Fractionation with Solid Ammonium Sulfate

Reprinted by permission of the Oxford University Press (Oxford) from Data for Biochemical Research, 2nd ed. Edited by R. M. C. Dawson, D. C. Elliott, W. H, Elliott, and K. M. Jones, (gt Oxford University Press (1969). c.. r TwnnJno Vrti. 1. n. 76 n955) for a similar table prepared for 25 C. 

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The SI70 supernatant (220 ml) was made to 40 % saturation with solid ammonium sulfate, stirred for 20 min, and then the precipitate was collected by centrifugation at 15,000 g for 15 min. The precipitate was suspended in small volume of buffer B-50 at pH 7.6 containing 20 mM HEPES/KOH, 0.1 mM EDTA, 1 mM dithiothreitol, 10 % (v/v) glycerol, and 50 mM potassium acetate. The 60 % saturated ammonium sulfate solution was prepared similarly. Protein concentrations for 0 - 40 % and 40 - 60 % ammonium sulfate fractions were 4.2 mg/ml and 4.7 mg/ml, respectively. 

Fraction 2 is brought to 0.4 saturation with solid ammonium sulfate (250 g. per liter). A white precipitate appears and the suspension is allowed to settle at 4° for 1 to 2 hours. The precipitate (Fraction 3) is then collected by centrifugation at 2500 r.p.m. for 1 hour and the supernatant solution (Fraction 3a) is discarded. Fraction 3 is washed once with 400 to 500 ml. of cold ammonium sulfate solution (250 g. per liter) and separated by centrifugation as before. Fraction 3 is dissolved in 600 ml. of 0.02 M cysteine (pH 7 to 7.5) and 60 g. of solid NaCl are added slowly. Papain is precipitated as a fine white solid by this procedure. This suspension is also allowed to stand for one hour at 4° it is then centrifuged in the cold for 1 hour at 2500 r.p.m. and the supernatant solution (Fraction 4a) is discarded. 

Purification of Red Iron-Sulfur Flavoprotein. The red iron-sulfur flavoproteinis eluted from the DEAE-Sephacel column shortly after (or sometimes together with) the cognate 2-oxoacid ferredoxin oxidoreductase activity, as described above. The red-colored fractions are combined and made to 1 A/ ammonium sulfate solution by addition of solid ammonium sulfate at 4°, with stirring. 

C. 7-Methoxyphthalide (4). The crude product from Step B (26.9 g), in a 1-L, one-necked, round-bottomed flask with a magnetic stirring bar, is dissolved in 465 mL of absolute methanol (Note 12). To this solution, cooled to 0°C, is added 7.4 g (0.20 mol) of powdered sodium borohydride, in small portions (Note 13), and the mixture is stirred at room temperature for 18 hr. The reaction mixture is cooled in an ice water bath and taken to pH 4-5 with about 35 mL of 6 M aqueous hydrochloric acid. A further 48 mL (0.288 mol) of 6 M hydrochloric acid is added, the flask is fitted with a reflux condenser and the reaction mixture is heated under reflux for 12 hr. The reaction mixture is then allowed to cool to room temperature and most of the methanol is evaporated under aspirator pressure. The residue is dissolved in 400 mL of methylene chloride, the phases are separated, and the organic fraction is washed with saturated ammonium chloride (3 x 200 mL) and water (1 x 100 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The pale yellow solid is recrystallized from ethyl acetate-hexane (1 1) (Note 10) to give 15.4 g... 

The only non-mammalian /8-glucuronidase that has been subjected to systematic purification is the enzyme from sheep-rumen microorganisms. After repeated ammonium sulfate fractionation, Marsh101 obtained a colorless preparation with a specific activity of 1,900 (400-fold purification and 2% recovery). The final product gave a linear, specific-property, solubility test from which a figure of 2,200 was derived for the ultimate specific activity of the enzyme, but it was considered that the enzyme may have formed a solid solution with inactive protein. 

Fig. 9. Preparative chromatography of bovine ehymotrypsinogen B (105). CM-cellulose column (3.0 X 9.0 cm) equilibrated with 0.05 M citrate, pH 4.2. Arrow indicates change to 0.05 M citrate, pH 4.6. Solid line, potential activity. Dotted line, proteins. The values along the peaks indicate the specific activity of some fractions. I. Chromatography of the precipitate obtained in 0.4 saturated ammonium sulfate (specific activity, 0.9). II. Second chromatography of the fractions having the lowest activity in diagram I. Ordinates, activity or proteins found in each fraction (1 ml) and expressed in per cent of the total activity and total proteins introduced into the column. Abscissas, volume of eluate in milliliters.

