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.     

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.     

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.     

Application of response surface methodology to the modeling of alpha-amylase purification by aqueous two-phase systems

Mathematical models concerning the purification of alpha-amylase from the cultivation supernatant of Bacillus subtilis in a polyethylene glycol-citrate aqueous two-phase system (ATP) are established with response surface methodology. The PEG3350, citrate and sodium chloride concentrations were selected as variables to evaluate the purification impact factors in aqueous two-phase system, including partition coefficients of alpha-amylase, total protein, purification factor and alpha-amylase yield. An experimental space with two-fold purification and over 90% yield of alpha-amylase is achieved through the optimized condition basing on the model. Two systems with low viscosity within said space were further selected to perform alpha-amylase purification and the experimental results coincide well with the calculation of the models, which indicates that the model provides a promising tool for experimental design of protein purification by aqueous two-phase system.     

Predicting the partition coefficients of a recombinant cutinase in polyethylene glycol/phosphate aqueous two-phase systems

A model for the prediction of protein partition coefficients in aqueous two-phase systems has been developed. This model accounts for both charge-independent and electrostatic effects. The determination of nonelectrostatic effects was based on the model of Eiteman and Gainer for uncharged solutes while the electrostatic contribution was computed using TITRA, a program that uses a continuum electrostatic model to treat charge interactions in proteins and considers the effect of pH and ionic strength. The partition coefficients of Fusarium solani pisi recombinant cutinase have been satisfactorily predicted in polyethylene glycol (PEG) 1000 and phosphate aqueous two-phase systems at a pH range of 6.0-9.0. The model failed to predict the enzyme partitioning behavior at pH 4.5. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 248-257, 1997.     

Dynamic quenchers in fluorescently labeled membranes. Theory for quenching in a three-phase system.

The theory for quenching of fluorescently labeled membranes by dynamic quenchers is described for a three-phase system: a fluorescently labeled membrane, a nonlabeled membrane, and an aqueous phase. Two different experimental protocols are possible to determine quenching parameters. Using the first protocol, partition coefficients and bimolecular quenching constants were determined for a hydrophobic quencher in carbazole-labeled membranes in the presence of an unlabeled reference membrane. These parameters determined for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) using this three-phase analysis were in good agreement with values determined by a two-phase analysis without the reference lipid. Hence, the theory was verified. In the second protocol, the quencher partition coefficient was determined for unlabeled membranes in the presence of a carbazole-labeled reference membrane. Partition coefficients for DDE determined by this method were the same as partition coefficients determined for carbazole-labeled membranes using the two-phase analysis. The greater ease in determining partition coefficients and bimolecular quenching constants by the three-phase analysis and, in particular, the ability to determine the partition coefficient in unlabeled membranes make the three-phase analysis especially useful. This method was used to study the effect varying the membrane lipid composition has on the partition coefficient. The data indicate that partition coefficients of DDE in fluid membranes are not dramatically dependent upon polar head group composition, fatty acid composition, or cholesterol content. However, partitioning into gel-phase lipids is at least 100-fold less than fluid-phase lipids.     

Partition of isomeric dipeptides in poly(ethylene glycol)/magnesium sulfate aqueous two-phase systems

A series of isomeric dipeptides, i.e., those containing identical residues but in different order such as Trp-Gly versus Gly-Trp, was partitioned in a poly(ethylene glycol) (PEG)/magnesium sulfate (MgSO4) aqueous two-phase system. Dipeptides having a more hydrophobic character favored the upper (PEG) phase. Moreover, the partition coefficients for isomeric dipeptides are different, with the partition coefficients for dipeptides containing the more hydrophobic residue in the C-terminal position being, in general, greater than the partition coefficients for corresponding isomers which contain the more hydrophobic residue in the N-terminal position. These observations can be attributed to the different interactions that the isomers have with specific two-phase systems.     

Reversing the amino acid sequence of a dipeptide changes its partition in an aqueous two-phase system

Summary Reversing the amino acid sequence of a dipeptide produces distinct differences in how they partition between a polyethylene glycol/potassium phosphate aqueous two-phase system. The partition coefficients for twenty-three dipeptides in this biphasic system are recorded.     

