Counter-current Distribution Technique Research Articles

Irritant and co-carcinogenic diterpene esters from the latex of Euphorbia cauducifolia L.

Ten (1–10) irritant and mild co-carcinogenic diterpene esters were isolated from the latex of Euphorbia cauducifolia L. using bioassay-guided countercurrent distribution and other chromatographic techniques. The isolated compounds were characterized on the basis of spectroscopic results and mass measurements. As an outcome, the ingenane-type esters were established with the following structures: 3-O-angeloyl-17-O-palmatoylingenol (1), 3-O-palmatoyl-5-O-angeloylingenol (2), 5-O-angeloyl-17-O-palmatoylingenol (3), 3-O-angeloyl-5-O-palmatoylingenol (4), 17-O-(2Z,4E,6Z)-2,4,6-tetradecatrienoyl-20-O-palmatoylingenol (5), 5-O-angeloyl-17-O-benzoylingenol (6), 5-O-angeloyl-17,20-diacetoxyingenol (7), 3-O-angeloyl-17-O-benzoyl-20-acetoxyingenol (8), 3-acetoxy-5-O-angeloyl-17-O-benzoylingenol (9), and 5-O-angeloyl-3,17,20-triacetoxyingenol (10). Their biological screening revealed that they are moderate irritants, and low to moderate tumor promoters compared to TPA, but hardly showed any solitary carcinogenic activity. The isolated esters represent new compounds and were not reported before from any source.

Surface and metabolic properties of microcytic and macrocytic human anaemic red blood cells detected in polymer aqueous two-phase systems

Microcytic and macrocytic red blood cells from anaemic patients have been fractionated as a function of cell surface properties by the countercurrent distribution technique using charge-sensitive dextran/poly(ethylene glycol) aqueous two-phase systems. As deduced from the fractionation profiles, microcytic cells constitute a heterogeneous cell population with decreased surface charge properties while, macrocytic cells constitute a homogeneous cell population with behaviour similar to that of the control red blood cells. The specific activity of pyruvate kinase, an age-dependent enzyme, did not change along microcytic red blood cells fractionation profiles, suggesting that such cells have altered ageing properties. However, pyruvate kinase specific activity decreases from the left- to the right-hand side of the fractionation profile of macrocytic red blood cells, indicating that these cells follow the normal ageing process. Bisphosphoglycerate mutase specific activity did not change along the fractionation profile of any cell population under study, thus providing 2,3-bisphosphoglycerate during the life-span of the red blood cells from anaemic patients.

Heterogeneity of hypotonically loaded rat erythrocyte populations as detected by counter-current distribution in aqueous polymer two-phase systems

Carrier rat erythrocytes loaded with exogenous substances ([ 125I]carbonic anhydrase) by hypotonic-isotonic dialysis become heterogeneous cell populations that can be fractionated using the counter-current distribution (CCD) technique. Two well-defined low- and high-partition ratio, G, subpopulations are obtained in charge-sensitive dextran-polyethylene glycol two-phase systems. Thelow- G subpopulation, which contains the most fragile and surface-altered cells, as deduced from their osmotic fragility curves and partition behaviour, respectively, presents a high amount of exogenous substance incorporated (134.6 cpm/10 6 cells). Thehigh- G subpopulation, that contains cells similar to the control or isotonically dialyzed cells presents a lower amount of exogenous substance incorporated (69.8 cpm/10 6 cells). Cells in thishigh- G subpopulation seem to be fractionated, like the controls, according to ageing as suggested by the decline of the pyruvate kinase specific activity from the left- to the right-hand side of the CCD profile.

Time-dependent effect of the partitioning of electroplax membranes in aqueous biphasic systems using counter-current distribution technique and hexaethonium-polyethylene glycol

Cholinergic receptor-rich membranes from the electroplax of Torpedo californica have been partitioned between the upper phase and the interface of an aqueous two-phase system consisting of water, dextran and polyethylene glycol. The partitioning of membranes containing the cholinergic receptors was influenced by including polyethylene glycol-bound hexaethonium in the upper phase. The partition behaviour was studied by using the two-phase systems for counter-current distributions with a varying number of steps. The membranes showed time-dependent distributions, which is assumed to be due to changes in the internal distribution of membrane constituents and/or formation of larger membrane aggregates. Despite this effect good purification of the cholinergic membranes could be obtained. The consequences of the time-dependent effect for the partitioning of membranes within aqueous two-phase systems as well as the possibilities to reduce it are discussed.

