Aqueous Polymer Two-phase Systems Research Articles

Synthesis and solution properties of a new sulfobetaine/sulfur dioxide copolymer and its use in aqueous two-phase polymer systems

The zwitterionic monomer, 3-(N,N-diallylammonio)propanesulfonate, was copolymerized with sulfur dioxide in dimethyl sulfoxide using azo-bis-isobutyronitrile (AIBN) to afford the polysulfobetaine (PSB) copolymer in excellent yields. The PSB was converted into the corresponding anionic polyelectrolyte (APE) by treatment with 1equiv. of sodium hydroxide. The solution properties of PSB and APE were investigated by potentiometric and viscometric techniques. The critical (minimum) salt concentrations (CSC) required to promote water solubility for the PSB at 23°C have been measured. Basicity constant of the amine functionality in APE is found to be 'apparent’ and as such follow the modified Henderson–Hasselbalch equation. The composition and phase diagram of the aqueous two-phase polymer systems of PSB (treated with 0.60equiv. sodium hydroxide) and poly(ethyelene glycol) (PEG) in 0.1N KCl has been studied for the first time for this class of PSB copolymer.

Synthesis and solution properties of a new ionic polymer and its behavior in aqueous two-phase polymer systems

The amine salt, N,N-diallyl-N-carboethoxymethylammonium chloride was copolymerized in dimethyl sulfoxide using ammonium persulfate or azo-bis-isobutyronitrile (AIBN) to afford the cationic polyelectrolyte (CPE) having five-membered cyclic structure on the polymeric backbone. The CPE on acidic (HCl) hydrolysis of the pendent ester groups gave the corresponding cationic acid salt (CAS) having the equivalent of chloride salt of N,N-diallylammonio ethanoic acid as monomeric unit. The CAS was converted into an anionic polyelectrolyte (APE) and polybetaine (PB) [having the monomeric unit equivalent of sodium N,N-diallylaminoethanoate and N,N-diallylammonioethanoate] by treatment with two and one equivalent of base, respectively. The solution properties of APE were investigated by potentiometric and viscometric techniques. Basicity constant of the amine functionality in APE is found to be ‘apparent’ and as such follow the modified Henderson–Hasselbalch equation; the protonation of the APE becomes more and more difficult as the degree of protonation (α) of the whole macromolecule increases. The composition and phase diagram of the aqueous two-phase systems of APE and poly(ethyelene glycol) (PEG) has been studied for the first time for this class of ionic polymers. The CAS and PB were found to be virtually insoluble in water.

SEPARATION OF CHICKEN EGG WHITE PROTEINS BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

Chicken egg white proteins were fractionated by high-speed counter-current chromatography (HSCCC) using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% polyethylene glycol (PEG) 1000 and 12.5% potassium phosphate buffer at different pH of 6.8, 8.0, and 9.2. From 20 g of the crude egg white solution, the best separation was achieved with a polymer phase system at pH 8.0 by eluting the lower phase at 1.0 mL/min. After the sequential elution of ovotransferrin, ovalbumin, and lysozyme from the column, ovomucin still retained in the column was eluted in the reverse direction with upper PEG-rich phase. All four proteins in the CCC fractions were identified by SDS gel electrophoresis with Coomassie brilliant blue staining.

Interaction between phosphofructokinase and aldolase from Saccharomyces cerevisiae studied by aqueous two-phase partitioning

Phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13) have been highly purified from Saccharomyces cerevisiae by improved protocols. Partitioning of the enzymes in aqueous polymer two-phase systems was used to detect complex formation. The partition of each enzyme was found to be affected by the presence of the other enzyme. AMP affected the partition of the individual enzymes as well as the mixture of the two. The activities of the respective enzymes were stimulated in the putative complex in an AMP-dependent manner. Two strictly conserved residues belonging to an acidic surface loop of class II aldolases, are a potential site for electrostatic interaction with the positively charged regions close to the active site in phosphofructokinase.

Acoustic field assisted demixing of aqueous two-phase polymer systems.

