Hydrophobic surface properties of erythrocytes studied by affinity partition in aqueous two-phase systems

Summary Erythrocytes from various species have been partitioned in aqueous two-phase systems consisting of water, dextran, poly-(ethylene glycol), salt and buffer. The terminal hydroxyl groups of the latter polymer were esterified with palmitic, oleic, linoleic and linolenic acids, as well as with deoxycholic acid. In a two-phase system containing unesterified poly(ethylene glycol) the erythrocytes are exclusively in the dextran-rich lower phase. When the poly(ethylene glycol) is esterified the red blood cells collect at the interface and/or in the poly(ethylene glycol)-rich upper phase depending on the type and concentration of esterified acid. Palmitate ester is most effective in increasing the affinity of the cells for the upper phase, followed by oleate, linolate, linolenate, and deoxycholate esters. The partition behaviour of erythrocytes from various species differs considerably. Two groups can be distinguished: one consisting of erythrocytes from dog, guinea pig and rat, the other from human, sheep and rabbit. This division can be correlated to the content of sphingomyelin and phosphatidyl choline in the erythrocyte membranes.     

Hydrophobic affinity partition of spinach chloroplasts in aqueous two-phase systems

The surface properties of spinach chloroplasts, both of intact chloroplasts with surrounding envelope and broken chloroplasts consisting of the inner lamellar system, have been studied by partitioning them between two aqueous phases, especially using counter-current distribution technique. The two-phase system consists of poly(ethyleneglycol), dextran and water. The two polymers are enriched in opposite phases and by binding deoxycholate or palmitate to one of the polymers the affinity of chloroplasts for the corresponding phase is strongly enhanced. The partition of the two classes of chloroplasts, however, is not affected to the same degree and the affinity of the chloroplast envelope for deoxycholate and palmitate is stronger than that of the lamellar system. This has been correlated to the chemical composition of the two types of membranes. By studying the effect of salts on the partition it has been found that the lamellar system bears a larger number of negative charges as compared to the envelope of the intact chloroplast.     

Relative hydrophobicity of surfaces of erythrocytes from different species as measured by partition in aqueous two-polymer phase systems

Erythrocytes from different species were subjected to partition in an aqueous, buffered Ficoll/Dextran two-phase system. The effects of different salt composition of the phase system on the distribution of erythrocytes was examined. Different ratios of sodium chloride to sodium phosphate buffer (pH 7.4) with the ionic strength varying from 0.176 to 0.288 M were used in the systems and similar relationship between the partition coefficients of the cells under study and the ionic strength were established. The relationships were treated according to a general equation previously established (Zaslavsky, B.Y., Miheeva, L.M., Metechkina, N.M., Pogorelov, V.M. and Rogozhin, S.V. (1978) FEBS Lett. 94, 77-80) and the results obtained were used to evaluate the relative hydrophobicity of the cells' surface.     

Hydrophobic interaction determined by partition in aqueous two-phase systems. Partition of proteins in systems containing fatty-acid esters of poly(ethylene glycol).

In this report we describe a new method which is useful for measuring hydrophobic interactions between aliphatic hydrocarbon chains and proteins in aqueous environment. The method is based on partition of proteins in an aqueous two-phase system containing dextran and poly(ethylene glycol) and different fatty acid esters of poly(ethylene glycol). The partition is measured under conditions where contributions from electrostatic interactions are eliminated. The difference in partition of proteins in phase systems with and without hyrocarbon groups bound to poly(ethylene glycol), deltalog K, where K is the partition coefficient, is taken as a measure of hydrophobic interaction. Deltalog K varies with size of hydrocarbon chain and type of protein. The length of the aliphatic chain should be greater than 8 carbon atoms in order to get a measurable effect in terms of deltalog K. Bovine serum albumin, beta-lactoglobulin, hemoglobin and myoglobin have been shown to have different affinities for palmitic acid ester of poly(ethylene glycol). No hydrophobic effect could be observed for ovalbumin, cytochrome c or alpha-chymotrypsinogen A.     

