The effect of KSCN on the partition of proteins in polyethylene glycol/(NH4)2SO4 aqueous two-phase system

The effect of potassium thiocyanate on the partitioning of lysozyme and BSA in polyethylene glycol 2000/ammonium sulfate aqueous two-phase system has been investigated. As a result of the addition of potassium thiocyanate to the PEG/ammonium sulfate system, the PEG/mixed salts aqueous two-phase system was formed. It was found that the potassium thiocyanate could alter the pH difference between the two phases, and, thus, influence the partition coefficients of the differently charged proteins. The relationship between partition coefficient of the proteins and pH difference between two phases has been discussed. It was proposed that the pH difference between two phases could be employed as the measurement of electrostatic driving force for the partitioning of charged proteins in polyethylene 2000/ammonium sulfate aqueous two-phase system.     

Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation

The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored. The partitioning behavior of P450 BM-3 in N-Me-HM-EO/buffer systems was measured. The influences of some factors such as total proteins concentration, pH, temperature and salt concentration, on the partitioning coefficients of P450 BM-3 were investigated. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to conduct the proteins with different charges to top or bottom phases by adjusting pH and salt concentration in the system. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction with N-Me-HM-EO/buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The recovery of P450 BM-3 was 73.3% with the purification factor of 2.5. The results indicated that the aqueous micellar two-phase system composed of hyamine modified polysoap has a promising application for selective separation of biomolecules depending on the enhanced electrostatic interactions between micelles and proteins.     

固定化青霉素酰化酶在光-pH敏感可回用两水相中裂解青霉素G为6-APA

两水相体系在发展中存在的关键问题是相体系回收困难.由于生产成本及降低污染的原因, 用过的相体系需要回收和重复使用.用环境敏感型溶解可逆聚合物形成可回用两水相体系是当前是为可行的回收方法。本文在光敏感可回用高聚物PNBC与pH敏感型可回用高聚物PADB形成的两水相体系中进行固定化青霉素酰化酶的相转移催化青霉素G产生6-APA的反应。在这个两水相体系中,通过优化,在1% NaCl 存在下,６－ＡＰＡ的分配系数可达5.78。催化动力学显示,达平衡的时间近7h,反应最高得率约85.3%（pH 7.8, 20℃）。较相近条件下的单水相反应得率提高近20%。在反应过程中,通过底物及产物的分配系数检测,发现底物分配系数变化不大,而产物6-APA及苯乙酸的分配系数发生很大变化,从而引起产物的得率变化。在两水相中,底物及产物主要分配在上相,固定化酶分配在下相,底物青霉素G进入下相经酶催化产生的6-APA及苯乙酸又转入上相,从而解除了青霉素酰化酶催化反应的底物及产物抑制作用,达到提高产物得率的效果。此外,采用固定化酶较固定化细胞效率高,占用下相体积小,较游离酶稳定性高,且完全单侧分配在下相。因此,在两水相中进行固定化酶的催化反应具有明显的优越性。形成两水相的高聚物PNBC通过488 nm 的激光照射或经滤光的450nm 光源照射得到回收;pH敏感型成相聚合物PADB可通等电点 4.1沉淀可实现循环利用,高聚物的回收率在95%-98%之间,按此回收率计算,聚合物可使用60次以上。     

牛血清白蛋白在聚乙二醇/葡聚糖双水相体系中分配特性的研究

采用考马斯亮蓝G250染色法测得室温下BSA在PEG/dextran双水相体系中的分配系数。以BSA在PEG/dextran体系的下相富集为目标,研究了PEG的分子量、浓度、dextran浓度以及所加入中性盐的种类与浓度、体系pH诸因素对其分配特性的影响。实验结果表明,在PEG4000/dextran体系中,采用PEG质量分数9%-dextran质量分数9%的浓度组成,同时在pH=7.0,NaC l浓度为0.2 mol.L-1或pH6.0,NaC l浓度为0.34 mol.L-1的工艺条件下萃取BSA均可达最小分配系数,其值为0.014。     

Conformational and electrostatic properties of unoccupied and liganded estrogen receptors determined by aqueous two-phase partitioning

