Fractionation of mucopolysaccharides by countercurrent distribution in aqueous polymer two-phase systems

A new procedure for the fractionation of mucopolysaccharides based upon differences in their partition behavior in aqueous polymer two-phase systems has been devised. Systems containing dextran, poly(ethylene glycol), trimethylamino-poly(ethylene glycol), potassium bromide and sodium phosphate buffer were employed. Countercurrent distributions were performed with a miniature countercurrent distribution device designed especially for use with aqueous polymer two-phase systems. An advantage over the widely used procedures involving precipitation of mucopolysaccharides as their quaternary ammonium detergent complexes is that the countercurrent distribution pattern of a particular mucopolysaccharides is not affected by the simultaneous presence of other mucopolysaccharides. Preliminary distributions of labelled mucopolysaccharides isolated from the cells and culture medium of monolayer cultures of rat tumor cells demonstrate that the procedure is particularly well suited for the fractionation of very minute quantities of mucopolysaccharides.     

Cross partition and determination of net charge of the isoenzymes of enolase.

Enolase from bakers' yeast was separated into three isoenzymes by countercurrent distribution. The isoenzymes were partitioned in aqueous polymer two-phase systems containing positively charged trimethylamino poly(ethylene glycol) or negatively charged poly(ethylene glycol) sulphonate. The plots of the partition coefficient of each isoenzyme versus pH in the two biphasic systems intersect at pH equal to the isoelectric point. From slopes of the plots, the net charge of the isoenzymes at pH 6.57 was determined to be +2, -3, and -8 respectively.     

Cross partition and determination of net charge of the isoenzymes of enolase

Enolase from bakers' yeast was separated into three isoenzymes by countercurrent distribution. The isoenzymes were partitioned in aqueous polymer two-phase systems containing positively charged trimethylamino poly(ethylene glycol) or negatively charged poly(ethylene glycol) sulphonate. The plots of the partition coefficient of each isoenzyme versus pH in the two biphasic systems intersect at pH equal to the isoelectric point. From slopes of the plots, the net charge of the isoenzymes at pH 6.57 was determined to be +2, −3, and −8 respectively.     

Changes in plasma membrane and microfilaments accompanying morphologic differentiation in Chinese hamster ovary (CHO) cells.

Studies on the application of the techniques of counter-current distribution (CCD) in aqueous two-phase systems and multiple sedimentation for the fractionation of metaphase chromosomes are presented. The two-phase systems were composed of aqueous solutions of Dextran 500 and poly(ethylene)glycol 6000 (PEG). It has been found that different groups of chromosomes differ in their distribution between the two phases and that the introduction of PEG with covalently attached positively or negatively charged groups provides a means of steering the distribution of chromosomes. A rough fractionation of chromosomes on the basis of size is possible by the technique of multiple sedimentation and this, in combination with CCD, yields 10 fractions of chromosomes. Partition and CCD in aqueous two-phase system separate chromosomes according to their surface properties and may prove useful for isolation of individual chromosomes in bulk.     

Separation of human rosette- and nonrosette-forming lymphoid cells by countercurrent distribution in an aqueous two-phase system.

Mixtures of aqueous solutions of dextran and of poly(ethylene glycol) form immiscible two-phase systems suitable for the separation (by partition) of cells based on subtle differences in membrane surface properties. Lymphoid cells from human tonsils were subjected to countercurrent distribution in such a system. Analysis of cells from different parts of the countercurrent extraction train by the sheep red blood cell rosette test indicates a separation of rosette-forming and nonrosette-forming lymphocytes with the former having the higher partition coefficient. Since a major determinant of cell partition in these phases is a membrane-charge associated property it appears likely that the rosette-forming cells have a higher surface charge than the nonrosette-forming cells.     

Subfractionation of human peripheral blood lymphocytes on the basis of their surface properties by partitioning in two-polymer aqueous phase systems.