Acoustic field assisted demixing was employed to decrease the demixing time in polymer-polymer (polyethylene glycol-maltodextrin) two-phase system. Application of acoustic field has decreased the demixing time in these systems up to 2-fold. Ultrasonication has induced mild circulation currents in the phase dispersion, which has enhanced the rate of droplet coalescence, eventually resulting in decreased demixing time. In polymer-polymer systems, phase demixing was found to depend greatly on which of the phases is continuous and viscosity of the continuous phase was observed to have a strong influence on the movement of the droplets and hence the phase demixing. Addition of NaCl increased the demixing time and presence of E. coli cells did not seem to have any influence on phase demixing.

Affinity partitioning of a poly(histidine)-tagged integral membrane protein, cytochrome bo3 ubiquinol oxidase, in a detergent–polymer aqueous two-phase system containing metal-chelating polymer

A system has been developed for selective partitioning of membrane proteins. For the first time, an integral membrane protein, cytochrome bo 3 ubiquinol oxidase from Escherichia coli, has been affinity partitioned in an aqueous two-phase system. The systems used were different detergent/polymer aqueous two-phase systems containing a metal-chelating polymer, such as poly(ethyleneglycol)–iminodiacetic acid–Cu(II) as well as dextran–iminodiacetic acid–Cu(II). Many non-ionic detergents, such as alkyl(polyethyleneoxide) (C mEO n), Triton, Tween and alkylglucosides, form two-phase systems in mixture with polymers, such as dextran and poly(ethyleneglycol), i.e., a micelle-enriched phase in equilibrium with a polymer-enriched phase are formed. In general, membrane proteins partition strongly to the micelle phase. We show that it is possible to selectively partition a poly(histidine)-tagged integral membrane protein into the polymer phase by metal affinity partitioning, with a shift in the partitioning coefficient from 0.015 to 4.8 (300-fold). The affinity partitioning was characterized and the effects of ligand concentration, pH, time, salts, buffer type, imidazole and charged detergent are discussed. Thus, a fast and mild affinity procedure for the purification of integral membrane proteins can be developed in affinity detergent/polymer aqueous two-phase systems, and the method is especially suitable for the purification of labile integral membrane proteins, such as receptors.

An Aqueous Polymer Two-Phase System as Carrier in the Spray-Drying of Biological Material

This investigation describes a novel concept in the formulation of carrier systems for the spray-drying of biological materials. As carrier material a system composed of poly(vinyl pyrrolidone) (PVP) and dextran was used. This system yields an aqueous two-phase system in which each phase is enriched in one of the polymers. By varying the composition of the system, the effective structure of a “stirred” system can be varied, covering the entire range from dextran continuous to PVP continuous. This facilitates encapsulation of either of these polymers in a spray-drying operation. In an attempt to investigate the spray-drying from such a system, the surface composition of the spray-dried powder obtained from various compositions of the two-phase system was analyzed by electron spectroscopy for chemical analysis (ESCA), providing information on the distribution of the polymers in the powder and thus also in the spray droplets. The two-phase system was applied for the spray-drying of live bacteria. The survival rate of the bacteria depended on the composition of the two-phase system. The storage stability of the bacteria in these formulations was investigated after storage at room temperature under dry conditions for 4 weeks, and it was found that the survival rate was 10–45%. The results therefore show that this type of formulation holds promise for future applications for micro-organisms as well as other sensitive biological materials such as proteins.

Purification of Rat C6 Glioma Plasma Membranes by Affinity Partitioning

We have studied the feasibility of purifying rat C6 glioma plasma membranes by a phase partitioning approach. The purification procedure involves cell homogenization and fractionation with an aqueous two-phase polymer system followed by selective affinity purification of plasma membranes by a wheat germ agglutinin-coupled polymer system. We demonstrate that the two-phase affinity partitioning technique is a simple and efficient method of isolating cell plasma membranes with high purity and yield. Furthermore, the isolated plasma membranes retain their functional integrity, as shown by the high-affinity insulin-like growth factor-I (IGF-I) binding capacity of IGF-I receptors.

Aqueous polymer two-phase systems formed by new thermoseparating polymers.

