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PubMed1983

Surface properties of lymphocyte subpopulations in autoimmune NZB/NZW F1 hybrid mice: alterations correlated with the immunodeficiency of aging.

Partitioning in a two-polymer aqueous phase system was used to probe the surface properties of lymphoid cell subpopulations in aged male NZB/NZW F1 hybrid (B/W) mice, an important model of autoimmunity, immunodeficiency, and lymphoid malignancy. Spleen cells were fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged dextran-polyethylene glycol system. CCD of spleen cells from young, clinically normal male B/W mice yielded several broad distribution patterns which frequently had two or more peaks. Analysis of differentiation antigens and functional properties of cells from different parts of the distribution revealed a subfractionation of the three major lymphocyte subpopulations. B lymphocytes had a low partition coefficient (K); T cells had an intermediate K and null cells had the highest K. To examine the partitioning behavior of T lymphocytes, spleen cells which were nonadherent to nylon wool columns were subjected to CCD. Nonadherent cells from young B/W mice consistently gave a single peak with high K. Aged mice (18 months) usually had nonadherent cells with a predominantly low K. In some experiments a systematic increase in the number of these cells could be demonstrated with increasing mouse age. An analysis of the adherence and partitioning behavior of lymphocyte subpopulations revealed no change in the adherence properties or proportions of B lymphocytes in aged mice. The large proportion of cells having a low partition coefficient in the nonadherent spleen cell population of old mice appears to be due to an increase in the number of null cells and in a decrease in the K of some T lymphocytes.

Aging↗

PubMed1976

Properties of chloroplasts isolated by phase partition.

Chloroplasts from spinach can be separated into at least three different populations by countercurrent distribution using polymer two-phase systems. The chloroplast particles of the three populations differ in protein/chlorophyll ratio, ultrastructure and metabolism. One population, peak I, consists of intact chloroplasts surrounded by the chloroplast envelope; the second population, peak II, consists of chloroplasts, which have lost their envelopes and much of their stromal material; the third population, peak III, consists of particles containing intact chloroplasts surrounded by a membrane-bound cytoplasmic layer including mitochondria and peroxisomes. Rapid batch procedures of peak I chloroplasts incorporated 14C almost entirely into glycolate and intermediates of the Calvin cycle and starch synthesis. Only small amounts were found in sucrose and amino acids. On the other hand preparations of peak III chloroplasts have a much broader spectrum of 14C-labelled products. Sucrose, malate and some amino acids contained about 40% of the 14C incorporated. It is concluded from these experiments that sucrose is formed not within the chloroplast but in the cytoplasm from intermediates exported by the chloroplast. The origin of peak III particles and their use for studying the cooperation between the chloroplast and the surrounding cytoplasm including mitochondria and peroxisomes is discussed.

Carbon Dioxide↗

PubMed1983

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. I. Human red blood cell subpopulations.

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning 51Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.

Cell Separation↗

PubMed1969

Studies on phenolic steroids in human subjects. IX. Role of the intestine in the conjugation of estriol.

In order to compare the enteric circulation of estriol-16alpha-glucosiduronate (see preceding paper) with that of estriol (E(3)), labeled estriol was administered to six women by several routes: both injection and infusion (300 min) into the cubital vein, injection into the portal vein system, ingestion and instillation into the jejunum and ileum. Urine, collected from 0-2, 2-4, 4-8, 8-12, and 12-24 hr, was analyzed by countercurrent distribution for its content of radioactive 3- and 16-glucosiduronate (E(3)-3Gl,E(3)-16Gl) and sulfoglucosiduronate (E(3)-3S,16Gl) of estriol. After peripheral injection of E(3), E(3)-16Gl was excreted rapidly and E(3)-3S,16Gl at a slower and more constant rate. E(3)-3Gl was barely detectable after infusion. After injection of E(3) into the portal vein, the excretion of E(3)-3S,16Gl was greater and quicker than after peripheral injection. Even in a subject with a complete bile fistula, the urinary excretion of E(3)-3S,16Gl was essentially unchanged. Ingestion also produced the same result. Only after instillation into the ileum was a large and rapid excretion of E(3)-3Gl obtained, whereas the excretion of E(3)-3S,16Gl, and E(3)-16Gl were depressed. These results together with those of the preceding paper suggest that E(3) does not readily appear in the small intestine except via a hepatoenteric circulation that produces very little E(3)-3Gl. When present in the distal segment of the small intestine, however, absorption, conjugation, and elimination proceed readily.