Partitioning of cells in dextran-poly(ethylene glycol) aqueous-aqueous two-phase systems is a sensitive method for separating cells and for obtaining information on their surface properties. Highly purified lymphocytes were obtained by velocity sedimentation of human peripheral blood mononuclear cells and fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged two-polymer aqueous phase system. The lymphocytes remained viable after separation (order of 90%) and the E-rosetting cells responded (after adding back monocytes) to mitogens (PHA, Con A, PWM). Not only was the total lymphocyte population found to be highly heterogeneous (as evidenced by a broad and skewed distribution curve), but we were able to show that cells that rosetted with E, or had complement or Fc receptors were composed of additional subpopulations as well. The bulk of complement-receptor-bearing cells had the lowest partition coefficient (K), E-rosetting cells an intermediate K, and Fc-receptor-containing cells the highest K. The largest lymphocytes were among the subpopulation having the highest K and neither responded to T cell mitogens nor rosetted with E. Our results thus demonstrate that human peripheral blood lymphocytes can be subfractionated by CCD. The fractions are differentially enriched with lymphocyte subpopulations having characteristic surface markers and functional abilities.     

Physico-chemical features of solvent media in the phases of aqueous polymer two-phase systems

Solvent polarity and pH in the coexisting aqueous phases of aqueous dextran-poly(ethylene glycol) and dextran-Ficoll two-phase systems of varied polymer concentrations were examined using the solvatochromic technique and potentiometric measurements, respectively. The relative solvent polarity of the phases, as measured by the solvatochromic technique, is suggested as a measure of the hydration power of water in the phases of aqueous polymer systems. Partitioning of a series of sulphonephthalein dyes in aqueous dextran-poly(ethylene glycol) and dextran-Ficoll two-phase systems of fixed polymer composition containing 0.01 mol/L universal buffer, pH 7.15, was studied. The results obtained are discussed together with those reported earlier on the physico-chemical features of aqueous media in the coexisting phases of the systems. It is suggested that the two phases of aqueous polymer systems should be viewed as two immiscible water-like solvents. The implications of the suggestion for the theoretical treatment of aqueous polymer two-phase systems are discussed.     

Purification of hyperthermophilic archaeal amylolytic enzyme (MJA1) using thermoseparating aqueous two-phase systems

Purification of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii, was investigated in the ethylene oxide-propylene oxide random copolymer (PEO-PPO)/(NH(4))(2)SO(4), and poly(ethylene glycol) (PEG)/(NH(4))(2)SO(4) aqueous two-phase systems. MJA1 partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. It was found that enzyme recovery of up to 90% with a purification factor of 3.31 was achieved using a single aqueous two-phase extraction step. In addition, the partition behavior of pure amyloglucosidase in polymer/salt aqueous two-phase systems was also evaluated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. This work is the first reported application of thermoseparating polymer aqueous two-phase systems for the purification of extremophile enzymes.     

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.     

Cell separations and subfractionations by countercurrent distribution in two-polymer aqueous phase systems depend on non-equilibrium conditions

Certain kinetic aspects of the partitioning behaviour of erythrocytes in dextran-poly(ethylene glycol) aqueous phase systems have been examined, and their implications for cell partitioning have been considered. It is concluded that the diverse and sometimes unique fractionations attained by use of multiple extractions (e.g., countercurrent distribution) of cells in two-polymer aqueous-phase systems depend on non-equilibrium conditions.     

Separation and subfractionation of small numbers of cells (approximately 10(6)) by countercurrent distribution in dextran-poly(ethylene glycol) aqueous-phase systems

Separation and subfractionation of cells on the basis of subtle differences in surface properties by partitioning in dextran-poly(ethylene glycol) aqueous phase systems is an established method. We report here that the incorporation of fetal bovine serum into such systems permits countercurrent distribution of small quantities of cells (approximately 10(6]. In the absence of serum such small quantities of cells are lost (probably by adherence) and cannot be recovered after countercurrent distribution.     

DNA fractionation by two-phase partition with aid of a base-specific macroligand

The application of a GC (guanosine-cytidine)-specific DNA macroligand, consisting of a GC-specific phenazinium dye covalently bound to one end of polyethylene glycol (6000–7500 $\text{M}{}_{\mathrm{r}}\text{)}$, for preparative DNA fractionation with base composition is described. The fractionation is performed in aqueous two-phase systems formed by polyethylene glycol and dextran in which the macroligand shows a strong affinity for the upper phase and shifts the partition coefficient of the DNA as a function of its GC content. Examples of fractionations by simple extractions and countercurrent distributions of calf thymus DNA over a few steps are given.     

