Purification of glucose-6-phosphate dehydrogenase from baker’s yeast in aqueous two-phase systems with free triazine dyes as affinity ligands

To improve the selectivity of glucose-6-phosphate dehydrogenase (G6PDH) extraction by an aqueous two-phase system, a simple and inexpensive affinity aqueous two-phase system using unbound reactive triazine dyes as ligands was introduced. In a polyethylene glycol (PEG)/hydroxypropyl starch (PES) system, the unbound free triazine dyes, Cibacron Blue F3GA and Procion Red HE3B, partitioned unevenly in the top PEG-rich phase and thus showed an affinity effect on G6PDH, but no influence on hexokinase. The various parameters investigated were pH of the system, buffers, molecular weight of PEG, and ligand type and concentration. A two-step affinity extraction process was established for the purification of G6PDH from baker’s yeast. The total yield of G6PDH was 66.9% and purification factor was 2.35.     

Affinity partitioning of human antibodies in aqueous two-phase systems

The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.     

蛋白质在表面活性剂与高分子共组双水相体系中 的分配

高分子和正负离子表面活性剂混合物可形成一种新型双水相体系。研究蛋白质在溴化十二烷基三乙铵/十二烷基硫酸钠与聚氧乙烯(EO)-聚氧丙烯(PO)嵌段共聚物(EO~2~0PO~8~0)共组双水相体系中的分配。通过在高分子接上亲和配基,研究蛋白质在带有亲和配基高分子的双水相体系中的分配。将表面活性剂富集相稀释或加热高分子富集相,又可形成新的双水相体系,由此可进行蛋白质的多步分配。在蛋白质的分配完成之后,通过将表面活性剂富集相进一步稀释或将高分子富集相加热至高分子浊点以上可将表面活性剂和高分子与目标蛋白质分离。正负离子表面活性剂和高分子还可以循环使用。     

Detection and purification of glucose 6-phosphate dehydrogenase, malic enzyme, and NADP-dependent isocitrate dehydrogenase by blue native polyacrylamide gel electrophoresis

We describe a blue native polyacrylamide gel electrophoretic technique that allows the facile detection, quantitation and purification of three NADPH-producing enzymes. Glucose 6-phosphate dehydrogenase, malic enzyme and NADP-dependent isocitrate dehydrogenase were detected simultaneously. Activity staining based on the formation of NADPH from the respective substrates and the subsequent precipitation of formazan enabled the relative quantitation of enzymatic activities, while Coomassie staining on one-dimensional or two-dimensional gels helped monitor the amount of protein associated with these enzymatic activities. This technique provides a simple and effective route to obtain homogeneous protein for further analyses and also enables the screening of these NADPH-producing enzymes in various cellular systems.     

Polyethyleneglycol-pepsin interaction and its relationship with protein partitioning in aqueous two-phase systems

The interaction between the acidic protein, pepsin, and the non-charged polyethyleneglycol polymer was studied by dynamic light scattering, fluorescence spectroscopy and measurements of the protein thermal stability at neutral pH. Polyethyleneglycol of average molecular mass 1450 showed a higher interaction capacity with the protein than polyethyleneglycol of average molecular mass 8000. Polyethyleneglycol of average molecular mass 1450 showed a molecular mechanism where the interpolymer interaction led to the complex formation. This fact can be explained taking into account that the extended form on this polymer molecule favours the interaction with the protein, which is highly dependent of the polymer total concentration. Polyethyleneglycol of average molecular mass 8000 showed a cooperative interaction between the polymer and protein molecules which was independent of the PEG concentration.     

Partitioning of lactate dehydrogenase from bovine heart crude extract by polyethylene glycol-citrate aqueous two-phase systems

Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG)-citrate have been used for enzyme partitioning studies. The behavior of lactate dehydrogenase (LDH) from bovine heart crude extract was analyzed using a two-level factorial design in which the PEG molar mass and concentration, the citrate concentration were selected as independent variables, while the purification factor, the partition coefficient (K) and the activity yield were selected as responses. The statistical analysis revealed the effect of PEG molar mass on K. LDH exhibited a better partitioning toward PEG-rich phase and the highest K value (1079.81) was obtained with 42% (w/w) PEG 400 and 7.5% (w/w) citrate concentration. PEG molar mass also influenced the purification factor of the enzyme in the top phase. Possibly these ATPS remove inhibitors present in the extract affording higher enzyme yield.     

Affinity partitioning of proteins tagged with choline-binding modules in aqueous two-phase systems

We present a novel procedure for affinity partitioning of recombinant proteins fused to the choline-binding module C-LytA in aqueous two-phase systems containing poly(ethylene glycol) (PEG). Proteins tagged with the C-LytA module and exposed to the two-phase systems are quantitatively localized in the PEG-rich phase, whereas subsequent addition of the natural ligand choline specifically shifts their localization to the PEG-poor phase by displacement of the polymer from the binding sites. The described procedure is simple, scalable and reproducible, and has been successfully applied to the purification of four diverse proteins, resulting in high yields and purity.     