Biliary Fistula↗

PubMed1975

Abnormal membrane surface properties during maturation of rat reticulocytes elicited by bleeding as measured by partition in two-polymer aqueous phases.

Partition of cells in two-polymer aqueous phases is an extremely sensitive method for the separation of cells and for tracing subtle changes in the cells' membrane surface properties (primarily surface charge) as a function of in vivo processes (e.g. differentiation, maturation, ageing). Combining isotope labelling and cell countercurrent distribution techniques we have examined the membrane surface properties of rat reticulocytes produced in response to erythroid stress by bleeding. It was found that the rapid increase in the partition coefficient of normal reticulocytes subsequent to release into the peripheral blood (which reflects a rapid increase in membrane surface charge) is absent in reticulocytes produced in response to bleeding. In this way the reticulocytes behave like those produced in response to repeated phenylhydrazine injections. 'Stress reticulocytes' never mature to erythrocytes having normal membrane surface properties as judged by partition. The experiments show that our previous results are not due to the phenylhydrazine per se but rather to the production of 'stress reticulocytes'. Further, if remodelling of the phlebotomy-induced reticulocyte cell membrane occurs in the circulation as has been suggested it does not lead to a cell with normal membrane surface properties. Whether the abnormal membrane surface properties of 'stress reticulocytes' and of the erythrocytes to which they give rise affect the cells' life-span is not clear since phenylhydrazine-induced 'stress reticulocytes' have been reported to have at least a component of short-lived cells while the phlebotomy-induced cells are said to have a virtually normal life-span. It is likely that reticulocytes produced in severe anaemias, in general, mature to erythrocytes having abnormal surface properties (i.e. lower membrane surface charge).

Animals↗

PubMed1969

Studies on phenolic steroids in human subjects. 8. Metabolism of estriol-16 alpha-glucosiduronate.

6,7-(3)H-Estriol-16alpha-glucosiduronate-(14)C was administered to eight women (nine studies) by several routes: both injection and infusion (300 min) into the cubital vein, injection into the portal vein system, ingestion and instillation into the duodenum, jejunum, and ileum. Urine, collected from 0-2, 2-4, 4-8, 8-12, and 12-24 hr, was analyzed by countercurrent distribution for its content of radioactive 3- and 16-glucosiduronate (E(3)-3Gl,E(3)-16Gl) and sulfoglucosiduronate (E(3)-3S,16Gl) of estriol as well as for (3)H/(14)C ratio of each conjugate. After peripheral injection 50-60% of the injected E(3)-16Gl was excreted unchanged along with about 5% as E(3)-3S,16Gl with an unchanged (3)H/(14)C ratio, indicating direct sulfation of the injected E(3)-16Gl. During a 300 min infusion, urinary excretion closely resembled that following injection. But 2-4 hr after the end of the infusion excretion of E(3)-3S, 16Gl stopped, excretion of E(3)-3Gl (17%/24 hr) with an elevated (3)H/(14)C ratio started, and excretion of E(3)-16Gl continued (70%/24 hr), but with a rapidly increasing (3)H/(14)C ratio. This indicated sequestration in a sluggishly metabolizing compartment where two processes occurred: (a) extensive hydrolysis of E(3)-16Gl followed by reconjugation at either C3 or C16 with unlabeled uridine diphosphate glucuronic acid (UDPGA), thereby increasing the (3)H/(14)C ratio; and (b) transconjugation from C16 to C3, thereby producing E(3)-3Gl with finite (3)H/(14)C ratios. Instillation into various segments of the small intestine produced results qualitatively similar to those after intravenous infusion, whereas ingestion and intraportal injection resembled peripheral intravenous injection. Therefore, we have postulated the possibility of an enteric circulation (in addition to an enterohepatic circulation) in which the steroid or its conjugates are transported into the small intestine in the succus entericus, modified, and then reabsorbed and excreted in the urine-a process which requires several hours.