Countercurrent Chromatography Using a Toroidal Coil Planet-Centrifuge: A Comparative Study of the Separation of Organelles Using Aqueous Two-Phase Partition

Abstract A toroidal coil planet centrifuge is described and compared with other countercurrent chromatography (CCC) and countercurrent distribution (CCD) techniques. The basis of separation is partition in aqueous two-phase polymer systems, with each method assessed by fractionating rat liver organelles. The size and ease of operation of the toroidal coil planet centrifuge gave significant advantages over conventional CCD systems achieving equivalent resolution in a fraction of the time.

Toroidal Coil Countercurrent Chromatography in the Affinity Partitioning of Nicotinic Cholinergic Receptor Enriched Membranes

Abstract A variation on the aqueous polymer phase partition method, affinity partitioning, has proved suitable for the preparative scale purification of binding site enriched membrane fragments. The full resolving potential of the affinity partitioning technique often requires the utilization of multiple extraction procedures such as countercurrent distribution. In this report, we evaluate the combination of a newly developed countercurrent purification technique, toroidal coil chromatography, with affinity partitioning. This approach provides an efficient method for purification and characterization of membrane bound nicotinic cholinergic receptors. The relative merits of the toroidal coil chromatography technology and the more conventional thin-layer countercurrent distribution techniques are compared.

Applications of Rotation Locular Countercurrent Chromatography in Natural Products Isolation

Abstract Purification of natural products and the fractionation of crude plant extracts are processes which traditionally involve adsorption and/or molecular exclusion chromatography. While countercurrent chromatography avoids the irreversible adsorption and decomposition frequently encountered in adsorption chromatography, the classical countercurrent distribution technique is both time- and solvent-consuming, consequently it is of limited value today. Rotation locular countercurrent chromatography (RLCC) is one of three new countercurrent techniques, the others being droplet contercurrent chromatography (DCCC)1 and planet coil countercurrent chromatography (PCCCC)2, which have revived the chromatographic application of liquid-liquid partition. We have employed a commercially available RLCC apparatus in the isolation of several natural products with a broad range of polarity demonstrating the versatility of the RLCC with regard to the functionality of the compounds to be isolated.

Liquid—liquid extraction of membranes from calf brain using conventional and centrifugal counter-current distribution techniques

Neural membranes isolated from calf brain have been partitioned in aqueous two-phase systems containing dextran and polyethyleneglycol. When the partition was repeated several times, using counter-current distribution technique, the distribution of the membranes between the upper phase and the interface changed in a non-ideal manner and in favour of the interface. By using a centrifugal counter-current distribution device the time for the experiment could be reduced by a factor of 7–8 and the distribution was similar to what could be expected for ideally behaving membranes. The time-dependent change of the membranes is discussed in terms of aggregation and lateral membrane perturbations. Despite this effect a certain fractionation has been achieved as deduced from analysis of cholesterol content, opiate receptor activity and acetylcholinesterase activity along the counter-current distribution row of fractions. Compared to the starting material these activities were enriched some two-fold in certain fractions.

Protein-protein interactions studied by counter-current distribution : III. simulation of self-associating systems

Biological macromolecules that undergo self-association in solution are widely encountered. As differences in surface properties, such as charge and hydrophobicity, between monomeric and polymeric forms, are very probable it should be possible to use the counter-current distribution technique in two-phase liquid systems for the study of self-association. The distribution behaviour of a self-associating solute has been simulated in order to establish the boundary conditions and limitations as well as potentials of the counter-current distribution technique as a tool for studying self-associations. The distributions have been calculated for a range of association constants, partition coefficients and initial solute concentration as well as for small zone and moving boundary counter-current distribution.

Binding of human carbonic anhydrase to human hemoglobin.

The ability of human carbonic anhydrases to interact with human CO-hemoglobin have been studied with the counter-current distribution technique in aqueous/aqueous biphasic systems. The experimental results show that human carbonic anhydrase II interacts with human CO-hemoglobin whereas human carbonic anhydrase I does not. THe interaction between CO-hemoglobin and carbonic anhydrase II was quantified using the theoretical model developed previously for one-to-one interacting systems. [Backman, L. and Shanbhag, V.P. (1979) J. Chromatogr. 171, 1-13]. The apparent association constant was estimated to be 4.1 x 10(5) l mol-1 at pH 8.0 and 21 degrees C for the association of carbonic anhydrase II and CO-hemoglobin.

Affinity partitioning of membranes. Evidence for discrete membrane domains containing cholinergic receptor.