Enhanced discrimination in the partition of proteins by aqueous polymer two-phase systems

Aqueous polymer two-phase systems containing dextran T-500 and PEG 4000 can be prepared which are biphasic below 18 degrees C and monophasic at higher temperatures. Both liganded and unliganded forms of glutamate dehydrogenase and troponin, which have similar partition coefficients if the protein is added to a two-phase system at 4 degrees C, have widely differing partition coefficients if added to the same system in the monophasic state at 20 degrees C and subsequently cooled to 4 degrees C.     

Partitioning of beta-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems.

Four different beta-galactosidase fusion proteins have been partitioned in poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase systems. The partition coefficients (K) of staphylococcal protein A-beta-galactosidase (SpA beta gal) (K = 3.5) and staphylococcal protein A-streptococcal protein G-beta-galactosidase (AG beta gal) (K = 2.8) were compared with the partition coefficients of their constituent molecules, beta-galactosidase, SpA, and protein AG. It was found that by fusing beta-galactosidase to the smaller proteins SpA and protein AG, their partition coefficients were increased four to five times. Experimental data were fitted into, and found to agree with, the Albertsson partition model of interacting molecules. The compatibility with PEG and potassium phosphate of beta-galactosidase, SpA, and two different versions of the SpA beta gal protein, displayed as precipitation curves, showed a relationship to the protein partition coefficients in PEG/potassium phosphate systems. High solubility in one phase component was accompanied by preferential partitioning to the phase rich in the same component in the PEG/potassium phosphate system. Also, a changed linker region in SpA beta gal resulted in a more soluble protein. This, together with the improved K values of the target proteins by fusion, shows that it is possible to use beta-galactosidase as an affinity handle.     

Peptide hydrophobicity and partitioning in poly(ethylene glycol)/magnesium sulfate aqueous two-phase systems.

Several amino acids and peptides were partitioned in poly(ethylene glycol) (PEG)/magnesium sulfate (MgSO4) aqueous two-phase systems. The partition coefficients measured for amino acids and peptides were proportional to the difference in PEG concentration between the phases. The partitioning data were used to calculate the relative hydrophobicities of individual amino acids, which were then used to estimate the hydrophobicities of peptides. The partition coefficients of several dipeptides were predicted from these estimated hydrophobicities. A series of peptide fragments that compose the pentapeptide leucine enkephalin was also partitioned in the PEG/MgSO4 system. Again, the partitioning depended upon the hydrophobicities of the individual exposed amino acids.     

Countercurrent distribution of L line monolayer and suspension cell cultures in a 2-phase polymer system

The distribution of two sulines of L cells, differing in adhesive properties (monolayer and suspension cultures), have been investigated using polymer two-phase system dextran-500-polyenthylenglycol-600. By counter-current distribution it has been shown that most of the cells growing in monolayer have low coefficients of partition, unlike cells grown in suspension having a high coefficient of partition. It is supposed that differences revealed in properties of the cell surface may be associated with the ability of cells to form adhesive contacts.     

Aqueous two-phase extraction for downstream processing of amyloglucosidase

A polymer/salt aqueous two-phase system has been successfully employed for separation and purification of amyloglucosidase. The effects of system pH, molecular weight of polymer and composition of the two-phase system on amyloglucosidase partition behaviour in polyethylene glycol (PEG 4000, 6000)/disodium hydrogen phosphate were investigated. Experimental data are explained based on Kim's theoretical model for the prediction of biomolecule partitioning in a PEG/salt system.     

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.     

Characterization of His-X3-His sites in alpha-helices of synthetic metal-binding bovine somatotropin.

Variants of bovine somatotropin have been engineered to contain synthetic metal-binding sites consisting of two solvent-exposed histidines separated by a single turn of an alpha-helix (His-X3-His variants). The affinities of these proteins for Cu(II) were characterized by measuring their partition coefficients in an aqueous two-phase polymer system. The partition coefficients were used to generate binding constants for formation of a complex between the engineered metal-binding site and Cu(II) chelated to an iminodiacetic acid derivative of polyethylene glycol. For three His-X3-His variants described here, these constants range from 2 x 10(4) to 1.6 x 10(6) M-1. The metal affinity of a His-X3-His site depends on the rigidity of the helix into which the site is engineered. The affinities of the His-X3-His sites for Cu(II) are large enough to dramatically increase not only the partitioning of these proteins in aqueous two-phase systems, but also their retention times on a metal-affinity chromatography column. Both these features can greatly facilitate the purification of engineered proteins. Criteria for choosing positions for incorporating metal-binding sites are discussed.     