The technique of aqueous two-phase partitioning (ATPP) has been used to characterize conformational and electrostatic properties of unoccupied and liganded rat uterine estrogen receptors. The adaptation of the hydroxylapatite receptor assay with ATPP systems has permitted estrogen receptor (ER) partition coefficients to be accurately determined, even when the partitioning process results in significant loss of ER binding capacity. The pH and salt dependences of estrogen receptor partition coefficients indicate that the theory governing partitioning behavior can be accurately applied to partitioning data obtained with crude cytosols. This technique has revealed a ligand-induced change in the properties of the unoccupied receptor that precedes the process of heat-induced transformation in vitro. The difference in partitioning behavior between unoccupied and nontransformed estrogen receptor is observed in all combinations of buffers and salts tested and is of equal magnitude as the difference between partition coefficients of nontransformed and transformed ER. The partition coefficients of both unoccupied and nontransformed ER are constant over the ER concentration range in which binding cooperativity has been previously demonstrated. The combined effects of salt and pH on ER partition coefficients indicate a pI of approximately 5.5 for both unoccupied and nontransformed estrogen receptors. However, the partition coefficients at the pI differ. It is concluded that estradiol binding to its unoccupied receptor results in a change in surface properties of the ER monomer that is independent of receptor transformation and makes the receptor less hydrophobic.     

Amino acid/water interactions study: a new amino acid scale

Partition ratios of 8 free l-amino acids (Gln, Glu, His, Lys, Met, Ser, Thr, and Tyr) were measured in 10 different polymer/polymer aqueous two-phase systems containing 0.15?M NaCl in 0.01?M phosphate buffer, pH 7.4. The solute-specific coefficients representing the solute dipole/dipole, hydrogen-bonding and electrostatic interactions with the aqueous environment of the amino acids were determined by multiple linear regression analysis using a modified linear solvation energy relationship. The solute-specific coefficients determined in this study together with the solute-specific coefficients reported previously for amino acids with non-polar side-chains where used in a Quantitative Structure/Property Relationship analysis. It is shown that linear combinations of these solute-specific coefficients are correlated well with various physicochemical, structural, and biological properties of amino acids.     

Influence of electrostatic interactions on partitioning in aqueous polyethylene glycol/dextran biphasic systems: Part I

To study the influence of charges on the partition of solutes in aqueous two-phase systems of polyethylene glycol and dextran, partition coefficients of dimethylaminoethyl-dextran, trimethylamino-dextran, and bis (alpha,omega)-amino-poly(ethylene glycol) were determined as a function of pH (range 2 to 12) and ionic strength. These polymers are derivatives of the phase forming components and carry ionizable groups that are charged or uncharged depending on the pH. Unexpectedly, the largest differences in the partition coefficients were found at high pH, where the modified polymers are uncharged. In addition, the partitioning of low-molecular-weight model compounds, ethylenediamine and iminodiacetic acid, as well as poly-L-lysine and poly(allylamine) was analyzed. A consistent pattern was observed in the partition of polyelectrolytes reflecting the influence of charge, but another property of aqueous phase systems unrelated to charge and changing with pH seems to be superimposed. (c) 1995 John Wiley & Sons, Inc.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.     

Protein sorption and recovery by hydrogels using principles of aqueous two-phase extraction

Use of the thermodynamic principles of aqueous two-phase extraction (ATPE) to drive protein into a crosslinked gel is developed as a protein isolation and separation technique, and as a protein loading technique for drug delivery applications. A PEG/dextran gel system was chosen as a model system because PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex(R)) are common chromatographic media. The effects of polymer concentrations and molecular weights, salts, and pH on the partitioning of ovalbumin matched ATPE heuristics and data trends. Gel partition coefficients (Cgel/Csolution) increased with increasing PEG molecular weight and concentration and decreasing dextran concentration (increased gel swelling). The addition of PEG to the buffer solution yielded partition coefficients more than an order of magnitude greater than those obtained in systems with buffer alone, or added salt. A combined salt/PEG system yielded an additional order of magnitude increase. For example, when ovalbumin solution (2.3 mg/mL) was equilibrated with Sephadex(R) G-50 at pH 6.75, the partition coefficients were 0.13 in buffer, 0.11 in buffer with 0.22M KI, 2.3 in 12 wt% PEG-10,000 and 32.0 in 12 wt% PEG-10, 000 with 0.22M KI. The effect of anions and cations as well as ionic strength and pH on the partitioning of ovalbumin also matched ATPE heuristics. Using the heuristics established above, partition coefficients as high as 80 for bovine serum albumin and protein recoveries over 90% were achieved. In addition, the wide range of partition coefficients that were obtained for different proteins suggests the potential of the technique for separating proteins. Also, ovalbumin sorption capacities in dextran were as high as 450 mg/g dry polymer, and the sorption isotherms were linear over a broad protein concentration range.     