Subsynaptic membrane domains from Torpedo californica electroplax contain nicotinic cholinergic receptor molecules at densities as high as 20,000 micrometers-2. Intense homogenization of the electroplax releases membrane fragments enriched in nicotinic receptor from basal lamina and other synaptic cleft and presynaptic elements. Ideally, preparations of membrane fragments, highly enriched in nicotinic receptor, should approach 125I-alpha-bungarotoxin-specific binding activities near the levels observed after receptor dispersal in detergents and subsequent affinity chromatography. We report the application of affinity partitioning, combined with multiple extraction techniques, to yield preparations of virtually homogeneous membranes enriched in nicotinic receptor alpha, beta, gamma, and delta subunits as well as the 43,000-dalton peripheral protein subunit. The countercurrent distribution technique serves to resolve three populations of receptor-containing membranes. One fraction is refractory to affinity partitioning and may represent aggregates of receptor-rich membranes with fragments derived from nonsynaptic membranes. The second and third fractions contain membrane fragments derived from the subsynaptic membrane and are highly enriched in nicotinic receptor (5.1 to 7.8 nmol of alpha-bungarotoxin binding sites/mg of protein). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of all three fractions indicates that alpha, beta, and gamma subunits are present in stable stoichiometric ratios, while the 43,000-dalton peripheral protein content varies by 33% between the fractions. However, removal of 90% of the 43,000-dalton component by mild alkali treatment does not result in conversion of one fraction into the other. The combination of affinity partitioning and counter-current distribution techniques utilized in this study should prove useful in the resolution of a variety of subcellular particles that contain specific binding sites.

Abnormal membrane lipid--related properties during maturation of reticulocytes from severely anaemic rats as measured by partition in two-polymer aqueous phases.

Aqueous solutions of dextran and of polyethylene glycol when mixed give rise to immiscible, liquid two‐phase systems. Such systems are suitable for separating cells by partition and for tracing subtle changes in the cells’membrane surface properties as a function of differentiation, maturation and age. By appropriate manipulation of polymer concentration and ionic composition and concentration one can select certain membrane properties (e.g. charge‐associated, lipid‐related) that will determine the cells’partition coefficient (i.e. the cells’relative affinity for the top or bottom phase or adsorption to the interface). Combining radioisotopic labelling and cell countercurrent distribution techniques we have examined the partition behaviour of reticulocytes from severely anaemic rats (made so through phenylhydrazine‐injection) in two‐polymer phase systems which reflect membrane lipid‐related properties. It was found that such stress reticulocytes are abnormal with respect to their membrane lipid which does not produce the changes in partition coefficients observed with normal reticulocytes during maturation. The mature erythrocytes derived from such stress reticulocytes also have abnormal lipid‐related membrane properties. Together with our earlier results it is clear that red blood cells produced in severe anaemia have and retain abnormal charge‐associated and lipid‐related membrane surface properties during their life‐span and do not undergo the membrane surface changes associated with normal maturation and ageing as discerned by partition in two‐polymer aqueous phase systems.

Surface properties of rat reticulocytes during maturation as measured by partition: membrane charge-associated changes discerned prior to membrane lipid-related changes.

SummaryPartition of cells in dextran‐polyethylene glycol aqueous phases is an extremely sensitive method for separating cells and for tracing subtle changes in the cells’membrane surface as a function of differentiation, maturation and ageing. By appropriate manipulation of polymer concentration and ionic composition and concentration one can select, to a great extent, the membrane properties (e.g. charge‐associated, lipid‐related) that will determine the cells’partition coefficient. Combining radioiosotopic labelling and cell countercurrent distribution techniques we have previously reported rapid increases in charge‐associated partition coefficients during rat reticulocyte maturation followed by slower decreases in the partition coefficients during the entire life span of the mature erythrocyte. In the present experiments red blood cell populations containing radioisotopically labelled cells of different and distinct ages have been subjected to countercurrent distribution in phase systems reflecting either membrane charge‐associated or lipid‐related characteristics. It was found that: (a) membrane lipid‐related surface properties change rapidly during reticulocyte maturation; (b) membrane charge‐associated changes during reticulocyte maturation are discerned prior to those that are lipid‐related; and (c) lipid‐related membrane changes continue slowly throughout the entire life‐span of the erythrocyte.The sensitivity with which red cell membrane lipid‐related differences or changes can be detected and measured by cell partition suggests great usefulness of this simple method in the study of abnormalities in red cell lipids.

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

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

Counter-current chromatography using a new coil planet centrifuge

Coil planet centrifugation has existed for a number of years, but its potential in superseding existing counter-current distribution and liquid-liquid chromatographic techniques has not been fully realised. The development of a new coil planet centrifuge that is capable of analytical or preparative separations of biological materials is described. Testing of solvent systems, fluid dynamic problems, and the running of a complete separation system are discussed, and the accuracy and repeatability of separations are demonstrated.