Place in an eight-inch tube 5 ml of the unsaturated hydrocarbon and 5 ml of cold 70 per cent sulfuric acid. Cool the mixture in tap water, place a solid rubber stopper in the mouth of the tube, and shake with cooling until the hydrocarbon dissolves and a clear liquid results. Add 6 g of ammonium sulfate dissolved in 8 ml of water. Insert the separatory stopper, and remove the aqueous acid layer. The liquid which is left in the reaction tube can be tested with bromine water or alkaline permanganate to show that it is not an olefin. If it is desired to purify the alcohol, add 1 g of anhydrous calcium sulfate and shake from time to time over a period of fifteen minutes. Pour the crude alcohol into a distilling tube. Heat with a small flame and collect the proper fraction. If amylene is used, and the water is not completely removed by the drying agent, a constant boiling mixture will be formed which boils at 87°. 

Phenyl mercuric nitrate, iodoacetate, thiourea, and ammonium sulfate stimulated arsenite-oxidizing activity, whereas cupric ion, and p-chloromercuriben-zoate inhibited it significantly. Legge (8) showed that cytochromes associated with the solid fraction from broken cells of P. arsenoxydans-quinque played a role in aerobic oxidation of arsenite. 

Fig. 6. Effects of substrate, isoleucine, valine, and pH on the activity of maize threonine dehydratase. The enzyme was isolated from shoots of etiolated seedlings and partially purified by ammonium sulfate fractionation. Activity was measured as described by Datta (1971) under conditions in which velocity was proportional to the amount of enzyme added and linear over the time of measurement (10 min). The assay mixtures contained EPPS (0.1 M) as buffer and 0.2 M KCI. The pH of complete reactions mixtures was determined with an Orion Model 70IA pH meter and did not change during the incubations at 30°C. Further information will be published elsewhere by E. Lissik and J. Bryan. Solid circles represent control measurements. L-Valine (1.0 mM) was added to the assays indicated by open symbols and dashed lines. Solid and open triangles represent measurements in the presence of L-isoleucine. Isoleucine concentration was 10 txM for the assays at pH 7.45 and 20 juAf during measurements at pH 7.95 and 8.70.

The reaction mixture is cooled in a water-ice bath, and a saturated aqueous ammonium chloride solution is added at such a rate as to maintain the temperature below 35°C. Ammonium chloride solution is added in portions until addition produces no further exothermic reaction (Note 3). The supernatant solution is decanted through glass wool onto 400 g of ice in a 4-L separatory funnel. The residual solids are washed with three portions of hexane, approximately 1000 nt total, and the washes are decanted into the separatory funnel. After the phases are separated, the aqueous phase is washed with an additional 500-mL portion of hexane. The combined organic extracts are washed with 500 nl of saturated ammonium chloride, and then with 500 nl. of brine. The organic layer is dried over anhydrous magnesium sulfate and filtered. Most of the solvent is removed by a rotary evaporator and the residual oil is distilled at reduced pressure using an ice water-cooled fraction cutting head. After a small forerun, approximately 390-392 g (94% of theory) is collected as a colorless oil, bp 116°C/1.6 nm (lit. 155°C/17 rim). ... 

Immediately after the addition of the last portion of ammonium hydroxide, the reflux condenser and stirrer are removed and the flask is connected to the steam-distillation assembly. The mixture is steam-distilled as rapidly as possible, and two 2S0-ml. fractions of distillate are collected during a period of 10-13 minutes (Note 2). The first fraction is saturated with sodium chloride, and the solution is stirred at 5° until precipitation appears complete. The solid is collected on a Buchner fimnel and dried in the air. The product weighs 2.72-3.11 g. (57-65%) and melts at 38-39°. The second fraction of the distillate is saturated with sodium chloride and combined with the filtrate remaining from the first fraction. The combined solution is extracted with two 45-ml. portions of ether. The combined ether extract is filtered, dried over anhydrous sodium sulfate, and concentrated by distillation, finally under reduced pressure. The residue solidifies on cooling and weighs 0.6-1.0 g. it can be purified by steam distillation from 40-50 ml. of saturated sodium chloride solution until 100 ml. of distillate is collected, saturation of the distillate with sodium chloride, cooling, and filtration. The pure product so obtained weighs 0.42-0.87 g. The total yield (Note 3) is 3.3-3.6 g. (69-75%). 

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