Preparation of benzoyl dextran and its use in aqueous two-phase systems.

The graft modification of dextran with benzoyl groups has been studied. The factors that affect the degree of substitution of benzoyl dextran were investigated. Phase diagrams for aqueous two-phase systems composed of polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran have been determined. Phase separation was also obtained in aqueous solution of two benzoyl dextran polymers with different degrees of substitution. A four-phase system was obtained with a mixture of polyethylene glycol, dextran and two kinds of benzoyl dextrans. The partitioning of methylene blue and a Procion yellow HE-3G dextran derivative were studied in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems and in systems of two benzoyl dextrans differing in degree of substitution. The proteins bovine serum albumin and glucose-6-phosphate dehydrogenase were partitioned in polyethylene glycol/benzoyl dextran aqueous two-phase systems and the effect of the degree of substitution of benzoyl dextran was studied. Chlorella pyrenoidosa, thylakoid membrane vesicles, plasma membrane vesicles and chloroplasts were partitioned in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems, and in a polyethylene glycol/dextran/benzoyl dextran four-phase system.     

Concentration of Rous Sarcoma Virus from Tissue Culture Fluids with Polyethylene Glycol

Concentration of Rous sarcoma virus from tissue culture fluids with polyethylene glycol, with and without NaCl or dextran sulfate, resulted in significant and highly variable losses caused by entrapment of virus particles in proteinaceous debris. Treatment of concentrated preparations with Pronase greatly enhanced the recovery of virions. Maximum recovery of virus particles was obtained by the addition of 8% polyethylene glycol and 0.4 M NaCl to tissue culture fluids, followed by Pronase treatment of the concentrated virus preparations.     

Dextran sulfates as a contaminant of DNA extracted from concentrated viruses and as an inhibitor of DNA polymerases.

Dextran sulfate is commonly used with polyethylene glycol to concentrate viruses before extraction of their DNA. However, dextran slulfate then easily contaminated such DNA and acted as a potent inhibitor of DNA polymerases from Bacillus subtilis (III), phage PBS2, and phage T4. Dextran sulfate only weakly inhibited Micrococcus luteus and Escherichia coli DNA polymerase I preparations.     

A rapid and simple quantification of human apolipoprotein E-rich high-density lipoproteins in serum.

A rapid and simple method for the quantitative determination of human serum apo E-rich high-density lipoproteins is described. A sample was divided into two parts; one part was mixed with an equal volume of 13% polyethylene glycol 6000, and the other part was mixed with a solution containing dextran sulfate, sodium phosphotungstate, and Mg2+, respectively. The mixed solutions were centrifuged (2000 g; 15 min). The supernate obtained by the former procedure contained both apo E-rich HDL and apo E-poor HDL, but that obtained by the latter procedure contained solely apo E-poor HDL. The serum apo E-rich HDL concentration in terms of apo E (E) and cholesterol (C), was given by the following equations: E = EP x 2, and C = (CP - CD) x 2, where EP and CP were the concentrations of apo E and cholesterol, respectively, in the supernate obtained with 13% polyethylene glycol, and CD was the concentration of cholesterol in the supernate obtained with the mixture solution of dextran sulfate, sodium phosphotungstate, and Mg2+. Normal serum apo E-rich HDL concentrations were 2.6 +/- 1.5 and 6.7 +/- 2.3 mg/dl (means +/- SD, n = 38) in terms of apo E and cholesterol, respectively. Apo E-rich HDL was increased strikingly in the sera from three patients with hepatobiliary diseases.     

Separation of Listeria monocytogenes and Salmonella berta from a complex food matrix by aqueous polymer two-phase partitioning

In the present study, the use of aqueous polymer two-phase systems for separation of pathogenic bacteria from a complex food sample was investigated. Three different two-phase systems, a polyethylene glycol 3350/dextran T 500, a methoxy polyethylene glycol 5000/dextran T 500 and a polyethylene glycol 3350/hydroxypropyl starch system, were compared at pH 3 and pH 6 for their capacity to separate the pathogenic bacteria Listeria monocytogenes and Salmonella berta from a Cumberland sausage. In all three phase systems, the food particles partitioned to the lower phase. Best performance was obtained by the polymer combinations, polyethylene glycol 3350/dextran T 500 and polyethylene glycol 3350/hydroxypropyl starch. In these systems, Salmonella berta partitioned to the hydrophobic upper phase both at pH 3 and pH 6 with an average partitioning ratio of 80% and a recovery of 56%. Listeria monocytogenes partitioned to the upper phase at pH 3 only with an average partitioning ratio of 72% and a recovery of 45%. This method may become a valuable tool for separation of bacteria from complex food matrices.     