Enhancement of riboflavin-mediated photo-oxidation of glucose 6-phosphate dehydrogenase by urocanic acid

We have investigated the riboflavin (RF)-sensitized inactivation of glucose 6-phosphate dehydrogenase (G6PD) in the presence and absence of trans-urocanic acid (UCA). The inactivation of the enzyme results from its direct oxidation by the excited triplet RF in a Type-I-photosensitized reaction whose efficiency increases at low oxygen concentration. The addition of histidine to the system produced no change in the inactivation rate, discarding the participation of singlet oxygen in the reaction. On the other hand, the presence of UCA results in its bleaching, with a significant enhancement of RF-mediated inactivation of G6PD. Both the consumption of UCA and G6PD are faster at low oxygen concentrations. UCA also produced a decrease in the sensitizer photodecomposition yield. These results indicate that the enhancement of the RF-mediated G6PD inactivation observed in the presence of UCA is not a singlet oxygen-mediated process. It is proposed that UCA consumption and its effect on G6PD inactivation are due to a complex reaction sequence initiated by a direct oxidation of UCA by the excited sensitizer triplet. The oxidation of the semireduced flavin gives rise to reactive oxygen species (ROS) responsible for the increased rate of the process. This is supported by the protection afforded by several additives with ROS removal capacity: benzoate, superoxide dismutase and catalase.     

Solvent properties governing protein partitioning in polymer/polymer aqueous two-phase systems

Distribution coefficients of various proteins were measured in aqueous Dextran-Ficoll, Dextran-PES, and Ficoll-PES two-phase systems, containing 0.15M NaCl in 0.01 M phosphate buffer, pH 7.4. The acquired data were combined with data for the same proteins in different systems reported previously and known solvatochromic solvent properties of the systems to characterize the protein-solvent interactions. The relative susceptibilities of proteins to solvent dipolarity/polarizability, solvent hydrogen bond acidity, solvent hydrogen bond basicity, and solvent ability to participate in ion-ion and ion-dipole interactions were characterized. These parameters, which are representative of solute-solvent interactions, adequately described the partitioning of the proteins in each system. It was found that the relative susceptibilities of proteins to solvent dipolarity/polarizability are interrelated with their relative susceptibilities to solvent hydrogen bond acidity and solvent hydrogen bond basicity similarly to those established previously for small nonionic organic compounds.     

Effects of organic solvents on the partitioning of enzymes in aqueous two-phase systems

Organic solvents (ethylene glycol, glycerol, dimethyl sulphoxide, dimethylformamide, dioxane, methanol and propanol-2, as well as sucrose and urea) have been included in aqueous two-phase (liquid-liquid) systems comprised of water, dextran and poly(ethylene glycol). The concentration of the organic solvent was in most cases 20% (w/w). The influence of these solvents on the phase-forming properties, the volume ratio, the freezing point and the partitioning of a polymer-bound ligand, Procion Red HE-3B poly(ethylene glycol), has been studied. The partition coefficients for alkaline phosphatase decrease with ethylene glycol, glycerol, sucrose and urea (factors of 0.25-0.5), but increase with the other substances (factors of 1.2-1.6). The temperature effects on the partitioning of alkaline phosphatase from calf intestine as well as of phosphofructokinase from yeast in systems containing ethylene glycol have been studied and compared with partitioning in standard systems, not containing solvents. The possible uses of the above systems for partitioning studies of enzymes are discussed.     

Dual affinity method for plasmid DNA purification in aqueous two-phase systems

The DNA binding fusion protein, LacI–His₆–GFP, together with the conjugate PEG–IDA–Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600–DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG–IDA–Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG–dextran system as a second extraction system, with 80–90% of pDNA partitioning to the bottom phase. This represents about 7.4 μg of pDNA extracted per 1 mL of pUC19 desalted lysate.     

Radiation target analyses of free and immobilized glucose 6-phosphate dehydrogenase

The sensitivity of the enzyme glucose 6-phosphate dehydrogenase to ionizing radiation was examined under several conditions, including the presence of several free-radical scavengers. The enzyme was also irradiated when covalently bound to polyacrylamide beads whose structure is very similar to the polypeptide backbone of proteins. All the enzyme forms were irradiated in the frozen state with high-energy electrons from a linear accelerator. Surviving enzyme activity and surviving monomers were determined; the data were analyzed by target theory.Free-radical scavengers reduced the radiation target size of both the activity and monomers of the free enzyme, but not that of the immobilized enzyme activity. The target size of the activity of the free enzyme was that of a dimer mass, but in the case of the immobilized enzyme it was equal to the smaller mass of the monomer.Free-radical scavengers reduce the target size by modifying radiation energy transfer. The target size of the polyacrylamide-bound enzyme activity was expected to be very large since the connection between polyacrylamide and protein is a peptide bond which permits transfer of radiation-deposited energy. Several explanations concerning energy transfer are suggested for this result.     

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.     