A set of new polymers that can be used as phase forming components in aqueous two-phase systems is presented. All polymers studied have thermoseparating properties i.e. form one separate polymer enriched phase and one aqueous solution when heated above the critical temperature. This property makes the polymers attractive alternatives to the polymers used in traditional aqueous two-phase systems such as poly(ethylene glycol) (PEG) and dextran. The thermal phase separation simplifies recycling of the polymers, thus making the aqueous two-phase systems more cost efficient and suitable for use in large scale. Thermoseparating polymers studied have been copolymers of ethylene oxide and propylene oxide (EO-PO), poly (N-isopropylacrylamide) (poly-NIPAM), poly vinyl caprolactam (poly-VCL) and copolymers of N-isopropylacrylamide and vinyl caprolactam with vinyl imidazole (poly(NIPAM-VI) and poly(VCL-VI), respectively). In addition, the copolymer poly(NIPAM-VI) has the property to be uncharged at pH above 7.0 and positively charged at lower pH. This allows the partitioning of protein to be directed by changing the pH in the system instead of the traditional addition of salt to direct the partitioning. Hydrophobically modified EO-PO copolymer (HM-(EO-PO)) with alkyl groups (C14) at both ends forms two-phase system with for example poly(NIPAM-VI). The phase diagram for poly(NIPAM-VI)/HM-(EO-PO) was determined and the model proteins lysozyme and BSA were partitioned in this system. For BSA in poly(NIPAM-VI)/HM-(EO-PO) system a change in pH from 8.0 to 5.4 results in a change of partition coefficient from K = 0.8 to K = 5.1, i.e. BSA could be transferred from the HM-(EO-PO) phase to the poly(NIPAM-VI) phase. BSA partitioning in poly(NIPAM-VI)/HM-(EO-PO) system allows quantitative BSA recovery, and recoveries of poly(NIPAM-VI) and HM-(EO-PO) were 53% and 92%, respectively, after the thermoseparation step.

Redox activity ofHydrodictyon reticulatum plasmalemma vesicles

Plasmalemma vesicles with preserved redox activity were prepared from nets ofHydrodictyon reticulatum. Since the walls are mechanically very resistant, a combination of partial cell-wall enzyme digestion and ultrasonic homogenization had to be used for the disruption of cells. To isolate the plasma-membrane-enriched microsomal fraction separation in an aqueous two-phase polymer system was found to be most suitable. The right-side-out vesicles reduced added hexacyanoferrate(III) by electrons supplied either by a transmembrane flow or from external NADH.

In vivo survival of selected murine carrier red blood cells after separation by density gradients or aqueous polymer two-phase systems

In order to explore possibilities of using erythrocytes as carrier systems for delivery of pharmacological agents, we have studied the in vivo survival of murine carrier red blood cell populations enriched in young or old cells. Hypotonicisotonic dialysis has been used to modify the cells as carrier systems and Percoll/albumin density gradients or counter-current distribution in aqueous polymer two-phase systems to separate them according to age. Hypotonicisotonic dialysis produces a decrease in the red blood cell populations in vivo survival rate (from 9.5 to 7.8 days). Among the cells modified as carriers, the enriched young red blood cell populations show a higher in vivo survival (half-life 6.5–7.4 days) than populations made up of predominantly old red blood cells (half-life 4.7-6.2 days). Half-life of young or old circulating red blood cells was approximately one day longer when these cells were separated by counter-current distribution rather than by Percoll density gradients. Based on these results, hypotonic-isotonic dialysis of whole and enriched young or old red blood cell populations, with higher or lower survival rates, can be considered as a useful tool for modification of these cells as carriers. The final outcome of such changes can be translated into better control of plasma drug delivery during therapy.

Hyaluronic acid grafted with poly(ethylene glycol) as a novel peptide formulation

