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.     

Effect of poly(ethylene glycol) on the polarity of aqueous solutions and on the structure of vesicle membranes

The partitioning of TEMPO into phosphatidylcholine vesicle membranes is reduced upon addition of poly(ethylene glycol). This is caused by reduced polarity of the aqueous phase as well as decreased membrane fluidity in the presence of poly(ethylene glycol). The isotropic hyperfine splitting of TEMPO in aqueous poly(ethylene glycol) solutions was used as a measure of solvent polarity. The alterations of the membrane fluidity were detected by means of two different fatty acid spin labels. The influences of physicochemical properties of an aqueous poly(ethylene glycol) phase on the membrane structure of cells and vesicles are discussed in the light of membrane fusion.     

Liquid-liquid partitioning of some enzymes, especially phosphofuctokinase, from Saccharomyces cerevisiae at subzero temperature

The effects of low temperature (−18°C) on the stability and partitioning of some glycolytic enzymes within an aqueous two-phase system were studied. The enzymes were phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase present in a crude extract of bakers' yeast. The partitioning of pure phosphofructokinase, isolated from bakers' yeast, was also examined. The two-phase systems were composed of water, poly(ethylene glycol), dextran, and ethylene glycol and buffer. The influence on the partitioning of the presence of ethylene glycol, phenylmethylsulfonyl fluoride and poly(ethylene glycol)-bound Cibacron Blue F3G-A was investigated at −18, 0 and (in some cases) 20°C. The presence of ethylene glycol, phase polymers and low temperature stabilized all three enzyme activities. Cibacron Blue, an affinity ligand for phosphofructokinase, increased its partitioning into the upper phase with decreasing temperature. Depending on the conditions, various amounts of the enzymes were recovered at the interface, also in systems not containing ethylene glycol. The implications of the observed effects on the use of aqueous two-phase systems for the extraction and fractionation of proteins are discussed.     

Interaction of human serum albumin with fatty acids. Role of anionic group studied by affinity partition.

The interaction of human serum albumin with fatty acids has been determined using the method of affinity partitioning in aqueous biphasic systems containing dextran, poly(ethylene glycol) and esters of dicarboxylic acids with poly(ethylene glycol). The difference in the partition of albumin in phase systems with and without the poly(ethylene glycol)-bound fatty acid group provides a measure of the interaction of fatty acids with the protein. The relative contribution of the polar and non-polar interaction to the binding of fatty acids to albumin has been estimated by comparing the present data with that obtained earlier using poly(ethylene glycol)-bound straight chain aliphatic hydrocarbons. In both cases, the aliphatic chain should contain a minimum of 8 carbon atoms to affect the partition of albumin and that the maximum effect is obtained with chains containing 16 carbon atoms. The effect of the polymer-bound fatty acid group is higher than the corresponding hydrocarbon only when the number of carbon atoms in it exceeds 12. The relative effect of polymer-bound 16-carbon chains, with and without a carboxyl group in the terminal position is independent of pH in the range 5--9.     

Differential effect of neuraminidase-treatment on the surface charge-associated properties of rat reticulocytes and erythrocytes. Studies by partitioning in two-polymer aqueous phases

Rat reticulocytes undergo charge-associated surface changes, detectable by cell partitioning in charged dextran-poly(ethylene glycol) aqueous phase systems, as they become mature erythrocytes. Young reticulocytes have a lower partition coefficient, i.e., quantity of cells in the top phase as a percentage of total cells added, than do mature erythrocytes. Sialic acid is the main charge-bearing group on red blood cells and, in the case of the rat, most of the sialic acid can be removed by treatment of the cells with neuraminidase (Vibrio cholerae). By combining isotopic 59Fe-labeling of reticulocytes with countercurrent distribution of the entire red blood cell population in charged dextran-poly(ethylene glycol) aqueous phases we have now studied the relative effect of neuraminidase-treatment on rat reticulocytes and mature erythrocytes. It was found that neuraminidase-treatment (a) does not eliminate surface differences, detectable by partitioning, between rat reticulocytes and erythrocytes and (b) reduces the partition coefficient of mature erythrocytes to a greater extent than the partition coefficient of reticulocytes indicating a differential effect of this enzyme on the two cell populations.     

Partitioning of caseinomacropeptide in aqueous two-phase systems

This study evaluates the influence of type of salt and temperature on the partition coefficient of caseinomacropetide (CMP) to determine the best conditions for the recovery of CMP in aqueous two-phase systems (ATPS) composed by poly(ethylene glycol) (PEG) 1500 and an inorganic salt (potassium phosphate, sodium citrate, lithium sulfate or sodium sulfate). In all systems, CMP presented affinity for the PEG-rich phase. The PEG1500+lithium sulfate showed the highest values of partitioning coefficient. In addition, thermodynamic parameters (DeltaH degrees , DeltaS degrees , DeltaG degrees) as a function of temperature, were calculated for the system PEG1500-sodium citrate at different PEG concentrations and the results imply thermodynamic differences between partitioning of CMP in this system.     

