Solubility In Ammonium Sulfate Solution Research Articles

Preparative Centrifugal Precipitation Chromatography Using Dialysis Membrane Inserted into Convoluted Tubing

The method uses preparative centrifugal precipitation chromatography comprising a seal‐free continuous‐flow centrifuge machine equipped with a plastic disk and a spiral groove which accommodates convoluted tubing. A dialysis membrane is inserted into the convoluted tubing to separate proteins according to their solubility in ammonium sulfate solution. The system relies on the original model of centrifugal precipitation chromatography, which employs the centrifugal force and a concentration gradient of precipitants to perform a repetitive process of precipitation and dissolution of protein molecules. Compared to the previous model, the current model results in a higher yield of separated compounds.

Preparative Separation of Proteins Using Centrifugal Precipitation Chromatography Based on Solubility in Ammonium Sulfate Solution

A preparative modification of the centrifugal precipitation chromatography (CPC) is described. The sample‐loading capacity is improved in the present system by the use of convoluted tubing containing dialysis tubing instead of a dialysis membrane placed between a pair of disks equipped with mirror‐imaged spiral grooves as in the original design. The system uses, basically, the same principle of as the original CPC, in that a concentration gradient of precipitant is generated under a centrifugal force field. The protein sample injected into the CPC column is exposed to an increasing concentration of the precipitant where it precipitates at various portions of the column according to its solubility. The gradient is then gradually lowered so that the sample undergoes dissolution and precipitation many times within the column; the proteins finally elute from the column according to their solubilities. A basic study was performed using this machine to separate human albumin and 3‐globulin using ammonium sulfate (AS) as precipitant. Preliminary results indicate that this method can separate 500 mg of protein.

Gradient formation in membrane unit for differential precipitation of proteins

AbstractA new protein fractionation technique presented transfers ammonium sulfate to create a gradient of ammonium sulfate inside a membrane unit for differential precipitation of proteins called “centrifugal precipitation chromatography.” Because it does not require any solid support, it should provide a better alternative to the conventional chromatography with solid stationary phase. To understand the phenomena and achieve a better separation, a mathematical model explaining the ammonium sulfate gradient formation inside the stationary membrane unit is investigated. The model is extended with empirical correlation to the centrifugal membrane unit—a new approach for protein purification. The model agreed well with the experiments for both stationary and centrifugal units. Upon using the model to calculate the ammonium sulfate gradient formation in a membrane unit, this new technique can be useful in separating a mixture of proteins whose solubility in ammonium sulfate solution differs. To demonstrate the technique experimentally, mixtures of proteins are loaded into the column; the partial resolution between proteins is achieved with the step concentration switch of the inlet ammonium sulfate solution.

CENTRIFUGAL PRECIPITATION CHROMATOGRAPHY APPLIED TO FRACTIONATION OF PROTEINS WITH AMMONIUM SULFATE

A novel chromatographic system introduced here internally generates a concentration gradient of ammonium sulfate (AS) along a long channel to fractionate proteins according to their solubility in AS solution. The separation column consists of a pair of discs with mutually mirror-imaged spiral channels that are separated by a semipermeable membrane. The disc assembly is mounted on a seal-less continuous flow centrifuge. A concentrated AS solution is introduced into the upper channel while a water solution is passed through the lower channel in the opposite direction in a rotating column. A mixture of proteins injected into the water channel moves along an AS gradient of increasing concentration that has been established in the water solution. Each protein species precipitates at a different AS concentration along the gradient. By decreasing the concentration of the AS solution its concentration in the water is decreased, causing the protein to redissolve and to reprecipitate further down the channel. The eluate is continuously monitored and collected using a fraction collector. The method has been demonstrated on separation of serum proteins and applied to purification of a recombinant ketosteroid isomerase from a crude E. coli lysate by adding an affinity ligand to the sample solution. A possible mechanism involved in this affinity separation is discussed. The method may be applied to other biopolymers such as DNA and RNA.

Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic behavior of glycosaminoglycans on phenyl-sepharose CL-4B and their solubility in the presence of high concentrations of ammonium sulfate

The effect of bound sulfate groups and uronic acid residues of glycosaminoglycans on their behavior in chromatography on hydrophobic gel was examined by the use of several pairs of depolymerized chondroitin, chondroitin 4- or 6-sulfate, and dermatan sulfate having comparable degree of polymerization. Chromatography on Phenyl-Sepharose CL-4B in 4.0-2.0 m ammonium sulfate containing 10m m hydrochloric acid showed that: (a) The retention of depolymerized chondroitin 4- or 6-sulfate on the gel varies with the temperature, whereas the depolymerized samples of chondroitin and dermatan sulfate does not show a temperature dependence (this is not the case for hyaluronic acid or dextrans). (b) Among depolymerized samples of chondroitin and chondroitin 4- and 6-sulfate that have a similar degree of polymerization, chondroitin 4- and 6-sulfate showed the highest retention. (c) The retention on the gel of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate decreased in this order. The solubility in ammonium sulfate solution of the polysaccharides agreed well with the chromatographic behavior, suggesting that the fractionation by the hydrophobic gel largely depends on the ability to precipitate on the gel rather than on the hydrophobic interaction between gel and polysaccharide.

Improvement of rapeseed protein nutritional value

Although the efficiency ratio (PER) of vegetable proteins can be improved by supplementation with the limiting free amino acids, the digestibility of these proteins remains lower than that of animal proteins. The digestibility of a rapeseed protein concentrate measured in rats was improved from a value of 81 to 87% by water solubilization and ammonium sulfate precipitation. 2S and 12S fractions were also prepared from a rapeseed protein concentrate by a method based on differences of solubility in ammonium sulfate solutions. Protein digestibility of the rapeseed protein concentrate was found to be 82%, as compared to 87% and 89% for 2S and 12S fractions. A reduction of protein efficiency ratio was also recorded and was found to result from losses in amino acids.

Studies on histone oligomers. IV. Reassociation of chromatin from histones of various conformations.

Chicken erythrocyte chromatin, whole or (H1,H5)-depleted, was dissociated in 2 M NaCl in the presence or absence of 5 M urea at pH 8 or pH 5, and reconstituted by decreasing the concentration of NaCl and urea. The reassociated products were characterized by their solubilities in 0.1 mM EDTA, micrococcal nuclease digestion, cross-linking of histones, and fractionation of histone oligomers by solubility in ammonium sulfate solution. In the absence of urea, the nucleosome structure and the histone octamer were reconstituted perfectly at both pH 5 and pH 8. When chromatin was exposed to urea, no nucleosome structure or histone octamer was obtained at pH 5 either decreasing the concentration of salt first or that of urea first. At pH 8, the chromatin structure was regained fairly well by decreasing the concentration of urea first, but only partially by decreasing the concentration of salt first. Solubility in 0.1 mM EDTA was found to be a good criterion for monitoring the proper reassociation of chromatin.

Antibody-Dependent Cell-Mediated Cytotoxicity in X-Irradiation-Induced Adenocarcinoma of the Rat Small Bowel

Antibody-dependent cell-mediated cytotoxicity (ADCC) was detected in the serum of Holtzman rats which had only their hypoxic ileum and jejunum exposed to 1700 to 2000 R of x rays, among which 10 to 25% would develop visible lesions in 4 to 6 months. All irradiated animals possessed serum which induced the in vitro injury of allogeneic cultured cells of an x-ray-induced rat small bowel adenocarcinoma by effector peripheral-blood lymphoid cells isolated from normal unexposed rats. The active serum component was apparently in IgG, on the basis of its solubility in ammonium sulfate solution, reactivity with anti-rat IgG and matrix-bound Protein A, molecular weight, and inactivity of F(ab')/sub 2/ fragments. The in vivo significance of this ADCC is presently unclear since it existed in the serum of all rats exposed to the ionizing radiation regardless of whether they developed visible lesions. However, the results do suggest that if this immunological responsiveness plays a role in host tumor defense, then the deficiency in those rats developing the cancer may lie in their circulating effector cells.

