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.     

Extraction of penicillin-G by aqueous two-phase partition

Summary Pure potassium penicillin-G solution and penicillin-G fermentation broth were extracted using the aqueous two-phase systems of poly-ethylene glycol and salts. The partition coefficients of penicillin-G were above 10 in both pure solution and broth systems. The partition coefficients of phenyl acetic acid were about 0.25. Cell mass and solid residue in the broth system were partly concentrated around the interfacial region and partly settled to the bottom when under 1000 × g centrifugal force. Accordingly, this technique is a promising alternative for the recovery of penicillins.     

Temperature dependence of the partition coefficient of proteins in aqueous two-phase systems.

We report the partition coefficients of lysozyme, chymotrypsinogen-A, albumin and catalase in sixty four Polyethyleneglycol/Dextran/Water systems at 4, 25 and 40 degrees C. We found that the partition coefficients of the four proteins generally increase with increasing temperature. The influence of temperature on the partition coefficient seems to be highly dependent on the kind of protein which is partitioned and on the total polymer concentration, but does not, in general, depend on the molecular weight of the polymers. The partition coefficients of small and hydrophilic proteins like lysozyme and chymotrypsinogen-A are only slightly affected by changes in temperature, while the partition coefficients of bigger and more hydrophobic proteins like albumin and catalase are strongly affected by changes in temperature. The results suggest the incorporation of attractive forces (possible electrostatic) into a model previously reported by us.     

Characterisation of anaerobic mixed cultures by partition in an aqueous two-phase system

A method to characterise a dynamic microbial consortium is described. By exploiting differences in surface properties between different cells and between cells of different physiological status, it was possible to develop a partition pattern for a mixed culture under different conditions. The separation method used was partition in aqueous two-phase systems and when using a counter current extraction process one could clearly differentiate the partition profile between resting, active and overloaded biomethanation cultures.     

Extraction of amino acids by aqueous two-phase partition

Summary Amino acids, including lysine, glutamic acid, and phenylalanine, in pure solution or in fermentation broth, were extracted with the aqueous two-phase system consisting of polyethylene glycol and salts, giving a very sharp separation. The partition is influenced by the type and the amount of salts used, pH and components of the broth.     

Separation of presumptive plasma membranes from mitochondria by partition in an aqueous polymer two-phase system

Light-induced absorbance changes (LIAC), indicating the reversible reduction of a b-type cytochrome, and with a possible connection to blue light photomorphogenesis, have been found in a presumptive plasma membrane rich centrifuge fraction from LIAC could be due to plasma membrane vesicles turned inside out or to cytochromes localized in other organelles. Phase partition proved to be a rapid method (results technique membrane particles are separated according to differences in surface properties rather than size and density. LIAC could be separated into two fractions: one partitioning into the polyethylene glycol rich upper phase and another preferring the dextram rich lower phase. Mitochondria (cytochrome c oxidase) were recovered in the lower phase. A dual distribution of LIAC was found with all materials tested: corn coleoptiles, corn shoots, barley shoots and cauliflower inflorescences. About 80–90% of the cytochromes in the upper phase were related to LIAC, whereas only 10–15% of those in the lower phase were of this kind. The LIAC preferring the upper phase was probably bound to the plasma membrane, since plasma membrane vesicles are known to have a high partition in these phase systems. The lower phase LIAC could be due to plasma membrane vesicles turned inside out or to cytochromes localized in other organelles. Phase partition proved to be a rapid method (results within one hour after the initial pelleting) for purification of presumptive plasma membranes, yielding a preparation which contained five times less mitochondrial contamination than the preparation obtained with sucrose gradient centrifugation (the 33/45% w/w sucrose interface fraction).     

Choice of an aqueous polymer two-phase system for cell partition

Partition of human erythrocytes in aqueous two-phase polymer systems produced by Ficoll and different molecular weight fractions of dextran and polyethylene glycol and the influence of the ionic composition on the cells' partition in the systems was studied. It is found that the Ficoll-dextran-40 system is characterized by a number of advantages as compared with the common dextran-polyethylene glycol system or the others systems under study. The main advantage of the system appears to be that it is possible to concentrate the red cells in the top phase or in the bottom phase of the system, depending on the system ionic composition. The influence of the nature and the concentration of salt additives on this two-phase system formation is examined.     