Polyethyleneglycol–pepsin interaction and its relationship with protein partitioning in aqueous two-phase systems

The interaction between the acidic protein, pepsin, and the non-charged polyethyleneglycol polymer was studied by dynamic light scattering, fluorescence spectroscopy and measurements of the protein thermal stability at neutral pH. Polyethyleneglycol of average molecular mass 1450 showed a higher interaction capacity with the protein than polyethyleneglycol of average molecular mass 8000. Polyethyleneglycol of average molecular mass 1450 showed a molecular mechanism where the interpolymer interaction led to the complex formation. This fact can be explained taking into account that the extended form on this polymer molecule favours the interaction with the protein, which is highly dependent of the polymer total concentration. Polyethyleneglycol of average molecular mass 8000 showed a cooperative interaction between the polymer and protein molecules which was independent of the PEG concentration.     

Purification of human red cell glucose 6-phosphate dehydrogenase by affinity chromatography.

Human glucose 6-phosphate dehydrogenase associated with NADPH was efficiently bound with agarose-bound NADP, whereas the enzyme associated with NADP was poorly bound with agarose-bound NADP. After the elimination of haemoglobin from haemolyzate by treatment with DEAE-cellulose, the enzyme was converted into the NADPH-bound form and was applied on an affinity column. The enzyme was specifically eluted from the column by NADP in the elution buffer. A homogeneous enzyme preparation was obtained in high yield.     

Horseradish peroxidase extraction and purification by aqueous two-phase partition

The effect of poly(ethyleneglycol) (PEG) molecular weight, system pH, and sodium chloride concentration on the partitioning behavior of horseradish peroxidase fromArmomcia rusticana root extract was investigated in poly(ethyleneglycol)/sodium phosphate systems. PEG molecular weight strongly affects the enzyme partition coefficient, whereas pH variation from 5.5 to 8.0 has little effect. The addition of sodium chloride (8% w/w) to a PEG 1540/phosphate system, pH 7.0, raises the peroxidase partition coefficient 13.5-fold without important changes in that of total horseradish root proteins. Moreover, these conditions allow direct homogenization of theA. rusticana roots with the selected aqueous two-phase system with the clear top phase containing over 90% of the enzyme and the purification factor being 4.8.     

Extraction of the antimicrobial peptide cerein 8A by aqueous two-phase systems and aqueous two-phase micellar systems

Cerein 8A is an antimicrobial peptide with potential application against food spoilage and pathogenic bacteria. The partitioning of cerein 8A was investigated in two liquid-liquid extraction systems that are considered promising for bioseparation and purification purposes. Aqueous two-phase systems (ATPSs) were prepared with polyethylene glycol (PEG) and inorganic salts, and the addition of NaCl was investigated in this system. The best results concerning partition coefficients (K (b)) were obtained with PEG?+?ammonium sulphate, and K (b) value significantly increases when NaCl was added. Cerein 8A was effectively extracted into the micelle-rich phase in a 4% Triton X-114 medium. Recovery yield was higher for ATPS compared to micellar systems. Cerein 8A can be isolated from a crude suspension containing the bioactive molecule by ATPSs. Successful implementation of peptide partitioning represents an important step towards developing a low-cost effective separation method for cerein 8A.     

Membrane protein isolation by in situ solubilization, partitioning and affinity adsorption in aqueous two-phase systems. Purification of the human type 1 11beta-hydroxysteroid dehydrogenase

Recently developed aqueous two-phase systems based on non-ionic detergents and polymers are suitable for the separation of membrane proteins. Moreover, within this relatively membrane protein "friendly" environment, changes in temperature can be controlled and stabilizing agents may be added to ensure integrity of the target protein during isolation. Here, we use aqueous two-phase partitioning for the isolation of membrane bound 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). Different detergents were used to find optimal conditions regarding solubilization and retaining target protein activity. We explored in situ solubilization by adding detergent directly to the aqueous two-phase system, as well as a batch metal affinity capture step of 6xHis tagged 11beta-HSD1 in the two-phase system. The use of detergent/polymer two-phase systems resulted in a specific enzyme activity of 3840 nmol mg(-1) min(-1) of the target membrane protein compared to a conventional purification protocol where a specific enzyme activity of 1440 nmol mg(-1) min(-1) was achieved.     

Phase equilibrium and insulin partitioning in aqueous two-phase systems containing block copolymers and potassium phosphate

In this work, phase diagrams of aqueous two-phase systems (ATPS) containing PEO–PPO–PEO block copolymers and potassium phosphate as well as the partitioning behavior of insulin in these systems are presented. Experiments aimed at the identification of the effects of copolymer structure (by varying the number of EO units per polymer molecule), temperature (283.15 and 298.15 K) and pH (5.0 and 7.0) on the phase behavior of these systems were carried out. The results indicated the enlargement of the two-phase region upon increasing the temperature, pH and copolymer hydrophobicity (expressed as the PO/EO ratio in the copolymer molecule). Experimental measurements of the partitioning of human insulin in these ATPS also indicated the dependency of the partition coefficients on temperature, pH, and copolymer hydrophobicity, with the latter being the most influential factor. Finally, experimental data on the phase behavior and insulin partitioning were correlated using an excess Gibbs energy virial-type model modified in order to account for coulombic interactions and ionization equilibrium between the various forms of the phosphate ion.