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.     

Differential effect of neuraminidase-treatment on the surface charge-associated properties of rat reticulocytes and erythrocytes: Studies by partitioning in two-polymer aqueous phases

Rat reticulocytes undergo charge-associated surface changes, detectable by cell partitioning in charged dextran-poly(ethylene glycol) aqueous phase systems, as they become mature erythrocytes. Young reticulocytes have a lower partition coefficient, i.e., quantity of cells in the top phase as a percentage of total cells added, than do mature erythrocytes. Sialic acid is the main charge-bearing group on red blood cells and, in the case of the rat, most of the sialic acid can be removed by treatment of the cells with neuraminidase (Vibrio cholerae). By combining isotopic ⁵⁹Fe-labeling of reticulocytes with countercurrent distribution of the entire red blood cell population in charged dextran-poly(ethylene glycol) aqueous phases we have now studied the relative effect of neuraminidase-treatment on rat reticulocytes and mature erythrocytes. It was found that neuraminidase-treatment (a) does not eliminate surface differences, detectable by partitioning, between rat reticulocytes and erythrocytes and (b) reduces the partition coefficient of mature erythrocytes to a greater extent than the partition coefficient of reticulocytes indicating a differential effect of this enzyme on the two cell populations.     

Generation of selective microenvironment at the cell periphery through bulk-phase hydrophilic polymers

In order to understand the previously demonstrated effect of poly(ethylene glycol) on the stimulation of lymphocyte responses to syngeneic tumor cells (Ben-Sasson, S.A. and Henkart, P.A. (1977) J. Immunol. 119, 227–231), we have investigated the effects of addition of poly(ethylene glycol) to the medium in a number of cellular systems. The binding of trimeric IgG to tumor-lymphocyte Fc receptors was greatly enhanced by poly(ethylene glycol); a substantial increase in binding of trimeric IgG to non-Fc-receptor-bearing tumor cells was also observed. Similarly, the binding of labeled bovine serum albumin to lymphocyte surfaces was increased by poly(ethylene glycol), implying that nonspecific binding of proteins to cells was generally enhanced. The dose-response curve of concanavalin A mitogenesis was shifted to the right, as would be expected from a local increase in concanavalin A concentration. Antibody binding to erythrocytes as detected by complement lysis was similarly increased. It was found that in aqueous two-phase mixtures created by poly(ethylene glycol) and dextran, erythrocytes partition into the dextran phase through exclusion into dextran-rich microdroplets. It is proposed that addition of poly(ethylene glycol) to cell culture media creates a similar separate phase around the cell surface in which the local concentration of proteins is greater than that in the bulk medium. This concept explains many of the diverse effects of addition of poly(ethylene glycol) to the medium. It also can partially explain the requirement for serum to observe the poly(ethylene glycol) effect on the lymphocyte response to syngeneic tumor cells.     

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.     

Polymer fractionation in aqueous two-phase polymer systems.

We consider the effects of the addition of poly(ethylene glycol) (PEG) of different molecular weights to aqueous two-phase system of PEG 8000 and dextran 500. The first purpose of this study was to determine the molecular weight partitioning of the polymers themselves so that, for example, aqueous two-phase separations using affinity ligands can be improved. The second purpose was to examine whether this molecular weight partitioning could be predicted by using solution thermodynamic models so that it would be possible to optimize affinity partitioning without extensive laboratory work. Experimentally, we find that, by increasing the PEG concentration of any molecular weight in the feed, the high molecular weight PEG concentration in the dextran-rich phase is reduced. This observation can be used to reduce the loss of expensive ligated PEG used in affinity partitioning. Further, there is generally good agreement between our experimental data and the predictions of a solution thermodynamic model.     

Coupling of poly(ethylene glycol) to albumin under very mild conditions by activation with tresyl chloride: characterization of the conjugate by partitioning in aqueous two-phase systems

Poly(ethylene glycol) activated with tresyl chloride has been covalently linked to albumin as a result of a 2-h incubation in 0.05 M sodium phosphate buffer, pH 7.5, containing 0.125 M sodium chloride (0.344 OSM). The coupling of poly(ethylene glycol) to albumin was demonstrated by the increase in the partition coefficient of the protein in poly(ethylene glycol)-dextran aqueous two-phase systems. A linear relationship between the log of the partition coefficient of the poly(ethylene glycol)-albumin conjugate and the degree of modification (measured as the amino groups consumed during the coupling step) has been demonstrated. Countercurrent distribution in the two-phase system showed that poly(ethylene glycol)-albumin was heterogeneous with respect to its partitioning behavior, indicating that the albumin was not uniformly modified with poly(ethylene glycol).     