Immobilized metal ion affinity partitioning of cells in aqueous two-phase systems: erythrocytes as a model

Immobilized metal ion affinity partitioning of erythrocytes from different species is described. We have explored the affinity between transition metal chelates and metal-binding sites situated on the cell surface by partitioning in aqueous two-phase system composed of poly(ethylene glycol) and dextran. Soluble metal-chelate-poly(ethylene glycol) was prepared by fixing metal ions to poly(ethylene glycol) via the covalently bonded chelator, iminodiacetic acid. The partitioning behaviour of erythrocytes in systems at different concentrations of the ligand was tested. The copper-chelate-poly(ethylene glycol) was quite effective in the affinity extraction of human and rabbit erythrocytes, while the zinc-chelate-poly(ethylene glycol) displayed significant affinity only to the rabbit cells. Furthermore, the influence of various effectors such as imidazole, sialic acid on immobilized metal ion affinity partitioning of erythrocytes was examined.     

Features of the acid protease partition in aqueous two-phase systems of polyethylene glycol-phosphate: chymosin and pepsin

The partitioning of chymosin (from Aspergilus niger) and pepsin (from bovine stomach) was carried out in aqueous-two phase systems formed by polyethyleneglycol-potassium phosphate. The effects of polymer concentration, molecular mass and temperature were analysed. The partition was assayed at pH 7.0 in systems of polyethyleneglycol of molecular mass: 1450, 3350, 6000 and 8000. Both proteins showed high affinity for the polyethyleneglycol rich phase. The increase of polyethyleneglycol concentration favoured the protein transfer to the top phase, suggesting an important protein-polymer interaction. Polyethyleneglycol proved to have a stabilizing effect on the chymosin and pepsin, increasing its protein secondary structure. This finding agreed with the enhancement of the milk clotting activity by the polyethyleneglycol. The method appears to be suitable as a first step for the purification of these proteins from their natural sources.     

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.     

Liquid-liquid partition chromatography of biopolymers in aqueous two-phase systems.

The theoretical and practical principles of liquid-liquid partition chromatography (LLPC) applying aqueous two-phase polymer systems are presented. The method is based on support materials which bind one of the two aqueous phases with high preference and reject the other. This selectivity is obtained by making use of incompatibilities between polymers grafted on support particles and polymers in solution. Applications of the separation technique to the fractionation of protein and nucleic acid mixtures are shown. For the DNA-fractionation according to base composition an affinity partition chromatography using polyethylene glycol-bound base-specific complexing agents has been developed which exhibits a resolution superior to all other methods known.     

Fractionation of chromosomes : II. Use of hydrophobic affinity partition in aqueous two-phase system

A simple method for separation of large quantities of isolated metaphase chromosomes in Single-Tube Partition (STP), using hydrophobic ligand in an aqueous two-phase system is presented. The two-phase system is composed of an aqueous solution of Dextran 500 and poly(ethylene) glycol 6000 (PEG). The concentration of chromosomes to be separated has no influence on the distribution behaviour in the partition system and up to 10(7) chromosomes can be used in a phase system as small as 3-5 g (5 ml tube). Different groups of chromosomes differ in their distribution in the two phases and the introduction of PEG with covalently attached hydrophobic ligand provides a means of controlling the distribution of chromosomes. A combination of positively charged trimethylaminomethane PEG (TMA-PEG) together with palmitat PEG (P-PEG) gives a fairly good condition for separating chromosomes on the basis of their net surface charge differences.     

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.     

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.     

Fractionation of chromosomal deoxyribonucleoproteins by partition in two-phase aqueous polymer systems

Deoxyribonucleoprotein (DNP)¹ prepared by shearing chromatin of mouse cells may be fractionated in 2-phase aqueous Dextran-polyethyleneglycol mixtures. A partial separation of DNPs with different non-histone protein/DNA ratios may be obtained in a single-step partition. Separation of a spectrum of fractions of DNP has been obtained by countercurrent distribution using the same 2-phase polymer system. DNP fractions which bear nascent RNA (representing approximately $\text{1}\text{3}$ of the total DNA) may be separated from the major fraction of DNP; they are found in the same region of the distribution pattern as DNP fractions with the highest non-histone protein/DNA ratio.     

Purification of acid-proteases by affinity partitioning in aqueous two-phase systems

Summary An affinity polymer derivative was synthesized with the group specific acid protease inhibitor pepstatin attached to dextran (M.W. 500,0001). This derivative was used in an aqueous two-phase system with hydroxypropyldextran to purify crude solutions of chymosin and Endothia parasitica (EP) acid proteases. Chymosin was purified by a factor of 6.2 with an overall yield of 83%. EP protease was similarly purified. A new pepstatin binding protease was discovered in crude EP extracts.     