Hyaluronic acids (HA) grafted with poly(ethylene glycol) (PEG) (PEG-g-HA) were synthesized. The materials characterization, enzymatic degradability and peptide (insulin) release from solutions of the copolymers were examined. Distribution of bioactive peptides within the polymer chain is well-known for combinations of PEG and polysaccharides as aqueous polymer two-phase systems. Insulin was preferentially partitioned into the PEG phase in a PEG/HA solution system. Enzymatic degradation of the copolymers was strongly dependent on the PEG content. Thermal analysis revealed that PEG-g-HA exhibited a variation in phase-separated structures depending on the PEG content. The solution of PEG-g-HA enabled insulin to remain in the PEG moieties dispersed in the HA matrix. Leakage of insulin from the copolymers was dependent upon the PEG content. Leakage rate of insulin from copolymer containing between 7 and 39% by weight of PEG were similar. A dramatic increase in leakage rate occurred when the PEG content was increased to greater than 39% by weight. It is considered that the loaded insulin was partitioned into the PEG moieties and became entangled with the PEG chains. The conformational change of insulin was effectively prevented in PEG-g-HA solutions, although insulin was denatured in storage of both phosphate buffered solution and HA solution. Such a heterogeneous-structured polymeric solution may be advantageous as an injectable therapeutic formulation for ophthalmic or arthritis treatment.

Fractionation of the thylakoid membranes from tobacco. A tentative isolation of ‘end membrane’ and purified ‘stroma lamellae’ membranes

Thylakoids isolated from tobacco were fragmented by sonication and the vesicles so obtained were separated by partitioning in aqueous polymer two-phase systems. By this procedure, grana vesicles were separated from stroma exposed membrane vesicles. The latter vesicles could be further fractionated by countercurrent distribution, with dextran–polyethylene glycol phase systems, and divided into two main populations, tentatively named ‘stroma lamellae’ and ‘end membrane’. Both these vesicle preparations have high chlorophyll a/b ratio, high photosystem (PS) I and low PS II content, suggesting their origin from stroma exposed regions of the thylakoid. The two vesicle populations have been compared with respect to biochemical composition and photosynthetic activity. The ‘end membrane’ has a higher chlorophyll a/b ratio (5.7 vs. 4.7), higher P700 content (4.7 vs. 3.3 mmol/mol of chlorophyll). The ‘end membrane’ has the lowest PS II content, the ratio PS I/PS II being more than 10, as shown by EPR measurements. The PS II in both fractions is of the β-type. The decay of fluorescence is different for the two populations, the ‘stroma lamellae’ showing a very slow decay even in the presence of K 3Fe(CN) 6 as an acceptor. The two vesicle populations have very different surface properties: the end membranes prefer the upper phase much more than the stroma lamellae, a fact which was utilized for their separation. Arguments are presented which support the suggestion that the two vesicle populations originate from the grana end membranes and the stroma lamellae, respectively.

Pulsatile peptide release from multi-layered hydrogel formulations consisting of poly(ethylene glycol)-grafted and ungrafted dextrans

Multi-layered hydrogel formulations consisting of poly(ethylene glycol)-grafted dextran (PEG-g-Dex) and ungrafted Dex were investigated as a model of Pulsatile drug release. In these formulations, it is considered that the grafted PEG domains act as a drug reservoir dispersed in the Dex matrix based on aqueous polymer two-phase systems. The formulations exhibited surface-controlled degradation by dextranase, and insulin release was observed in a pulsatile manner because of the multi-layered structure, PEG-g-Dex hydrogel layers containing insulin and insulin-free Dex hydrogel layers. Thus, it is suggested that the multi-layered hydrogel formulations using PEG-g-Dex and Dex are feasible for chronopharmacological drug delivery systems.

The isolation and characterization of right-side-out plasma membrane vesicles from barley aleurone cells.