Adolescent↗

PubMed1975

Affinity partitioning. A method for purification of proteins using specific polymer-ligands in aqueous polymer two-phase systems.

We describe a method, called affinity partitioning, for the purification of proteins containing specific ligand binding receptor sites. This method adds specificity to the procedures for protein purification with aqueous polymer two-phase systems by introduction of a polymer derivative, coupled to an appropriate ligand. The addition of a polymer-ligand that partitions predominantly into one phase shifts the protein that binds this substance to the same phase. By performing countercurrent distribution in the presence of a polymer-ligand, the protein that binds the polymer-ligand can be separated from a heterogenous mixture. One example of affinity paritioning used dextran as the polymer-ligand. Dextran was chosen since it is a constituent of the most commonly used system for partitioning proteins. In a dextran-poly(ethylene oxide) system, concanavalin A bound dextran and partitioned predominantly into the dextran-rich phase. The addition of the specific competitor, D-mannose, displaced the partition coefficient toward unity, while the application of L-fucose, a noncompetitor, had little effect. Application of affinity partitioning to the purification of another protein required the synthesis of a specific polymer-ligand. To study this we synthesized dinitrophenyl-poly-(ethylene oxide), which binds specifically to S-23 myeloma protein. Addition of dinitrophenyl-poly(ethylene oxide) to the dextran-poly(ethylene oxide) phase system shifted the S-23 myeloma protein into the poly(ethylene oxide)-rich phase. epsilon-N-dinitrophenyl-L-lysine, by competing with binding of dinitrophenyl-poly(ethylene oxide), antagonized the latter's effect on the partition coefficient of S-23 myeloma protein. By adding various amounts of dinitrophenyl-poly-(ethylene oxide), we correlated the partition coefficient with concentration of polymer-ligand. A model of the action of polymer-ligand derivatives on the partition coefficient, derived from thermodynamic considerations, was found to be consistent with the experimental data relating the concentration of polymer-ligand and partition coefficient. Affinity partitioning should prove to be a useful complement to affinity chromatography in the purification of mixtures of proteins. Since cells and subcellular particles may be purified with aqueous polymer two-phase systems, affinity partitioning might be applied to their fractionation by using polymer-ligands specific for unique surface receptors.

Animals↗

PubMed1992

Effect of cell exposure to top or bottom phase prior to cell partitioning in dextran-poly(ethylene glycol) aqueous phase systems: erythrocytes as a model.

Cells exposed to dextran (Dx)-rich bottom phase prior to cell partitioning in Dx-poly(ethylene glycol) (PEG) aqueous two-phase systems have lower partition ratios than cells exposed to PEG-rich top phase. Aspects of this previously observed phenomenon were explored. In the present work charge-sensitive phases made with Dx T500 and PEG 8000 were used exclusively. It was found that: (1) even on countercurrent distribution (CCD) red cells (RBC) loaded in bottom phase have a lower apparent partition ratio, G, than the same cells loaded in top phase; (2) when part of the same cell population is loaded into top phase and part into bottom phase of the same load cavities for CCD, with the cells loaded into top or bottom bearing an isotopic tracer (51Cr), the cells loaded into top phase have a higher G value than the cells loaded into bottom phase; (3) the shift in the CCD curves of human or of rat RBC between cells loaded in top or bottom phase using systems having the same polymer concentration (though different salt compositions) shows no striking difference and is, for the number of experiments run, not statistically significant; (4) when the quantity of cells loaded for CCD is reduced from 10(9) to 10(8), the G value of cells loaded in top phase is reduced slightly while that of cells loaded in bottom phase is diminished more appreciably; (5) increasing polymer concentrations yield larger differences in G values between (rat) RBC loaded in top or bottom phase; (6) when cells exposed to top or bottom phase, respectively, are centrifuged and suspended in bottom or top phase, respectively, their CCD patterns are qualitatively similar to cells exposed to these latter respective phases initially; (7) rat RBC populations containing 59Fe-labeled cells of different but distinct age are fractionated on CCD irrespective of whether loaded in top or bottom phase. An exception are populations containing very young mature labeled cells (e.g., 4-d old) which are resolved when loaded in top phase but not in bottom phase. Thus cell populations exist which can be resolved by CCD when loaded in one of the phases but not when loaded in the other. Glutaraldehyde-fixed rat RBC containing 4-d old labeled cells are fractionated by CCD irrespective of whether loaded in top or bottom phase.