Porcine pancreatic lipase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems

This research study examined porcine pancreatic lipase partition in aqueous two-phase systems formed by polyethylene glycol-potassium phosphate at pH 6.0, 7.0 and 8.0, the effect of polymer molecular mass, and NaCl concentration. The enzyme was preferentially partitioned into the polyethylene glycol rich phase in systems with molecular mass 4000-8000, while with polyethylene glycol of 10,000 molecular mass it was concentrated in the phosphate rich phase. The enthalpic and entropic changes found due to the protein partition were negative for all the polyethylene glycol molecular mass systems assessed. Both thermodynamic functions were shown to be associated by an entropic-enthalpic compensation effect suggesting that the water structure ordered in the ethylene chain of polyethylene glycol plays a role in the protein partition. The addition of NaCl increased the lipase affinity to the top phase and this effect was most significant in the system polyethylene glycol 2000-NaCl 3%. This system yielded an enzyme recovery more than 90% with a purification factor of approximately 3.4.     

Aspergillus oryzae alpha-amylase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems

The aim of this work is to study the partitioning of alpha-amylase from Aspergillus oryzae in polyethylene glycol-potassium phosphate systems formed by polymers of different molecular masses with different total concentrations, several NaCl concentrations and different volume ratio between the phases and at different temperatures. The enzyme was partitioned towards the top phase in the 2000-molecular-mass polyethylene glycol systems and towards the bottom phase in the other systems analyzed with higher molecular mass. The protein-medium interaction parameter (A) was determined; it increased in the same way as PEG molecular mass. The enthalpic and entropic changes found, in general, were negative and were shown to be associated by an entropic-enthalpic compensation effect suggesting that the ordered water structure in the chain of polyetyleneglycol plays a role in protein partition. The recovery in each of the phases was calculated in order to choose the best systems to be applied to enzyme isolation either from a polymer-rich or a polymer-poor phase.Enzymatic activity, circular dichroism and fluorescence were studied for the protein alone and in the presence of the different phases of the aqueous two-phase systems (ATPSs) in order to understand how they affect the enzymatic structure and the role of the protein-polymer interaction in the partitioning process. Secondary structure is not affected, in general, by the presence of the phases that do affect the enzymatic activity; therefore, there should be a change in the tertiary structure in the enzyme active site. These changes are more important for PEG 8000 than for PEG 2000 systems according to the results of the quenching of the intrinsic fluorescence. In a bio-separation process, the A. oryzae alpha-amylase could be isolated with ATPSs PEG 2000/Pi or PEG 8000/Pi with a high recovery, in the top or bottom phases, respectively.     

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.     

Purification of fusion proteins using affinity microspheres in aqueous two-phase systems

Affinity microspheres were prepared by immobilizing human -globulin (HGb) onto carboxylated poly (styrene/acrylamide) latex particles [P(St/AAm)-H; average diameter 0.33 m], which were prepared by emulsifier-free emulsion polymerization. HGB was covalently immobilized onto the latex particles with high efficiency by the carbodiimide method. A fusion protein (ZZB1B2) of immunoglobulin G and albumin-binding domains (ZZ and B1B2, respectively) was expressed intracellularly and extracellularly in Escherichia coli and was purified by the affinity microspheres. In poly (ethylene glycol) (PEG)/potassium phosphate aqueous two-phase system, the affinity microspheres were partitioned into the PEG-rich top phase, while cells and cell debris of E. coli were displaced into the salt-rich bottom phase. Therefore, ZZB1B2 was directly purified from cell disintegrate or culture broth by combining the affinity microspheres with the aqueous two-phase partitioning, and its purity was almost the same as that purified by conventional affinity chromatography. Therefore, by this purification method, the primary purification process and the subsequent high resolution purification process are combined, and the number of purification steps can be reduced. Correspondence to: A. Kondo     

Electromotive phenomena in partition of erythrocytes in aqueous polymer two phase systems

Measurement was made of the electrical potential between the two phases formed in an aqueous solution containing 5% dextran, 4% polyethylene glycol and varying concentrations of sodium chloride and sodium phosphate. Partition of the polycation DEAE-dextran-glycyltyrosine-¹²⁵I in such systems containing varying salt composition could be correlated with the measured electrical potential. Partion of human erythrocytes which have a negative surface charge was also correlated related with the measured electrical potential. Binding of DEAE-dextran-glycyltyrosine-¹²⁵I to human erythrocytes had less effect on their partitioning than might be expected from the number of positive charges bound to their surface.