Fractionation of cellulase and beta-glucosidase in a Trichoderma reesei culture liquid by use of two-phase partitioning

An aqueous two-phase system based on the two polymers poly(ethylene glycol) and dextran has been used for the fractionation of cellulase enzymes present in culture liquid obtained by fermentation with Trichoderma reesei. The activities of beta-glucosidase and glucanases were separated to high degree by using the two-phase systems for a counter-current distribution process in nine transfer steps. While the glucanases had high affinity to the poly(ethylene glycol) rich top phase the beta-glucosidase was enriched in the dextran-containing bottom phase. Multiple counter-current distribution performed indicates the heterogeneity of beta-glucosidase activities assuming at least four isoenzyme forms. One step concentration of beta-glucosidase by using system with 46:1 phase volume ratio resulted in 16 times higher enzyme activity.     

Separation and subfractionation of small numbers of cells (∼106) by countercurrent distribution in dextran-poly(ethylene glycol) aqueous-phase systems

Separation and subfractionation of cells on the basis of subtle differences in surface properties by partitioning in dextran-poly(ethylene glycol) aqueous phase systems is an established method. We report here that the incorporation of fetal bovine serum into such systems permits countercurrent distribution of small quantities of cells (∼10⁶). In the absence of serum such small quantities of cells are lost (probably by adherence) and cannot be recovered after countercurrent distribution.     

Bisphosphoglycerate mutase and pyruvate kinase activities during maturation of reticulocytes and ageing of erythrocytes

An increase in bisphosphoglycerate mutase (BPGM) and a decrease in pyruvate kinase (PK), i.e. a decrease in PK/BPGM ratio, was observed in red cell populations from anemic rats containing 95% down to 3% reticulocytes in blood. Such a ratio has been used to study the fractionation of recticulocytes, according to their degree of maturation, after counter-current distribution of those cell populations in dextrahpoly (ethylene glycol) two-phase systems. When applying this procedure to the fractionation according to age of erythrocytes from normal rats, the decrease of PK with cellular age was observed without a significant variation in BPGM activity.     

Aqueous polymer two-phase systems: effective tools for plasma membrane proteomics

Plasma membranes (PMs) are of particular importance for all living cells. They form a selectively permeable barrier to the environment. Many essential tasks of PMs are carried out by their proteinaceous components, including molecular transport, cell-cell interactions, and signal transduction. Due to the key role of these proteins for cellular function, they take center-stage in basic and applied research. A major problem towards in-depth identification and characterization of PM proteins by modern proteomic approaches is their low abundance and immense heterogeneity in different cells. Highly selective and efficient purification protocols are hence essential to any PM proteome analysis. An effective tool for preparative isolation of PMs is partitioning in aqueous polymer two-phase systems. In two-phase systems, membranes are separated according to differences in surface properties rather than size and density. Despite their rare application to the fractionation of animal tissues and cells, they represent an attractive alternative to conventional fractionation protocols. Here, we review the principles of partitioning using aqueous polymer two-phase systems and compare aqueous polymer two-phase systems with other methods currently used for the isolation of PMs.     

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.     

高速逆流色谱技术在生物大分子分离纯化中的应用

高速逆流色谱是一种连续液-液色谱技术,具有无固相载体、样品无需严格预处理等优点。近10年来,在设备结构和溶剂体系等方面进行了大量的研究开发,已推广应用于生物技术、医药、天然产物、环境监测、食品等领域。为适应生物大分子和活性细胞的分离,采用条件温和的双水相体系,研究开发相应的高速逆流色谱设备已成为热点。针对双水相体系的特点,已经开发出了多种具有较高固定相保留率的新型高速逆流色谱设备,通过优化实验条件,成功地进行了多种蛋白质的分离纯化。本对该领域的最新进展进行了综述与评价。     

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.     

Rat bone marrow erythroid cell fractionation by counter current distribution in non-charge-sensitive two-phase systems

Counter-current distribution in non charge-sensitive aqueous poly(ethylene glycol)-dextran two phase systems allows the fractionation of rat bone marrow cells into two broad cell subpopulations with different distribution coefficients in a relatively short time. Morphological identification and enzymatic studies suggest that erythroid cells are mainly present in the subpopulation with the higher distribution coefficient. The distribution coefficient and, therefore, surface hydrophobicity of these cells, apparently increase in parallel with an increase in their degree of differentiation and maturation.