Examination of organelle- and membrane-specific processes such as signal transduction necessitates the use of plasma membrane vesicles with cytoplasmic side-in orientation. We are interested in the structural identity and subcellular localization of in vivo [32P]phosphoric acid ([32Pi])-labeled phosphoinositides, including the recently discovered phosphatidyl-scyllo-inositol, for signal transduction studies. In the first part of this investigation, plasma membrane vesicles from barley aleurone cells were isolated employing the aqueous polymer (Dextran and polyethylene glycol) two-phase partition method. The membrane vesicles that partitioned into the upper and lower phases of the aqueous polymer two-phase system were characterized and the purity of the vesicles ascertained by assaying for two marker enzymes, K+-stimulated, Mg2+-dependent adenosine triphosphatase (EC 3.6.1.3, ATPase), localized in the plasma membranes, and cytochrome c oxidase, localized in the mitochondria. Inhibitors for ATPases such as azide, molybdate, and vanadate were used to distinguish between plasma membrane-associated and intracellular membrane-associated ATPases. These inhibitor studies suggest that the plasma membrane preparation contained about 7% of intracellular membrane vesicles and the intracellular membrane fraction contained about 6% of plasma membrane vesicles. Orientation of the plasma membrane vesicles was ascertained by measuring the latent ATPase activity. These latency studies suggest that about 95% of the plasma membrane vesicles were of cytoplasmic side-in orientation. In the second part of this investigation, intracellular distribution and in vivo [32Pi] labeling of phosphoinositides in the plasma membranes and intracellular membranes were investigated. Preferential accumulation of [32Pi]-labeled phosphatidyl-myo-inositol monophosphate (myo-PIP) and phosphatidyl-myo-inositol bisphosphate (myo-PIP2) was observed in the plasma membrane. However, scyllo-phosphatidylinositol (scyllo-PI) was detected in both the plasma membrane and the intracellular membranes. The cellular concentration of myo-phosphoinositides was determined, and, after 24 h of labeling with [32Pi], the ratio of radiolabel in myo-PI, PIP, and PIP2 paralleled the relative concentrations in aleurone cells.

Smart Polymers and Protein Purification

Smart polymers undergo fast and reversible changes in microstructure triggered by small changes of medium property (pH, temperature, ionic strength). These properties of smart polymers were exploited for the development of new protein purification techniques: affinity precipitation, affinity partitioning, and temperature-induced elution in dye-affinity chromatography. Different smart polymers were used for affinity precipitation: Eudragit S 100 (a copolymer of methacrylic acid and methyl methacrylate which precipitates on decreasing pH); stoichiomet-ric polyelectrolyte complexes of poly(methacrylate) and poly(N-ethyl-4-vinylpyridium bromide); poly(N-vinyl caprolactam), and poly(N-iso-propylacrylamide) (both precipitate on heating aqueous solutions to 35–40°C or increase in ionic strength). Recombinant thermostable lac-tate dehydrogenase from a thermophile Bacillus stear other mophilus was purified by affinity partitioning in an aqueous two-phase polymer system formed by dextran and a copolymer of N-vinyl caprolactam and 1-vinyl imidazole. The enzyme partitioned preferentially into the copolymer phase in the presence of Cu ions. The thermoprecipitation of the copolymer at 45°C resulted in an aqueous solution of the purified en- At elevated temperatures, poly-VCL molecules are in a compact globule conformation, capable of binding only to a few ligands on the matrix. The enzyme lactate dehydrogenase from porcine muscle has better access to the ligands and binds to the column. With a decrease in temperature, the polymer molecules undergo transition to a more expanded coil conformation. Polymer molecules interact now with more ligands and begin to compete with the bound enzyme for the ligands. Finally, the bound enzyme is replaced by the expanded polymer chains. This system was used for lactate dehydrogenase purification. Porcine muscle homogenate was applied on a column until breakthrough at 40°C. The foreign proteins were washed out with 0.1 M KC1; then the column was cooled to room temperature and virtually homogeneous lactate dehydrogenase was eluted with the same buffer. The purification factor was 17; recovery was 90% (13).

Lattice thermodynamics for aqueous salt-polymer two-phase systems

A lattice model is presented to compute salt-induced liquid–liquid phase separation in aqueous polymer solutions. The Gibbs energy of mixing contains an electrostatic contribution given by Pitzer's extension of the Debye–Hückel function, and the extended Flory–Huggins theory that uses empirical functions of temperature and composition as binary interaction parameters. Our Flory–Huggins theory assumes complete dissociation of salt into ions, but it does not distinguish between cation and anion; our theory represents the water–salt–polymer mixture as an incompressible ternary system consisting of water, ion, and polymer. In the extended Flory–Huggins theory, the binary interaction parameter between water and ion, and that between water and polymer, are obtained by correlating the observed activity of water in each of the two relevant binary systems. The electrostatic contribution does not contain adjustable parameters. We show that the electrostatic contribution to the Gibbs energy of mixing is responsible for inducing salt–polymer aqueous two-phase systems. Calculated phase diagrams are compared with experiment for aqueous solutions containing polyethylene glycol and a single salt at room temperature. The efficiency of a salt to form salt–polymer aqueous two-phase systems is discussed in terms of ion valence and the interaction parameter between ion and polymer. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci Inc. J Appl Polym Sci 68: 2007–2017, 1998