Studies on histone oligomers. II. Reconstitution of heterotype histone oligomers and pH dependency of oligomer formation.

Histone oligomers were reconstituted by annealing from dissociated calf thymus whole histones in 2 M NaCl-5 M urea. The products were fractionated in terms of solubility in ammonium sulfate solution. The oligomers formed at pH 5 were heterotype histone oligomers (H2A . H2B . H3 . H4)n at high ionic strength, and heterotype histone tetramer (H2A . H2B . H3 . H4) at low ionic strength. The pH dependency of oligomer formation was determined. Heterotype oligomers were formed at pH 4-6 and homotype oligomers at pH 7-9.

Studies on histone oligomers. I. Reconstitution and fractionation of homotypic histone oligomers.

Histone oligomers were reconstituted from calf thymus whole histones by annealing from 2 M NaCl-5 M urea-0.05 M Tris buffer (pH 8.0)-1% beta-mercaptoethanol, and were fractionated in terms of solubility in ammonium sulfate solution. Fractionation occurred according to the histone compositions of oligomers. The oligomers thus obtained were (1) pentamer and trimer of H1, (2) tetramer of H2A and H2B, and (3) tetramer and higher aggregates of H3 and H4, under the following ionic conditions: 0.5 M NaCl-0.05 M phosphate buffer (pH 7.0). The aggregates of H3 and H4 could easily be converted to the tetramer by lowering of pH.

Propriétés Physico-Chimiques et Composition en Acides Aminés des Fractions de Protéines de Soya, de Féverole, de Colza et de Feuilles de Luzeme

Fractions were prepared and purified from proteins of four different vegetable sources by a method based on differences of solubility in ammonium sulfate solutions. Sedimentation coefficient SW25, distribution of amino-acid residues, absorption coefficient at 280 and 260 nm and solubility in ammonium sulfate solutions of various concentrations have been determined with the isolated fractions. Results indicated that solubility decreased logarithmically with increases in concentration of ammonium sulfate. It is shown for the five fractions studied that solubility is related to the ratio of the number of polar/neutral residues.

Human pituitary growth hormone: Solubility in ammonium sulfate solutions

The solubility of human pituitary growth hormone in ammonium sulfate solutions has been investigated and compared under similar conditions with that of three related proteins: ovine pituitary growth hormone, bovine pituitary growth hormone, and human chorionic somatomammotropin.

Isolation and properties of sorghum α-amylase

α-Amylase was purified and prepared in good yield from sorghum malt by procedures based on ultracentrifugation, salt fractionation, and adsorption on starch granules. The purified enzyme had a specific activity comparable to crystalline barley α-amylase. The enzyme had a pH optimum of 4.6 and was inactivated at pH's below 3.5. It exhibited a minimum solubility in ammonium sulfate solutions at pH 5.8 and required Ca ++ ions for stabilization.

SOLUBILITY STUDIES ON PURIFIED TOBACCO MOSAIC VIRUS

Different samples of purified tobacco mosaic virus show a relatively wide variation in solubility in ammonium sulfate solution. This variation and the type of solubility curve obtained in the presence of varying amounts of solid phase show that the purified virus whether isolated by mild treatment with ammonium sulfate or by ultracentrifugation is not a homogeneous chemical substance but contains more soluble and less soluble virus fractions of comparable specific activities. Long contact with strong ammonium sulfate solutions or 0.1 M phosphate buffer results in a decrease in solubility. The variation in the solubility of samples isolated from different plants by the same method seems to depend in part on the length of time the plants are inoculated before they are cut, and probably also on the conditions under which they are grown. Virus preparations isolated from plants of different genera grown under the same conditions and inoculated at the same time, however, behaved like identical substances in solubility experiments.