Interactions in affinity partition studied using fluorescence spectroscopy.

Fluorescence titration has been used to determine the binding constant and number of binding sites for the textile triazine dye Procion Yellow HE-3G to lactate dehydrogenase from rabbit muscle (E.C. 1.1.1.27). Triazine dye was either free in solution or attached to one of the polymer carriers, polyethylene glycol or dextran. Titrations were performed in solutions of buffer, dextran, and polyethylene glycol. Aqueous two-phase systems composed of polyethylene glycol and dextran were prepared and the binding constant and number of binding sites for ligand polyethylene glycol-Procion Yellow to lactate dehydrogenase were determined in both upper and lower phases of these systems. Affinity partition of lactate dehydrogenase in a PEG-dextran system was also performed using PEG-Procion Yellow as ligand, and partition coefficients of lactate dehydrogenase showed good agreement with theoretical partition coefficients calculated from the binding constant and number of binding sites obtained from fluorescence titration. The advantage of using fluorescence titration to determine affinity of a polymer ligand for a protein is that measurement of binding strength can be made in the actual environment encountered by protein-ligand complex during the purification process.     

Phase separation in an aqueous quaternary system

(1) We have measured the incompatible phase separation that occurs in a polyethylene glycol-sodium dextran sulphate-sodium chloride-water system and have determined a critical point. (2) We have measured the activity coefficients of sodium chloride in critical-point concentrations of polyethylene glycol and sodium dextran sulphate respectively, and the osmotic coefficient of sodium dextran sulphate at the critical-point concentration. (3) With use of the relevant thermodynamic equations for a quaternary ionic system, we have determined the interaction coefficients between polyethylene glycol and dextran sulphate and between polyethylene glycol and sodium chloride. The former could be due mainly to volume exclusion, but the latter is too large to be explained on that basis.     

The effects of plant polysaccharides and buffer additives on PCR.

A survey of the inhibitory effects of various plant polysaccharides on PCR amplification of a 974-bp section of rbcL in spinach revealed that most of the polysaccharides tested (arabinogalactan, carrageenan, dextran, gum guar, gum karaya, gum locust bean, inulin, mannan, pectin, starch and xylan) were not inhibitory. In contrast, two of the acidic polysaccharides (dextran sulfate and gum ghatti) were inhibitory. The addition of 0.5% Tween 20 reversed the inhibitory effects of gum ghatti (polysaccharide:DNA ratio of 500:1). The inhibitory effect of dextran sulfate (50:1) could be reversed by the addition of Tween 20 (0.25% or 0.5%), DMSO (5%) or polyethylene glycol 400 (5%), but none of these three additives were effective at a 100:1 ratio of dextran sulfate/DNA.     

Decrease in intestinal permeability to polyethylene glycol 1000 during development in the pig

Changes in intestinal permeability during postnatal development in the pig were investigated by using different-sized polyethylene glycols in the Mr 766-1338 range (polyethylene glycol 1000) as permeability probes. Pigs of varying age, newborn (Oh), 36-45 h old and 22-28 days old, were gavage fed polyethylene glycol 1000 together with the macromolecular markers bovine serum albumin, ovalbumin or FITC-labelled dextran 70,000. The 4-h blood serum concentrations of the different markers were determined and taken as an estimate of their intestinal transmission. In the newborn pigs, high serum levels of polyethylene glycols were obtained, concomitant with high serum levels of bovine serum albumin and FITC-dextran. After intestinal macromolecular closure in the 36-45 h-old pigs, lower serum polyethylene glycol levels were found, especially of those with a Mr greater than 1100 Da. In the 22-28 days-old pigs, polyethylene glycol levels were reduced to one-tenth or less of those in the 36-45 h-old pigs, with the levels decreasing markedly with increasing molecular size. These results show that there is a correlation between the intestinal permeability of polyethylene glycols, especially those larger than 1100, and macromolecules in the newborn pig around intestinal closure, suggesting that such polyethylene glycols traverse the gut by the macromolecular route. During later development, further intestinal maturation results in a markedly reduced permeability to polyethylene glycol 1000.     