Lattice thermodynamics for aqueous salt–polymer two‐phase systems

A lattice model is presented to compute salt-induced liquid–liquid phase separation in aqueous polymer solutions. The Gibbs energy of mixing contains an electrostatic contribution given by Pitzer's extension of the Debye–Hückel function, and the extended Flory–Huggins theory that uses empirical functions of temperature and composition as binary interaction parameters. Our Flory–Huggins theory assumes complete dissociation of salt into ions, but it does not distinguish between cation and anion; our theory represents the water–salt–polymer mixture as an incompressible ternary system consisting of water, ion, and polymer. In the extended Flory–Huggins theory, the binary interaction parameter between water and ion, and that between water and polymer, are obtained by correlating the observed activity of water in each of the two relevant binary systems. The electrostatic contribution does not contain adjustable parameters. We show that the electrostatic contribution to the Gibbs energy of mixing is responsible for inducing salt–polymer aqueous two-phase systems. Calculated phase diagrams are compared with experiment for aqueous solutions containing polyethylene glycol and a single salt at room temperature. The efficiency of a salt to form salt–polymer aqueous two-phase systems is discussed in terms of ion valence and the interaction parameter between ion and polymer. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci Inc. J Appl Polym Sci 68: 2007–2017, 1998

Comparison of Mg 2+-dependent ATP hydrolase activities of pea nodule symbiosomes and of pea root plasmalemma, obtained by an aqueous polymer two-phase system

The characteristics of the Mg 2+-dependent ATPase activity from the peribacteroid membrane of pea symbiosomes was compared with that from pea root plasma membranes. Enzyme inhibitors, optimum reaction pH, substrate specificity and antibody recognition were the main parameters examined. Both the symbiosomes and the root plasma membrane were purified with an aqueous polymer two-phase system (APS). The final concentration of the APS for the purification of symbiosomes were: 6.3% w/w dextran T500, 6.3% w/w poly(ethylene glycol) 3350, 5 m M KH 2PO 4–K 2HPO 4, 5 m M KCl, 0.33 M sucrose, (pH 7.85); for the root plasma membrane was: 6.2% (w/w) dextran T500, 6.2% poly(ethylene glycol) 3350, 330 m M sucrose, 5 m M K 2HPO 4 and 4 m M KCl (pH 7.8). The lack of contamination of pea symbiosomes with endoplasmic reticulum, mitochondria, broken bacteroids and/or tonoplast vesicles was established. Similarly, the aqueous two-phase system used here provided a fairly enriched root plasma membrane with low cross-contamination from other sources. Both symbiosomal and root plasma membrane ATPase activities were highly specific to ATP. The symbiosome ATPase apparently corresponds to an E 1E 2–ATPase mechanism, similar to that found at the plasma membrane. The similarity between these two ATPases was further supported by immuno-analysis. Mg 2+-ATPase of pea symbiosome and root plasma membranes were very similar, by all parameters tested.

Surface and metabolic properties of microcytic and macrocytic human anaemic red blood cells detected in polymer aqueous two-phase systems

Microcytic and macrocytic red blood cells from anaemic patients have been fractionated as a function of cell surface properties by the countercurrent distribution technique using charge-sensitive dextran/poly(ethylene glycol) aqueous two-phase systems. As deduced from the fractionation profiles, microcytic cells constitute a heterogeneous cell population with decreased surface charge properties while, macrocytic cells constitute a homogeneous cell population with behaviour similar to that of the control red blood cells. The specific activity of pyruvate kinase, an age-dependent enzyme, did not change along microcytic red blood cells fractionation profiles, suggesting that such cells have altered ageing properties. However, pyruvate kinase specific activity decreases from the left- to the right-hand side of the fractionation profile of macrocytic red blood cells, indicating that these cells follow the normal ageing process. Bisphosphoglycerate mutase specific activity did not change along the fractionation profile of any cell population under study, thus providing 2,3-bisphosphoglycerate during the life-span of the red blood cells from anaemic patients.