Ca2+ and pH dependence of hydrophobicity of alpha-lactalbumin: affinity partitioning of proteins in aqueous two-phase systems containing poly(ethylene glycol) esters of fatty acids.

Hydrophobic affinity partitioning in an aqueous two-phase system, composed of dextran and poly(ethylene glycol), has been used to study the hydrophobic binding capacity of bovine alpha-lactalbumin. The hydrophobicity of the poly(ethylene glycol)-containing phase was adjusted by including varying amounts of fatty acids bound to the polymer via an ester linkage. The change in the logarithmic partition coefficient of the protein in such systems was used as a measure of the hydrophobic binding. This value was strongly influenced by the amount of Ca2+ present as well as the pH value. The results are discussed in terms of the exposure of hydrophobic binding sites on alpha-lactalbumin and their relation to the conformational change in this protein due to Ca(2+)-binding, chelation of Ca2+ and pH dependence.     

Influence of the polymeric morphology of sorbents on their properties in affinity chromatography.

The influence of the polymeric morphology of different types of Fe(3+)-containing sorbents and their properties in retention of phosphoamino acids is presented in this paper. Poly(hydroxylated polybutadienic-hydroxyethyl methacrylate) [poly(PB-HEMA)] and poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) [poly(EGDMA-HEMA)] base supports were submitted to chemical modifications to attain metal ion-containing sorbents. Properties such as specific surface area, pore volume, equilibrium volume swelling ratios, extent of conversion rate of functional groups, amount of chelated metal ion, ligand occupation, as well as quantity of phosphoamino acid retained, were used as comparative parameters for those different base matrices. Results suggest that Fe(3+) immobilized on poly(EGDMA-HEMA) base support are more efficient as a group-specific sorbent to retain phosphoamino acids than those obtained using poly(PB-HEMA) base support.     

Adsorbed surfactants for affinity chromatography. end-group modification of ethylene glycol polymers

The hydroxyl end-groups of Pluronic F108 [a tri-block copolymer surfactant of poly(ethylene glycol) and poly(propylene glycol) [PEG-PPG-PEG]] were converted into primary amine and quaternary ammonium equivalents for use in a new approach to affinity chromatography. The preparation of sulphonic acid end-groups was also attempted.     

Cultivation of Lactococcus lactis in a polyelectrolyte-neutral polymer aqueous two-phase system

The possibility to cultivate Lactococcus lactis in aqueous polymer two-phase system has been investigated. The phase system was made up of poly(ethylene imine) and (hydroxyethyl) cellulose. Long lag phases were needed for the microorganism to adapt to the polymer rich media. Cells favoured the (hydroxyethyl)cellulose rich top phase or they accumulated at the interface, while lactic acid showed affinity for the poly(ethylene imine) rich phase.Abbreviations PEG poly(ethylene glycol) - PEI poly(ethylene imine) - HEC (hydroxyethyl)cellulose     

New polymers forming aqueous two-phase polymer systems

A new type of polymer, starch-modified by acrylamide, has been developed for application in aqueous two-phase systems (ATPS) for protein separation. Partial hydrolysis and acrylamide modification of starch to different degrees make it suitable for forming ATPS with poly(ethylene glycol) in a moderate concentration range. The potential of the polymer to form ATPS with the thermoprecipitating copolymer of 1-vinylimidazole with N-vinylcaprolactam (poly-VI/VCL) has been evaluated. The thermoprecipitation properties of poly-VI/VCL and Cu(II)-loaded poly-VI/VCL have been studied for application in metal affinity partitioning. The formation of ATPS with Cu(II)-loaded thermoprecipitating copolymer was critically achieved for poly-VI/VCL (10/90) copolymer in under-loaded metal concentrations. With the Cu(II)-loaded copolymer, poly-VI/VCL in the top phase and modified starch in the bottom phase, the ATPS formed was used for the purification of alpha-amylase inhibitor from wheat meal. The protein partitioned in the top phase and phase-separated polymer-protein complex could be precipitated by salt. The protein inhibitor was recovered with a yield of 75%.     

Aging of erythrocytes results in altered red cell surface properties in the rat,but not in the human Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have now found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, counter-current distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter.These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Aging of erythrocytes results in altered red cell surface properties in the rat, but not in the human. Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have not found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, countercurrent distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter. These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin-dextran

A new concept for affinity two-phase partitioning was tested. The partitioning was based on the interaction of target membranes with a primary antibody which, in turn, interacted with a biotinylated secondary antibody and NeutrAvidin-dextran in a poly(ethylene glycol)/dextran two-phase system. Caveolae selectively redistributed from the top phase to the NeutrAvidin-dextran-containing bottom phase by employing anti-caveolin as the primary antibody. This immunoaffinity approach was more selective than the established sucrose gradient centrifugation method and resulted in highly purified caveolae from Triton X-100-treated liver and lung plasma membranes. The same approach, employing other selective primary antibodies, should facilitate the purification also of other membrane fractions.