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.     

Partition coefficient of bovine serum albumin in two-phase aqueous systems

Summary We measured partition coefficients of bovine serum albumin in the following two-phase aqueous systems: polyethylene glycol-dextran and polyethylene glycol-potassium phosphate. We report the effects on partition coefficients of variables such as relative molecular masses of polyethylene glycol and dextran, phase composition and temperature.Contribution of National Bureau of Standards, not subject to copyright.     

Evaluation of viscosities of aqueous two-phase systems containing protein

The dynamic viscosities of aqueous polyethylene glycol, aqueous bovine serum albumin, and polyethylene glycol-bovine serum albumin-water solutions were measured at temperatures of 15, 20, 25, 30 and 35 degree C. To estimate the viscosity values of polyethylene glycol-bovine serum albumin-water solutions, a one parameter Grunberg-like model which was satisfactorily used earlier by the present author for polyethylene glycol-dextran-water solutions was employed. The disposable parameter a for our temperature range was estimated as 3.71. The relative errors varying from 0.29 to 18.98 in absolute value indicates that the Grunberg-like model works perfectly for polymer-protein solutions as well.     

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.     

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.     

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.     

双水相电泳分离蛋白质的研究

近几年来,随着生物技术的迅速发展,制备型电泳技术的研究得到了重视。然而由于技术上的原因,大规模的制备型电泳技术的研究还未能取得突破。阻碍电泳放大的一个主要问题是由于电加热作用而导致的热对流对电泳分离的破坏。为解决这一问题,人们提出了许多方法。例如,在太空的微重力环境下进行电泳,应力稳定自由流动电泳,循环等电聚焦和区带电泳,色谱电泳和等电膜等电聚焦等。这些方法在电泳放大上都取得了一定的进展,但各有其局限性。最近,Clark提出利用双水相的液液界面阻止热对流的设想,为开发大规模的制备型电泳技术开辟了一条新途径、Raghava Rao等在两种双水相体系上施加电场后成倍地缩短了分相时间。Levine和Bier采用U型管电泳装置研究了双水相体系中血红蛋白的电泳迁移率,观测到界面有阻滞作用。Clark在柱型电泳装置中进行了一组双水相萃取肌红蛋白的简单实验。在10mA的恒电流下电泳40min之后,肌红蛋白的分配系数为7.5,而当电场反向后,分配系数变为0.04,界面阻力并不显著,两者结论并不一致。     

Hydrodynamics and mass transfer in two-phase aqueous extraction using spray columns

Spray columns can be used to isolate and purify proteins using the two-phase aqueous extraction technique based on polyethylene glycol (PEG) and dextran. The fractional dispersed phase (PEG) holdup and overall mass transfer coefficients were measured in a 9.7 mm i.d. spray column. We found that the dispersed phase holdup increased with increasing PEG phase velocity. The overall mass transfer coefficients for bovine serum albumin, normalized for the PEG holdup, were found to be independent of the PEG phase velocity. This result was expected, since true mass transfer coefficients do not vary with phase velocity.     

Theoretical and experimental studies on freezing point depression and vapor pressure deficit as methods to measure osmotic pressure of aqueous polyethylene glycol and bovine serum albumin solutions

For survival in adverse environments where there is drought, high salt concentration or low temperature, some plants seem to be able to synthesize biochemical compounds, including proteins, in response to changes in water activity or osmotic pressure. Measurement of the water activity or osmotic pressure of simple aqueous solutions has been based on freezing point depression or vapor pressure deficit. Measurement of the osmotic pressure of plants under water stress has been mainly based on vapor pressure deficit. However, differences have been noted for osmotic pressure values of aqueous polyethylene glycol (PEG) solutions measured by freezing point depression and vapor pressure deficit. For this paper, the physicochemical basis of freezing point depression and vapor pressure deficit were first examined theoretically and then, the osmotic pressure of aqueous ethylene glycol and of PEG solutions were measured by both freezing point depression and vapor pressure deficit in comparison with other aqueous solutions such as NaCl, KCl, CaCl(2), glucose, sucrose, raffinose, and bovine serum albumin (BSA) solutions. The results showed that: (1) freezing point depression and vapor pressure deficit share theoretically the same physicochemical basis; (2) theoretically, they are proportional to the molal concentration of the aqueous solutions to be measured; (3) in practice, the osmotic pressure levels of aqueous NaCl, KCl, CaCl(2), glucose, sucrose, and raffinose solutions increase in proportion to their molal concentrations and there is little inconsistency between those measured by freezing point depression and vapor pressure deficit; (4) the osmotic pressure levels of aqueous ethylene glycol and PEG solutions measured by freezing point depression differed from the values measured by vapor pressure deficit; (5) the osmotic pressure of aqueous BSA solution measured by freezing point depression differed slightly from that measured by vapor pressure deficit.     

New hydrogel based on polyethylene glycol cross-linked with bovine serum albumin

Summary A new and rapid method for preparing a protaneous-polymeric hydrogel is described. The gel matrix is obtained by covalent cross-linking between preactivated polyethylene glycol of 3350 molecular mass and bovine serum albumin in aqueous solution at basic pH. The polyethylene glycol-albumin hydrogel showed high protein retention and a very good swelling capacity.     

Affinity separation of proteins in aqueous three-phase systems

Aqueous polymer three-phase systems composed of dextran-Ficoll-polyethylene glycol-water have been used for affinity partition of proteins. The upper, middle, and lower phases are rich in polyethylene glycol, Ficoll, and dextran, respectively. Affinity partition was performed using the reactive dyes Cibacron Blue F36-A and Remazol Yellow GCL which are known as specific ligands for albumin and prealbumin from human serum. When the ligands were bound alternatively to polyethylene glycol, Ficoll, or dextran the target proteins were directed toward the upper, middle, or lower phase, respectively. In the presence of two ligands immobilized to two different polymers the distribution of two proteins could be steered to different phases at the same time. Serum albumin and prealbumin could be separated by using Cibacron Blue-Ficoll and Remazol Yellow-dextran or Cibacron Blue-polyethylene glycol and Remazol Yellow-dextran as polymer ligands.     