Phase diagrams for dextran-PEG aqueous two-phase systems at 22°C

Summary We have determined phase diagrams at 22°C for the aqueous two-phase systems composed of dextran, polyethylene glycol, and water. The effects of polyethylene glycol and dextran molecular weight on phase separation are reported. These phase diagrams provide more complete data for dextran/PEG/water system, and will be needed for the correlation of biomolecule partitioning.     

Biochemical method for molecular weight determinations of proteins and other macromolecules

The invertase of Ricinus communis complexes with proteins, polyvinylpyrrolidone, polyethylene glycol, heparin and dextran sulfate. This association produces an increase of invertase activity. The minimal concentration of activator giving the maximal activation was attained at the same molarity for a given amount of enzyme for all macromolecules studied. These conditions are used for the molecular weight determination of the activating substance. The method may be used for the molecular weight determination of polymeric substances with a molecular weight in the range from 5000 to 1000,000 Da.     

Reaction patterns of monoclonal antibodies to DNA

Starting with spleen cells from MRL/lpr, NZB/W, and graft-vs-host-diseased mice, we prepared a total of 57 hybridomas that produce antibodies to DNA. Using various approaches, we studied the avidity of these monoclonals in relation to their behavior in four anti-DNA assays. From the results obtained, we postulate that on the basis of anti-DNA avidity the anti-DNA ELISA, the polyethylene glycol assay, the indirect immunofluorescence test on Crithidia luciliae, and the Farr assay (in this order) detect a decreasing amount of anti-dsDNA, the Farr assay being strictly selective for high avidity anti-dsDNA. mAb selected by the anti-DNA ELISA generally were of a low avidity toward DNA. Using cardiolipin and dextran sulfate, a polyanion that bears a resemblance in charge to DNA, we studied the cross-reactivity of the monoclonals. A total of 6 of the 57 monoclonals were found to cross-react with cardiolipin, and 26 with dextran sulfate. We observed an inverse relationship between anti-DNA avidity and cross-reactivity: the lower the avidity of the antibody, the more cross-reactive it is. Based on these findings, we postulate that it is at least questionable whether low avidity, cross-reactive (monoclonal) anti-DNA is representative for the anti-DNA found in patients with SLE.     

Purification of interferon from mouse Ehrlich ascites tumor cells

Interferon production was induced in mouse Ehrlich ascites tumor cells by infection with Newcastle disease virus. The interferon produced was purified by precipitation with ammonium sulfate, chromatography on carboxymethyl-Sephadex, treatment with blue dextran and polyethylene glycol, gel filtration on Bio-Gel P-60 and Bio-Gel P-200, chromatography on phosphocellulose, isoelectric focusing, and chromatography on octyl-Sepharose. The specific activity of the product was 1.6 x 10(9) NIH mouse interferon reference standard units/mg of protein. Electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate indicated that the apparent molecular weight of the interferon-active material ranged from 25,000 to 35,000. As revealed by staining the gels with Coomassie brilliant blue, the interferon activity co-migrated with the major, broad protein band. Minor, stainable bands of proteins were free of interferon activity and their apparent molecular weight was smaller than 12,000.     

An approach to the study of phase separation in ternary aqueous systems

1. Simple thermodynamic expressions are used to describe the properties of uncharged binary and ternary polymer solutions, in particular the sedimentation equilibrium of binary systems and the osmotic pressures and `incompatible' phase separations of ternary systems. 2. Sedimentation-equilibrium experiments were performed on four samples of dextran and two of polyethylene glycol. The critical points of the phase diagrams were determined for the mixed solutions of polyethylene glycol–dextran–water and of polyethylene glycol–bovine serum albumin–0·2m-sodium chloride solution. Osmotic pressures were measured on a single-phase mixed solution of a polyethylene glycol and a dextran. By use of the simple thermodynamic expressions consistent values of second virial and interaction coefficients for the materials used were obtained from these experiments. 3. The interpretation of the values of the second virial and interaction coefficients, on the basis of three models of molecular interaction, is discussed.     