Glucose-6-phosphate dehydrogenase partitioning in two-phase aqueous mixed (nonionic/cationic) micellar systems

The enzyme glucose-6-phosphate dehydrogenase (G6PD) plays an important role in maintaining the level of NADPH and in producing pentose phosphates for nucleotide biosynthesis. It is also of great value as an analytical reagent, being used in various quantitative assays. In searching for new strategies to purify this enzyme, the partitioning of G6PD in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated both experimentally and theoretically. Our results indicate that the use of a two-phase aqueous mixed micellar system composed of the nonionic surfactant C(10)E(4) (n-decyl tetra(ethylene oxide)) and the cationic surfactant C(n)TAB (alkyltrimethylammonium bromide, n = 8, 10, or 12) can improve significantly the partitioning behavior of G6PD relative to that obtained in the two-phase aqueous C(10)E(4) micellar system. This improvement can be attributed to electrostatic attractions between the positively charged mixed (nonionic/cationic) micelles and the net negatively charged enzyme G6PD, resulting in the preferential partitioning of G6PD to the top, mixed micelle-rich phase of the two-phase aqueous mixed micellar systems. The effect of varying the cationic surfactant tail length (n = 8, 10, and 12) on the denaturation and partitioning behavior of G6PD in the C(10)E(4) /C(n)TAB/buffer system was investigated. It was found that C(8)TAB is the least denaturing to G6PD, followed by C(10)TAB and C(12)TAB. However, the C(10)E(4)/C(12)TAB/buffer system generated stronger electrostatic attractions with the net negatively charged enzyme G6PD than the C(10)E(4)/C(10)TAB/buffer and the C(10)E(4)/C(8)TAB/buffer systems, when using the same amount of cationic surfactant. Overall, the two-phase aqueous mixed (C(10)E(4)/C(10)TAB) micellar system yielded the highest G6PD partition coefficient of 7.7, with a G6PD yield in the top phase of 71%, providing the optimal balance between the denaturing effect and the electrostatic attractions for the three cationic surfactants examined. A recently developed theoretical framework to predict protein partition coefficients in two-phase aqueous mixed (nonionic/ionic) micellar systems was implemented, and the theoretically predicted G6PD partition coefficients were found to be in reasonable quantitative agreement with the experimentally measured ones.     

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH₄)₂SO₄], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.     

Protein partitioning in weakly charged polymer-surfactant aqueous two-phase systems

The study includes partitioning of proteins in aqueous two-phase systems consisting of the polymer dextran and the non-ionic surfactant C₁₂E₅ (pentaethylene glycol mono-n-dodecyl ether). In this system a micelle-enriched phase is in equilibrium with a polymer-enriched phase. Charges can be introduced into the micelles by the addition of charged surfactants. The charge of the mixed micelles is easily varied in sign and magnitude independently of pH, by the addition of different amounts of negatively charged surfactant, sodium dodecyl sulphate (SDS), or positively charged surfactant dodecyl trimethyl ammonium chloride (DoTAC). A series of water-soluble model proteins (BSA, β-lactoglobulin, myoglobin, cytochrome c and lysozyme), with different net charges at pH 7.1, have been partitioned in non-charged systems and in systems with charged mixed micelles or charged polymer (dextran sulphate). It is shown that partition coefficients for charged proteins in dextran-C₁₂E₅ systems can be strongly affected by addition of charged surfactants (SDS, DoTAC) or polymer (dextran sulphate) and that the effects are directly correlated to protein net charge.     

The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II

In aqueous polyethylene glycol/dextran two-phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly-L-glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro- molecules. (c) 1995 John Wiley & Sons, Inc.     

Enzymatic hydrolysis of cellulose in aqueous two-phase systems. I. Partition of cellulases from Trichoderma reesei

The partitioning of endo-beta-glucanase, exo-beta-glucanase, and beta-glucosidase from Trichoderma reesei QM 9414 in aqueous two-phase systems has been studied with the object of designing a phase system for continuous bioconversion of cellulose. The partitioning of the enzymes in two-phase systems composed of various water soluble polymeric compounds were studied. Systems based on dextran and polyethylene glycol (PEG) were optimal for one-sidedly partitioning the enzymes to the bottom phase. The influence of polymer molecular weights, polymer concentration, ionic composition of the medium, pH, temperature, and adsorption of the enzymes to cellulose on the enzyme partition coefficients (K) were studied. By combining the effects of polymer molecular weight and adsorption to cellulose, K values could be reduced for endo-beta-glucanase to 0.02 and for beta-glucosidase to 0.005 at 20 degrees C in a phase system of Dextran 40-PEG 40000 in the presence of excess cellulose, At 50 degrees C, K values were increased by a factor of two. In a phase system based on inexpensive crude dextran and PEG, the partition coefficient for endo-beta-glucanase was 0.16 and for beta-glucosidase was 0.14 at 20 degrees C with excess cellulose present.     