A rapid purification of rat alpha 1-foetoprotein by phase partition

The phase partition system dextran/polyethylene glycol (2 : 1, w/w) was chosen to separate rat alpha 1-foetoprotein from rat serum albumin, which is its main contaminant due to their having close physicochemical properties. The optimization of this method necessitated a systematic study of the behaviour of rat serum albumin in the system under consideration. This article describes the optimum conditions, in terms of pH, ionic strength and the concentration of polymer solutions, for the purification and recovery of alpha 1-foetoprotein. After a prepurification of rat foetal serum by CM-cellulose chromatography, a single partition step permitted the recovery of 15% of the total alpha 1-foetoprotein present in the rat serum. The purity of this alpha 1-foetoprotein was demonstrated by its binding parameters and by analytical gel electrophoresis.     

Fractionation of proteins from human serum by counter-current distribution

Proteins of human serum have been fractionated by counter-current distribution using aqueous two-phase systems. These were composed of either polyethylene glycol and dextran or polyethylene glycol and the new water soluble starch polymer Aquaphase PPT. The distribution of serum proteins in the polyethylene glycol-Aquaphase PPT system resembles that in the polyethylene glycol-dextran system.The partition of a number of proteins could be changed by introducing polymer-bound reactive dyes into one of the phases. Due to affinity for the dyes several proteins were transferred into the phase containing the polymer-bound ligand leading to an improved separation of individual proteins.Furthermore, the effect of two different dyes, immobilised in the opposite phases, on counter-current distribution of serum proteins was demonstrated. The applicability of this method for fractionation of serum proteins is discussed.     

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.     

The thermodynamics of interaction between Sephadex and penetrating solutes

1. We have measured the partition coefficients of bovine serum albumin with Sephadex grades G-100, G-150 and G-200, and of a dextran ([unk]_n 19700) and a polyethylene glycol ([unk]_n 8000) with Sephadex G-200. We have also measured the effects of these solutes on the inner volumes of the grades of Sephadex. 2. The results can be described with fair consistency by means of a simple thermodynamic treatment that makes use of the virial coefficients of Sephadex and of the solute, and of a coefficient that expresses their interaction. This coefficient is related to the `exclusion volume' of Sephadex for the solutes. 3. The Sephadex G-200–polyethylene glycol system shows anomalies of behaviour that are ascribed to the occurrence of `incompatible' phase separation within the Sephadex beads.     

Phase behavior of aqueous solutions of bovine serum albumin in the presence of dextran, at rest, and under shear

The demixing conditions for aqueous solutions of bovine serum albumin (BSA, fraction V) and for joint solutions of BSA plus dextran (DEX, M(w) = 2000 kg/mol) were determined by turbidimetric measurements as a function of composition, temperature, and shear rate. Aqueous solutions of BSA phase separate upon heating. Within the region of BSA concentrations between 0.05 and 32 wt %, the demixing temperature, T1, falls from ca. 65 degrees C to an almost constant value of 45 degrees C. Adding DEX to the BSA solutions reduces the homogeneous region of the mixture drastically where the amount of DEX required to lower T1 to 25 degrees C decreases rapidly as the concentration of BSA is raised. Experiments concerning the influences of shear have been performed for the ternary system up to 500 s(-1). They demonstrate that the content of dextran determines the sign of the effect. At low DEX concentrations, the mechanical field favors the homogeneous state (shear-induced mixing), whereas the opposite effect (shear-induced demixing) is observed at high DEX concentrations. Possible reasons for this observation are discussed.     

Stabilization of Photosystem II (O(2) Evolution) of Spinach Chloroplasts by Radiation-induced Immobilization

Spinach chloroplasts were immobilized with vinyl monomers by radiation-induced polymerization at low temperature and stored in buffer containing bovine serum albumin. The lifetime of O₂ evolution activity in photosystem II was prolonged remarkably in immobilized chloroplasts. Thermostability of immobilized chloroplasts stored in buffer containing bovine serum albumin was far better than that of immobilized chloroplasts in pure buffer and that of intact chloroplasts. When immobilized chloroplasts were stored in buffer including polyethylene glycol, the lifetime of O₂ evolution activity was longer than for those stored in buffer containing bovine serum albumin.     

Triplet excited states of polycyclic aromatic compounds as probes of their microenvironment in serum albumin complexes.

Using flash photolysis techniques, the triplet excited states of benzo(a)pyrene, pyrene, benz(a)anthracene and other aromatic hydrocarbons have been detected in complexes of bovine (and human) serum albumin dissolved in aqueous solutions at room temperature. The triplet lifetimes can be adjusted to any value within the microsecond-millisecond time domains by varying the partial pressure of oxygen from zero to one atmosphere, thus providing a useful probe on these time scales. Local oxygen concentrations as low as ～ 2 × 10^?7M can be detected. In air saturated solutions, the triplet lifetimes are sensitive to pH dependent conformational changes of the host bovine serum albumin molecules.     

The contribution of endogenous and exogenous effects to radiation-induced damage in the bacterial spore

Radical scavengers such as polyethylene glycol 400 and 4000 and bovine albumin have been used to define the contribution of exogenous and endogenous effects to the gamma-radiation-induced damage in aqueous buffered suspensions of Bacillus pumilus spores. The results indicate that this damage in the bacterial spore is predominantly endogenous both in the presence of 1 atmosphere of oxygen, and in anoxia.