Separation of precultured pollen from Nicotiana tabacum by aqueous polymer two-phase partition

Pollen isolated from cold treated and precultivated anthers of tobacco (Nicotiana tabacum L. var. Wisconsin 38) were separated into different fractions with counter-current distribution using an aqueous Dextran-polyethylene glycol two-phase system. It was possible to distinguish among eight pollen classes differing in developmental stage and in partitioning. A part of each fraction was cultivated for analysis of embryo formation. This was highest in a fraction with an intermediate to high partition in the phase system. Enriched in this fraction were also pollen that were fairly well stained with acetocarmine, contained several nuclei and had a relatively low mitochondrial activity. The enrichment of embryogenic pollen offers several advantages especially to physiological studies on embryogenesis.     

Preparation of mammalian plasma membranes by aqueous two-phase partition

Plasma membranes are readily purified from crude mixtures by the technique of aqueous two-phase partition. This procedure has been used widely to prepare plasma membrane fractions, highly purified, from both green and dark-grown plant materials. Only recently, however, has the method been applied to animal cells and tissues to supplant previous protocols where preparative sucrose and other gradient procedures were employed. The method based on aqueous two-phase partition, is rapid, reproducible and facile. It is especially useful for tissue culture cells since gradient methods often are complicated by alterations in plasma membrane density with different culture conditions and the presence of extensive cytoskeleton-membrane interactions. Homogenates prepared either in dilute 1 mM bicarbonate or isotonic sucrose are first centrifuged to concentrate the plasma membrane vesicles. The concentrated membranes are then combined with a mixture of dextran and polyethylene glycol that will of itself spontaneously separate into a polyethylene glycol-rich upper phase and a dextran-rich lower phase. The mixture is usually centrifuged to accelerate phase separation. The plasma membranes enter the upper, polyethylene glycol-rich phase, whereas contaminating membranes remain with the dextran of the lower phase. The yield of plasma membranes is 20% or more of those present in homogenates and the fraction purity is 90% or greater.     

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.     

Extraction of proteins from material rich in anionic mucilages: partition and fractionation of vanadate-dependent bromoperoxidases from the brown algae Laminaria digitata and L. saccharina in aqueous polymer two-phase systems

For various reasons extraction of proteins from plant material is difficult. In particular phenolic compounds and polyanionic cell-wall mucilages render conventional procedures of extraction and purification much more difficult. In this respect, aqueous polymer two-phase systems are presented as a powerful technique in extraction of vanadate-dependent bromoperoxidases from the brown macroalga Laminaria digitata, a seaweed extremely rich in mucilages. Little bromoperoxidase activity was obtained when fresh thallus material was extracted in Tris buffer. Extraction from freeze-dried and powdered material was more efficient but only satisfactory when partitioning in an aqueous polymer two-phase system was employed. Among several two-phase systems tested, one composed of poly(ethylene glycol) (PEG 1550) and potassium carbonate proved most successful (phase system-1). A rapid and efficient extraction procedure was developed with special regard for suitability in large scale processes. Staining for catalytic activity after PAGE revealed a pattern of several bromoperoxidase isoforms. Bromoperoxidases extracted in phase system-1 were fractionated into two groups of isoforms by partitioning in a second system (phase system-2) indicating that isoforms from both groups differ significantly in surface properties. Subsequently, one purification step by hydrophobic interaction chromatography was sufficient to remove residual non-peroxidase proteins as well as remaining polysaccharides from bromoperoxidases of both groups. Thus, consideration of aqueous two-phase systems as a technique for extraction and purification of plant proteins can be recommended, whenever inconveniant amounts of phenolic compounds, mucilages or pigments are present.     

Relative hydrophobicity of mononucleotides and nucleosides determined by partition in aqueous two-phase polymeric system ficoll-dextran

The effect of ionic strength on the partition of several mononucleotides, deoxyribonucleosides and the corresponding bases in aqueous buffered (pH 7.4) ficoll--dextran biphasic system was examined. The relative hydrophobicity of the compounds at zero ionic strength was estimated in terms of an equivalent number of CH2 groups. It is found that the effect of the ionic strength on the relative hydrophobicity of the phosphate group is similar for all the mononucleotides examined but for the adenosine derivative.     