Optimization of bovine serum albumin sorption and recovery by hydrogels

Aqueous two-phase systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. Partitioning of proteins in such systems provides a powerful method for separating and purifying mixtures of biomolecules by extraction. If one of the phase forming polymers is a crosslinked gel, then the solution-controlled gel sorption may be considered as a modification of aqueous two-phase extraction. Since PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex) are common chromatographic media, we choose a PEG/dextran gel system as a model system in this study. The partitioning behavior of pure bovine serum albumin (BSA) in PEG/dextran gel systems is investigated to see the effects of variations in PEG and NaCl concentrations on the partition coefficient K. By making use of the Box-Wilson experimental design, K is shown to be maximized at 9.8 (%, w/w) PEG and 0.2 M NaCl concentrations, respectively, as 182.     

Effects of polyethylene glycol and dextrans on immunoprecipitations: a two-cross immunodiffusion study

Precipitating titers and immunochemical titers obtained in a wide range of antigen-to-antibody concentration ratios by the two-cross immunodiffusion technique are compared with the corresponding laser light scatter precipitin curves. The two-cross immunodiffusion technique has also been applied to investigate whether polyethylene glycol of molecular mass 6000 and dextrans of molecular masses from 10,000 to 2,000,000 enhance the immunoprecipitation processes of the system human serum IgG-rabbit immune serum at pH 5.5 and 8.1 at 20 degrees C. It was found that the significant increase of precipitating titers of both precipitating components in the presence of polyethylene glycol is a consequence of a strong decrease of solubility of the primary antigen-antibody complex. The decrease of solubility does not affect the immunochemical titer of the immune serum, indicating stoichiometrical invariance of the precipitate at the equivalence. The apparent strong decrease of diffusion coefficients of both antigen and antibody in 20- and 40-g/liter polyethylene glycol solution is attributed to increase of viscosity of the solutions and to a partial self-association of protein molecules due to steric exclusion. In 40-g/liter polyethylene glycol solutions at pH 5.5 every fourth molecular entity of antigen and every third molecular entity of antibody are present in the form of a two-molecular self-associate, whereas in 20-g/liter polyethylene glycol solutions only 1% of antigen molecules and 8% of antibody molecules are associated. With the increase of pH to 8.1 the self-association of protein molecules is strongly further enhanced. Dextrans in 20-g/liter solutions, without regard to their relative molecular masses, do not influence precipitating titers and solubility of the antigen-antibody system at equivalence and do not enhance self-association of protein molecules. The strong decrease of diffusion coefficients of immunoglobulin G antigen and antibodies in dextran solutions is solely attributed to the increase of viscosity of the dextran solutions; hence there was no evidence of interaction of dextrans with serum IgG proteins.     

Two phase aqueous extraction

Rheological properties have been measured for aqueous solutions of dextran, polyethylene glycol and bovine serum albumin. Mixtures of these materials have also been studied. A rotating concentric cylinder viscometer was used to study the rheological properties of these materials over the temperature range 10 to 40°C. Over the range of concentrations, molecular weights, temperature and shear rates covered in this work, all aqueous solutions exhibited Newtonian behaviour. Correlations have been reported for viscosities of dextran, polyethylene glycol, and bovine serum albumin. The viscosity of mixtures of these materials is not linear with respect to concentration.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.     

A rapid method for the isolation of circular DNA using an aqueous two-phase partition system.

A method of isolating circular plasmid DNA from cleared lysates of E. coli is described. Purification is achieved by virtue of the rapid re-annealing kinetics or supercoiled DNA. After a brief denaturation step, double stranded plasmid DNA is separated from denatured chromosomal DNA and RNA in a two-phase partition system using dextran and polyethylene glycol. The method is much more rapid than the conventional dye-centrifugation technique and plasmid DNA of comparable purity and yield is obtained.     

Purification of rabbit lacrimal gland plasma membranes by aqueous two-phase affinity partitioning

We describe the purification of lacrimal gland plasma membranes by affinity partitioning using a two-phase system containing polyethylene glycol and dextran in which wheat germ agglutinin conjugated to dextran is used as affinity ligand. When partitioning a microsomal fraction, the plasma membrane marker 5′-nucleotidase was obtained in the affinity ligand-containing bottom phase, whereas the endoplasmic reticulum marker NADH-ferricyanide reductase remained in the top phase. The affinity partitioning behaviour of components involved in exocytosis and cellular signalling was also examined.