Derivative spectrophotometry as a tool for the determination of drug partition coefficients in water/dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) liposomes.

The partition coefficients (K(p)) between lipid bilayers of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) unilamellar liposomes and water were determined using derivative spectrophotometry for chlordiazepoxide (benzodiazepine), isoniazid and rifampicin (tuberculostatic drugs) and dibucaine (local anaesthetic). A comparison of the K(p) values in water/DMPG with those in water/DMPC (dimyristoyl-L-alpha-phosphatidylcholine) revealed that for chlordiazepoxide and isoniazid, neutral drugs at physiological pH, the partition coefficients are similar in anionic (DMPG) and zwitterionic (DMPC) liposomes. However, for ionised drugs at physiological pH, the electrostatic interactions are different with DMPG and DMPC, with the cationic dibucaine having a stronger interaction with DMPG, and the anionic rifampicin having a much larger K(p) in zwitterionic DMPC. These results show that liposomes are a better model membrane than an isotropic two-phase solvent system, such as water-octanol, to predict drug-membrane partition coefficients, as they mimic better the hydrophobic part and the outer polar charged surface of the phospholipids of natural membranes.     

Determination of Partition Coefficients of Labile Compounds Subject to Dissociation Extraction Analysis of Partitioning

A novel method for the determination of partition coefficients in aqueous-organic two-phase systems is described. It is applicable for characterizing the distribution of substances undergoing proton dissociation in the aqueous phase. The method is based on titration by means of the organic phase. A linear form of the respective model is given for parameter estimation by linear regression. This strategy is used for the determination of the partition coefficients of some N-unprotected n-alkyl esters of phenylalanine in various aqueous-organic biphasic systems. These esters are of interest for the optical resolution of phenylalanine with chymotrypsin.     

Understanding viral partitioning in two-phase aqueous nonionic micellar systems: 1. Role of attractive interactions between viruses and micelles

The partitioning behavior of viruses in the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C10E4) micellar system cannot be fully explained by considering solely the repulsive, steric, excluded-volume interactions that operate between the viruses and the nonionic C10E4 micelles. Specifically, an excluded-volume theory developed recently by our group is not able to quantitatively predict the observed viral partition coefficients, even though this theory is capable of providing reasonable quantitative predictions of protein partition coefficients. To shed light on the discrepancy between the theoretically predicted and the experimentally measured viral partition coefficients, a central assumption underlying the excluded-volume theory that the viruses and the C10E4 micelles interact solely through repulsive, excluded-volume interactions was challenged in this study. In particular, utilizing bacteriophage P22 as a model virus, a competitive inhibition test and a partitioning study of the capsids of bacteriophage P22 were conducted. Based on the results of these two experimental studies, it was concluded that any attractive interactions between the tailspikes of bacteriophage P22 and the C10E4 micelles are negligible. Another experimental study was carried out wherein the partition coefficients of the model viruses, bacteriophages P22 and T4, were measured at various temperatures, and compared with those previously obtained for bacteriophage phiX174. This comparison also indicated that possible attractive, electromagnetic-induced interactions between the bacteriophage particles and the C10E4 micelles cannot be invoked to rationalize the observed discrepancy between the theoretically predicted and the experimentally measured viral partition coefficients.     

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.     

Differences in the surface properties of the mating types of Physarum polycephalum

In the slime mold Physarum polycephalum, formation of a diploid plasmodium occurs when compatible haploid amoebae fuse. To study cell surface changes associated with the fusion process, a non-destructive method known as aqueous, two-phase partitioning was employed. Using a two-phase system of dextran and polyethylene glycol, we observed that the two mating types (RSD4 and MA185) have different partition coefficients and hence different surface properties. Based on their partitioning behavior, MA185 cells appear to have a more hydrophobic surface than RSD4 amoeba. The partition coefficient of both cell types decreased with time. If amoebae were maintained in culture until they encysted, differences in their surface were not detectable.