-weight distribution of polydisperse polymers on the LLE predictions. Accounting for the polydispersity in our calculations, the prediction of the compositions as well as the molecular-weight distributions in the coexisting phases is in good agreement with our experimental results, as illustrated for the system PEG 3000 + dextran 110000 + water at 293.15 K. It should be stressed, that these calculations are true predictions, since the LLE were calculated using model parameters determined from VLE measurements alone.

Supports for liquid-liquid partition chromatography in aqueous two-phase systems: a comparison of Superdex and LiParGel

The ability of Superdex 200, a gel filtration matrix consisting of dextran-grafted beads, to act as a support for liquid-liquid partition chromatography (LLPC) in aqueous polyethylene glycol (PEG)-dextran two-phase systems was examined. The gel adsorbed the dextran-rich bottom phase readily and retained it during elution with the PEG-rich top phase. In contrast to LiParGel 650, a matrix designed for LLPC, the entire Superdex matrix seemed to form an immobilized stationary phase. Ideal partitioning of proteins was observed only for molecules partitioning towards the stationary phase on Superdex and for those favouring the mobile phase on LiParGel. Hence, the choice of matrix depends on the separation problem at hand.

Ligand-receptor interactions in affinity cell partitioning: Studies with transferrin covalently linked to monomethoxypoly(ethylene glycol) and rat reticulocytes

The partitioning of rat reticulocytes in poly(ethylene glycol)(PEG)-dextran two-phase systems increases into the PEG-rich top phase when the cells are incubated with transferrin covalently modified with monomethoxy-PEG (MPEG-transferrin) prior to partitioning. Two observations support the suggestion that such an increase in top-phase partitioning is due to the specific interaction of the MPEG-transferrin conjugate with the transferrin receptor on the surface of the reticulocyte: first, the MPEG-transferrin conjugate completes with [ 125I]transferrin for the transferrin receptor on reticulocytes ( K a = 6.28 · 10 6 l mol −1); and second, the MPEG-modified transferrin is unable to change the partitioning of rat erythrocytes, cells lacking the transferrin receptor. This example illustrates the feasibility of manipulating the partitioning of a selected cell population when ligand-receptor interactions are exploited. The increase in the partitioning of the reticulocytes takes place when a narrow range of MPEG-transferrin bound per cell, viz., 10.2–11.3 fg per cell. The latter range corresponds to ca. 80 000–89 000 molecules of MPEG-transferrin bound per cell.

Cell separation by immunoaffinity partitioning with polyethylene glycol-modified protein a in aqueous polymer two-phase systems

Previous work has shown that polyethylene glycol (PEG)-bound antibodies can be used as affinity ligands in PEG—dextran two-phase systems to provide selective partitioning of cells to the PEG-rich phase. In the present work we show that immunoaffinity partitioning can be simplified by use of PEG-modified Protein A which complexes with unmodified antibody and cells and shifts their partitioning into the PEG-rich phase, thus eliminating the need to prepare a PEG-modified antibody for each cell type. In addition, we provide a more rigorous test of the original technique with PEG-bound antibodies by showing that it is effective at shifting the partitioning of either cell type of a mixture of two cell populations.

Partition of rat erythrocytes in aqueous polymer twophase systems

The partition of rat erythrocytes between the top phase and interface of aqueous poly(ethylene glycol)-dextran two-phase systems containing 0.15 M NaCl and 0.01 M sodium phosphate depends on the association of the cells with microscopic globules of dextran that persist in the poly(ethylene glycol)-rich top phase after the horizontal interface between the two phases has formed.