Animals↗

PubMed1992

Aqueous two-phase systems for biomolecule separation.

Over the past thirty years, aqueous polymer two-phase technology has evolved, both experimentally and theoretically, into a separation science with many useful applications in biomolecule purification and bioconversion. This paper summarizes the developments in the applications of aqueous two-phase systems to biotechnology. The main topics to be considered are the phase diagram and its characteristics, fundamentals of biomolecule partition, large-scale and multi-stage aqueous two-phase biomolecule purification, and extractive bioconversions. The first topic involves a discussion of the thermodynamics of aqueous polymer two-phase formation and how it is influenced by such factors as polymer molecular weight and concentration, temperature, and salt type and concentration. Next, the theoretical and experimental aspects of biomolecule partition in aqueous two-phase systems will be discussed in light of the factors which influence biomolecule partition: polymer concentration and molecular weight; temperature; salt type and concentration; the addition of charged, hydrophobic and affinity derivatives. Having reviewed the fundamentals of phase diagram formation and biomolecule partition, the next two topics are applications of aqueous two-phase technology. The first set of applications involve the large-scale extraction of proteins using one to three equilibrium stages and multi-stage purifications using countercurrent distribution, liquid-liquid partition chromatography and continuous countercurrent chromatography. The second application, and very promising area for future aqueous two-phase technology, is the extractive bioconversion which permits the simultaneous production and purification of a biomolecule.

Biotechnology↗

PubMed1990

Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA class 1, HLA class 2, and neoplastic epithelial antigens) in the apical cell membrane.

We have studied the effect of maturation to small intestinal-like epithelial cells of the human colonic carcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21-day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM-Gal. In comparison to control cells grown in DMEM-Glu medium, the more small intestinal-like cells grown in DMEM-Gal displayed no alteration of the lateral mobility of either cholera toxin (B subunit)-labelled ganglioside GM1 (diffusion coefficient, D [x 10(8)] = 0.8-0.9 cm2s-1; mobile fraction, R = 50-60%) or antibody-stained Class 2 histocompatibility (HLA-DR) antigen (D [x 10(9)] = 2 cm2s-1; R = 60-70%). However, antibody-labelled beta 2-microglobulin of HLA Class 1 antigen displayed increased mobility in HT29-Gal cells; D was x 1.4 and R x 1.8 larger in the HT29-Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were x0.60 and x0.69 of the values seen in HT29-Glu cells. It is thus concluded that DMEM-Gal-induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobility of different membrane components.

Carcinoma↗

PubMed1987

Phase partitioning in space and on earth.

In aqueous solution at low concentrations, the neutral polymers dextran and poly(ethylene glycol) (PEG) rapidly form a two-phase system consisting of a PEG-rich phase floating on top of a dextran-rich phase. Biological particles and macromolecules tend to partition differentially between the phases and the liquid-liquid phase interface in these systems. Bioparticle partitioning has been shown to be related to physiologically important surface properties such as membrane charge or lipid composition. Affinity partitioning into the PEG-rich phase can be accomplished by coupling PEG to a ligand having affinity for specific cells or macromolecules. Subpopulations can be identified or separated using multi-step countercurrent distribution (CCD). Incomplete understanding of the influence of gravity on the efficiency and quality of the impressive separations achievable by partitioning, and appreciation for the versatility of this efficient technique, have led to its study for low-gravity biomaterials processing. On Earth, two-phase systems rapidly demix because of density differences between the phases. In low-gravity, demixing has been shown to occur primarily by coalescence. Polymer surface coatings, developed to control localization of demixed phases in low-g, have been found to control electroosmosis which adversely affects electrophoretic separation processes on Earth and in space. In addition PEG-derivatized antibodies have been synthesized for use in immunoaffinity cell partitioning.