Enzyme mass-transfer coefficient in aqueous two-phase systems using static mixer extraction column

Recent technical advances in aqueous two-phase systems (ATPS) have made this a sound technique for the extraction of biomacromolecules. The extraction of alpha-amylase was investigated using aqueous two-phase systems formed by sodium sulphate-polyethylene glycol (PEG) in water in a 47-mm inner diameter spray column packed with three types of static mixers. The effects of dispersed-phase flow rate, phase composition, column height and diameter were studied. The extraction column was operated in a semi-batch manner. It was found that the hold-up and volumetric mass transfer coefficients increased with an increase in dispersed (PEG-rich) phase velocity and decreased with increasing phase composition. Empirical correlations were developed for fractional dispersed-phase hold-up and volumetric mass transfer coefficients.     

Aqueous two-phase metal affinity extraction of heme proteins

An iminodiacetic acid derivative of poly(ethylene glycol) (PEG-IDA) that chelates metal cations has been synthesized and used to extract proteins in metal affinity aqueous two-phase PEG/dextran systems. With less than 1% of the PEG substituted with chelated copper, partition coefficients are shown to increase by factors of up to 37 over extraction with unsubstituted PEG. The proteins studied are preferentially extracted into the Cu(II)PEGIDA phase in proportion to the number of accessible histidine residues on their surface. The affinity contribution to partitioning is proportional to the number of exposed histidine over a very wide range. The partition coefficients of heme-containing proteins measured in the Cu(II)PEG-IDA/dextran systems increase with the pH of the extraction mixture from pH 5.5 to pH 8.0, while partition coefficients in the unsubstituted PEG/dextran systems are very nearly independent of pH. The strong pH dependence of the metalaffinity extraction can be utilized in the recovery of the extracted protein.     

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.     

Partitioning of low molecular combination peptides in aqueous two-phase systems of poly(ethylene glycol) and dextran in the presence of small amounts of K2HPO4/KH2PO4 buffer at 293 K: experimental results and predictions

Buffered aqueous two-phase systems are effective extraction systems for separating amphoteric hydrocarbons like, for example, polypeptides from aqueous phases. The design and basic engineering of such processes requires the knowledge of the liquid-liquid equilibrium. The study presented here aims to contribute to the development of methods to predict the partitioning of peptides in aqueous two-phase systems. Experimental results are reported for the partitioning of small amounts ( approximately 0.001 g solute per gram of solution) of low molecular combination peptides of glycine, L-glutamic acid, L-phenylalanine, and L-lysine (9 dipeptides, gly-glu, gly-phe, gly-lys, glu-gly, phe-gly, phe-glu, lys-gly, lys-glu, lys-phe; 7 tripeptides, gly-gly-phe, gly-phe-gly, glu-gly-phe, phe-gly-gly, lys-gly-lys, lys-glu-gly, lys-phe-lys) in aqueous two-phase systems of high molecular weight dextran (molecular weight about 500,000) and poly(ethylene glycol) (molecular weight about 6,000 and 35,000, respectively) in the presence of small amounts (about 0.05 mol/kg) of K2HPO4/KH2PO4 buffer at about 293 K. The new data are compared to predictions. Partition coefficients are predicted applying a group contribution excess Gibbs energy model. The model is an osmotic virial equation. It uses surface fractions to encounter for the probability of interactions between solutes. All model parameters were taken from the literature. They were determined exclusively from experimental data for the phase forming systems and for the partitioning of amino acids and their di- and tripeptides (containing only a single amino acid), but no experimental data for the partitioning of combinations peptides were used. In most cases predicted partition coefficients agree favourably with the experimental data.     

A model for the prediction of partition coefficients in aqueous two-phase systems.

A mathematical model has been developed to predict partition coefficients for aqueous two-phase systems. The model is based on a previously-developed equation for partitioning which arises from an osmotic pressure viral expansion. The model suggests that the properties of importance are the concentration difference of one of the phase-forming components, such as a polymer, and the hydrophobicity of the solute relative to the hydrophobic-hydrophilic difference between the two phases. Several two-phase systems have been studied, with a particular emphasis on the poly(ethylene glycol)/magnesium sulfate system. Numerous solutes, including peptides, were used in this system and their partition coefficients show good agreement with the model.