Different functional states of ram spermatozoa analysed by partition in an aqueous two-phase system

The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing-thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen-thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen-thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk-yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk-yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.     

Surface heterogeneity of bovine sperm revealed by aqueous two-phase partition

Ejaculated, bovine sperm have been subjected to multiple partition in aqueous two-phase systems. This partition, carried out in a countercurrent fashion, reveals heterogeneity of the sperm population with respect to surface properties. The sperm, when partitioned in phase systems that detect non-change associated surface properties (change-insensitive) are largely distributed as two distinct populations. In charge-sensitive phase systems (which principally detect cell surface molecules carrying charge) the sperm do not show any obvious surface heterogeneity. Considerable heterogeneity is revealed in affinity-ligand phase systems containing palmitic acid coupled to one of the phase components-poly(ethylene glycol). There is a difference in surface heterogeneity between sperm which have been washed in buffer or left unwashed, direct from the ejaculate. This is indicative of weak adsorption of proteins to the sperm surface in seminal fluid. These results show that bovine ejaculated sperm is a heterogeneous cell population having unequal distributions of a number of different surface molecules.     

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.     

Seasonal differences in ram seminal plasma revealed by partition in an aqueous two-phase system

Seminal plasma plays an important role in maturation of spermatozoa through hormonal, enzymatic and surface-modifying events. We have previously shown that adsorption of seminal plasma proteins (SPPs) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we investigate the hypothesis that seasonal differences in seminal plasma could affect its ability to recover membrane integrity of cold-shocked sperm. The effect of seminal plasma proteins, obtained in breeding (bsSPPs) and non-breeding (nbsSPPs) season, on cold-shocked ram spermatozoa previously freed from seminal plasma, was analysed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system as well as membrane integrity determination by fluorescence markers. Cold-shock treatment greatly lowered cell viability in both breeding and non-breeding season spermatozoa. The cold-shocked sperm viability obtained was approximately 20%. The loss of heterogeneity and the decrease in viability revealed by CCCD analysis was reversed by the addition of increasing amounts of bsSPP, which induced restoration of the surface characteristics of viable-like spermatozoa, as well as an increase in the number of recovered viable sperm. However, this restoring effect was much lower when nbsSPPs were added, even in a sixfold higher concentration than used with bsSPPs. Incubation of cold-shocked cells with both kinds of proteins performed in both seasonal periods, showed that the recovering effect was related to the season when the plasma sample was obtained rather than to the semen season. The addition of bsSPPs to cold-shocked sperm accounted for a nearly 50% reversion for both studied breeding seasons. However, the reversion percentages obtained with nbsSPPs were significantly lower (P<0.05) than those found with bsSPPs in both studied seasonal periods. This different reversion capacity of bsSPPs and nbsSPPs was related to a different protein composition, as revealed by comparative sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The bands of 20, 21, 24, 36 and 67 kDa of the bsSP sample profile decreased in winter–spring SP, and were even less intensely stained in summer SP. Densitometric analysis of the stained gel patterns allows automatic comparison among the separated bands, and revealed an important decrease in the content of several bands. The 21.5 kDa band showed the highest decrease, lowering to 14% in June–August plasma with respect to the value obtained in September–December plasma.     

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.     

Determination of the dissociation constant of valine from acetohydroxy acid synthase by equilibrium partition in an aqueous two-phase system

An aqueous polyethylene glycol/salt two-phase system was used to estimate the dissociation constant, K_dis, of the Escherichia coli isoenzyme AHAS III regulatory subunit, IlvH protein, from the feedback inhibitor valine. The amounts of the bound and free radioactive valine in the system were determined. A Scatchard plot of the data revealed a 1:1 valine–protein binding ratio and K_dis of 133±14 μM. The protein did not bind leucine, and the ilvH protein isolated from a valine resistant mutant showed no valine binding. This method is very simple, rapid and requires only a small amounts of protein compared to the presently used equilibrium dialysis method.     

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.