Dextrans↗

PubMed1986

Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands.

Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.

Adenine↗

PubMed1988

Column chromatographic separation of cells using aqueous polymeric two-phase systems.

Cell separation using aqueous polymeric two-phase systems is well established. For separations of cells having similar partition coefficients a multistep countercurrent distribution procedure has to be used. However, its operation is limited by time and apparatus constraints. As an alternative strategy we have developed a chromatographic technique in which the dextran-rich phase of a dextran/polyethylene glycol (PEG) phase system is immobilized onto derivatized agarose beads. The PEG-rich phase is used as the eluent. Inclusion of PEG-fatty acid affinity ligand gradients into the eluent produces separations of mammalian erythrocytes based on the differential interaction between the fatty acid and the erythrocyte membranes. A model separation of dog and human erythrocytes has been carried out.

Cell Separation↗

PubMed1991

Separation of cell mixtures by immunoaffinity cell partitioning: strategies for low abundance cells.

The partitioning of cells in aqueous two-phase systems formed by poly(ethylene glycol) (PEG) and dextran can be changed by incubating the cells with a PEG-modified antibody directed specifically against its surface. We have developed a new approach for immunoaffinity cell partitioning (IACP) in which the antibodies are first reacted with tresylated monomethoxy PEG (TMPEG) in sodium phosphate buffer, pH 7.5, the excess TMPEG is quenched by reaction with bovine serum albumin, and the resulting preparation is used directly for incubation with the cells without any isolation of the monomethoxyPEG (MPEG)-antibody conjugates. We have demonstrated the specificity of this IACP method by showing that MPEG-modified anti-human red blood cell antibody increases the partition of human erythrocytes from the interface to the PEG-rich top phase (up to 100%) but not the partitioning of either neutrophils or HL60 cells. Irrelevant antibodies do not affect the partitioning of red blood cells. The partitioning behaviors of erythrocytes and HL60 cells in mixtures varying from 75 to 10% red blood cells subjected to IACP are similar to those of the pure cell population, i.e., erythrocytes ca. 100% and HL60 cells 3% in top phase. Thus, the population of erythrocytes can be almost completely extracted into the top phase in a single step. The contaminant cells represent only a small percentage (less than 5% in most of the cases) of the cell mixture recovered in top phase. Both cell populations can be completely separated by countercurrent distribution (CCD).(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody Affinity↗

PubMed1986

Detection of surface differences between two closely related cell populations by partitioning. Erythrocytes from inbred and out-bred rats and rat strains.

We have recently developed a new and powerful method capable of detecting, by purely physical means, surface difference between closely related red (or other) cell populations. The procedure consists of isotopically labeling (with [51Cr]chromate) aliquots of red blood cell populations. Such labeled cells are mixed with an excess of unlabeled red cells to which they are to be compared. The mixtures are subjected to countercurrent distribution in either a charge-sensitive or a non-charge-sensitive dextran-poly(ethylene glycol) aqueous phase system. The distribution curves are analyzed for total cells (in terms of hemoglobin absorbance) and labeled cells (in terms of cpm). Changes in the relative specific activities through the distribution curves are indicative of subtle differences in surface properties between such cell populations. Using this method we have found that erythrocytes from arbitrarily chosen (presumably hematologically normal) individuals differ. In the current work we have examined the surface properties of erythrocytes from Sprague-Dawley and from Lewis rats. This was done with a view to determining whether (a) differences of the type found between different humans can also be detected in other species and (b), if such differences do exist, to examine, by study of the highly inbred Lewis rat strain, whether the differences appear to have a genetic or an acquired basis. It was found that the surface properties of erythrocytes from Lewis and Sprague-Dawley rats differ as do erythrocytes among rats of the Sprague-Dawley strain. No difference was found between red blood cells from different rats of the inbred Lewis strain. These results indicate that the surface differences between red blood cells from different rats detected by partitioning have a genetic rather than acquired origin.