Separation of subchloroplast membrane particles by counter-current distribution

Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity. The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique. Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks. The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicates that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae.

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).

Improved high-performance liquid chromatographic method for polypeptide antibiotics and its application to study the effects of treatments to reduce microbial levels in bacitracin powder

Improvements were made in the high-performance liquid chromatographic (HPLC) method to obtain baseline separation of chromatographic peaks of structurally similar polypeptide components in bacitracin. The improved method uses a 30-cm-long stainless-steel column packed with μBondapak C 18. The theoretical plates of the column are approximately 140,000 per meter for the bacitracin A peak. The resolution function between bacitracins B 1 and B 2 and that between bacitracins A and B 2 have been improved 418 and 225%, respectively. The components of bacitracin, bacitracins A, B, C, D, E, F, and G, were fractionated by the countercurrent distribution technique. These components, together with Compound X, a compound separated on a carboxymethylcellulose column, and bacitracin F, obtained by degrading bacitracn A sample at neutral pH, were used to identify peaks in the HPLC chromatogram. Effects of processing methods used to reduce microbial contamination levels in bacitracin powders were evaluated. Heat treatment caused a significant loss of anti-microbial activity (35% reduction), bacitracins A, B 1, and B 2 were reduced by 37, 22, and 21%, respectively. A significant increase (2.8 times) of bacitracin F, an oxidative degradation compound, was shown. Irradiation by 60Co at 1.8 Mrad caused no loss of potency nor change in any of the bacitracin components. Ethylene oxide treatment, on the other hand, aused considerable (46%) reduction of potency. Substantial reduction of areas under the peaks of bacitracins A, B 1, and B 2 (50, 24 and 37%, respectively) were noted. The chromatograms showed numerous unresolved peaks around bacitracins A, B 1, and B 2; however, no significant increase in the bacitracin F peak, nor appearance of non-UV absorbng peaks were observed. Peptide antibiotics of the polymyxin group, circulin, colistin, and polymyxin, were also analyzed using the μBondapak C 18 column with a linear-gradient elution. A UV monitor was used for polymyxin. A moving-wire flame ionization detector was used to monitor circulin and colistin. A sample of polymyxin, circulin, and colistin may be analyzed in less than 20 min of chromatographic time.

16] Determination of equilibrium constants by countercurrent distribution

Three procedures have been outlined which may be used to obtain chemical equilibrium constants from countercurrent distribution experiments. The first, which has been used experimentally and is likely to be the most sensitive, depends on fitting predictions computed from a model to the experimental zonal countercurrent distribution pattern. The fit is optimized by variation in succession of the unknown parameters, such as equilibrium constants. For this procedure, it is necessary to perform iterative computations with the aid of a digital computer, to make the necessary predictions. The second procedure consists of measuring directly the weight-average rate factor { K (1+K) } w from the countercurrent distribution pattern in moving boundary experiments. This is done at different loading concentrations or compositions, and the data are combined with individual rate factors for the species present, to obtain equilibrium constants. The third procedure is to study the movement of differential moving boundaries between two large regions of constant composition. Such data, in the case of only one reacting solute, which is a single thermodynamic component, gives rise to the Z -average rate factor. In the case of a monomer-single polymer equilibrium, the use of this quantity, together with the weight-average rate factor and the rate factor for monomer, leads very directly to the equilibrium constant for the reaction. There is an important potential advantage in the use of the counter-current distribution technique rather than continuous transport methods in such determinations. Even if the reactions are quite slow, one can allow the system to come to equilibrium at each stage in countercurrent distribution with no loss in resolution whatsoever, and one can also test quite carefully to assure that equilibrium has been achieved. However, due to the effects of diffusion in continuous transport methods, resolution is impaired by decreasing the field responsible for migration. Moreover, if the speed of reaction is slow enough so that appreciable migration takes place before chemical equilibrium is locally reestablished, a wide variety of artifacts can also occur, which seriously complicate the possibility of achieving any meaningful equilibrium measurements. 23

Specificity of Accepted Procedures for Urine Creatinine

Abstract Specificities of various commonly used procedures for creatinine in urine were evaluated after subjecting pure creatinine or urine (with or without added creatinine) to counter-current distribution (CCD) between acetate buffer (pH 4.6) and n-butanol. After 10 transfers, each phase of each tube was analyzed for creatinine by each of the various procedures, and the resulting data were compared with the theoretical distribution curve for pure creatinine. The data indicate that the original or automated Jaffé reaction is the most nearly specific of the procedures investigated. Thus, the CCD technique, besides being a powerful separatory tool, also permits a physicochemical approach to the evaluation of specificities of standard procedures in clinical chemistry.