Animals↗

PubMed1975

Incorporation of ortho[32P]phosphate into phosphatidylcholines and phosphatidylethanolamines in rat skeletal muscle.

1. The specific radioactivities of individual molecular species of muscle phosphatidylcholine and phosphatidylethanolamine have been measured by a combination of argentation thin-layer chromatography and countercurrent distribution. 2. The specific radioactivities of individual molecular species of muscle phosphoglycerides have been determined 3 h after intraperitoneal injection of ortho[32 P] phosphate. Under these conditions the specific radioactivities of the species present in rat muscle were found to be measures of the relative turnover times of these molecules. 3. The specificity radioactivity of phosphatidylcholine was approx. three times that of phosphatidylethanolamine. The 1-palmitoyl-2-oleoyl and 1-oleoyl-2-linoleoyl phosphatidylcholines had the fastest turnover and the 1-palmitoyl-2-arachidonoyl the slowest. Of the phosphatidylethanolamines, the linoleoyl and the docosahexaenoyl species showed the fastest turnover and 1-stearoyl-2-arachidonoyl the slowest. 4. The results indicate that phosphoglycerides in muscle turn over more slowly and more evenly than do liver phosphoglycerides.

Animals↗

PubMed1992

Erythrocyte partitioning in dextran-poly(ethylene glycol) aqueous phase systems. Events in phase and cell separation.

Early events in the partitioning process which involve characteristic kinetics of cell- and phase-specific interactions and phase separation have been described previously. This paper reports on red cell-phase droplet interactions pertaining at the time of usual phase sampling (i.e., the time at which a clear bulk interface is first apparent) and beyond in cell partitioning and countercurrent distribution experiments. In non-charge-sensitive phase systems close to the critical point, cells can be free or attached to phase droplets. Cells that are free are virtually completely in the top phase, whereas different cell populations that show essentially complete binding to droplets can nevertheless have different partition ratios and be separated, thus reflecting the effects of the difference in the cells' avidity for the phase droplets during the early, elapsed events in partitioning. At higher polymer concentrations (i.e., higher interfacial tensions), the cell populations, completely bound to phase droplets, partition completely to the interface, and consequently cannot be separated. When such systems are made charge-sensitive by the generation of a Donnan potential between the phases or made into affinity systems by the incorporation of PEG ligands (e.g., PEG-palmitate), there is a decrease in the avidity of the cells for phase droplets. The resulting increase in the ratio of free to droplet-bound red cells in the top phase at the time of sampling correlates with an increase in the partition ratio, P, observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Effect of rapidity of phase separation on the efficiency of cell fractionation by partitioning in aqueous two-phase systems.

Partitioning in two-polymer aqueous phase systems is an established method for the separation, purification and characterization of biomaterials. Because of the relatively slow settling rates of these phases, a consequence of the slight difference in density between them, effort has been directed to speeding up phase separation by various means (e.g., the development of a thin-layer countercurrent distribution apparatus). This has resulted in the more rapid processing of materials. Unlike soluble materials, biological particulates (e.g., cells) generally partition between one of the bulk phases and the interface. The mechanism of cell partitioning involves cell-specific adsorption to droplets of one phase suspended in the other, subsequent to phase mixing, and the delivery of adsorbed cells to the bulk interface as the droplets settle. In this communication we show, using erythrocytes as a model, that speeding up phase separation is counterproductive when partitioning cells and results in reduced efficiency of their separation or subfractionation. The most likely reason for this result is that increasing the speed of phase settling removes the droplets of one phase suspended in the other more rapidly than cells can attach to them, thereby interfering with the mechanism whereby cells partition.

Animals↗