Fractionation with Saturated Ammonium Sulfate Solution

FRACTIONATION WITH SATURATED AMMONIUM SULFATE SOLUTION 

The table gives the milliliters of saturated ammonium sulfate solution to be added to one liter of solution to produce the desired change in percent saturation. The volume changes on mixing are negligible. The pH of a saturated ammonium sulfate solution is about 5.5. The pH can be adjusted to 7 by adding a few drops of concentrated NH4OH. 

Initial concentration of ammonium sulfate in the preparation (percent saturation) 

Kepnnted by permission of the Oxford University Press (Oxford) from Data for Biochemica Research, 2nd ed, Edited by R. M. C, Dawson, D. C. Elliott, W. H. Elliott, and K. M. Jones, Oxford University Press ([969). 

Listed below are some acids and bases that are useful in preparing buffers for enzyme assays. The choice of a particular compound depends on many factors. For example, multicarboxylic acids would be poor choices for reactions involving metal ions as cofactors amino acids may be poor choices for reactions involving amino acids as substrates. The number of buffer components can be kept to a minimum by using an acid and a base to cover the desired region. For example, maleic acid and Tris can be mixed to produce Tris-maleate buffers of pH 5.7 to 8.6 (rather than using maleic acid-NaOH and Tris-HCl). 

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FRACTIONATION WITH SATURATED AMMONIUM SULFATE SOLUTION... 

The above solution is stored in an icebox for 2 days. During this time, most of cytochromes b and c become insoluble and precipitate. After centrifugation (8,000 X g, 20 minutes) the supernatant fluid is made up to 35% saturation with saturated ammonium sulfate solution buffered at pH 7.5. Cytochrome a and the rest of the cytochromes b and Cj are precipitated and only a little cytochrome Cj remains in the supernatant. The precipitate is dissolved in 50 ml. of the above 0.1 M phosphate buffer (containing 25% saturation of ammonium sulfate and 0.5% cholic acid) and by addition of more saturated ammonium sulfate solution the fraction between 25 and 35% saturation is obtained (Fraction S3). This fraction has no cytochrome b and little c. To remove cytochrome Cj from the fraction, the above fractionation is repeated two or three times. The fraction obtained after each fractionation should always be stored overnight before the next treatment. In this way, a clear, deep-reddish colored concentrated solution of cytochrome a is obtained which contains no other cytochrome components (Fraction S4). 

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. 

Ammonium sulfate precipitation. Mixing of the ammonium sulfate with the antibody solution is not instantaneous and takes time, which depends on the properties of the components as well as on the processing conditions, i.e., mixing intensity, the volume, density and viscosity of the medium as well as on the power input for mixing. The volume of saturated ammonium sulfate solution in mL that has to be added to a sample to salt out proteins at any concentration is given by the equation V (mL) = 100 mL X (S2-S1)/(1-S2) where SI and S2 are expressed as fractions of the saturated solution, 100 is the volume of the sample and V(mL) is the volume of saturated ammonium sulfate that has to be added to the sample to obtain the desired concentration. For example A 100 mL sample is precipitated sequentially first to an initial saturation of 25% and then to a final 50% ammonium sulfate saturation. V(mL) = 100x(0.25-0)/(l-0.25). V(mL) = 33.3 mL. The volume of 33.3 mL of saturated ammonium sulfate is added... 

After cooling to about 40°C (note 2) the viscous brown liquid was poured into a vigorously stirred solution of 50 g of ammonium chloride in 250 ml of 4 N HCl, which was kept at 0-5°C. The flask was also rinsed with this solution. The product was extracted 5-7 times with a 1 1 mixture of diethyl ether and pentane. The combined extracts were washed with saturated NHi Cl solution and subsequently dried over magnesium sulfate. The residue remaining after removal of the solvents in a water-pump vacuum, was carefully distilled through a 30-cm Widmer column. The desired nitrile, b.p. 84°C/15 mmHg, n 1.4487, was obtained in 72% yield. The first fraction (about 5 g) consisted mainly of the 1,3-substitution product n-C,HgC(CsN)=C=CH2. 

Purification of photoprotein. The dialyzed photoprotein solution was centrifuged to remove precipitates, and then subjected to fractional precipitation by ammonium sulfate, taking a fraction precipitated between 30% and 50% saturation. The protein precipitate was dissolved in 50 ml of 10 mM sodium phosphate, pH 6.0, containing 0.1 mM oxine ( pH 6.0 buffer ), dialyzed against the same buffer, and the dialyzed solution was adsorbed on a column of DEAE-cellulose (2.5 x 13 cm) prepared with the pH 6.0 buffer. The elution was done by a stepwise increase of NaCl concentration. The photoprotein was eluted at 0.2-0.25 M NaCl and a cloudy substance (cofactor 1) was eluted at about 0.5 M NaCl. The photoprotein fraction was further purified on a column of Sephadex G-200 or Ultrogel AcA 34 (1.6 x 80 cm) using the pH 6.0 buffer that contained 0.5 M NaCl. 

After two fractionations between 0.33 and 0.6 saturated ammonium sulfate, several precipitations at 0.6 saturation were made throughout a period of two or three days, during which the preparation was stored in the refrigerator. Any denatured, insoluble proteins were removed by centrifugation, and a solution of the desired activity was prepared by dilution with buffer or distilled water. 

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... 

Sepharose and elution with lactose (Fig. 23). The UV-absorbing fraction that was eluted with the lactose solution was collected and the protein precipitated by addition of an equal volume of saturated ammonium sulfate. On refrigeration of the sample overnight, a white precipitate formed. This precipitate was collected by centrifugation and redissolved in 0.2 mL of 0.02 M phosphate buffer of pH 7 in saline. The agar-diffusion test shown in the inset of Fig. 23 showed that high-titer antibodies were produced that formed a precipitin complex with the antigen. Several affinity runs were made. The antibody samples were combined and lyophilized. The yield of anti-lactose antibody was 50 mg from 20 mL of serum. 

The first peak of enzyme solution was dialyzed against 10 mM sodium succinate buffer, pH 6.0, without EDTA and with 2-mercaptoethanol, then applied to a hydroxyapatite column equilibrated with the same buffer. Stepwise elution with ammonium sulfate in this buffer yielded a single peak in 0.1 M ammonium sulfate fraction. The pooled enzyme was concentrated with 0.8 saturated ammonium sulfate to give 1% protein concentration in imidazole-glycylglycine buffer, pH 7.1. Addition of ammonium sulfate to between 0.5 and 0.55 saturation yielded a turbid solution which in turn yielded crystals after 24 hours at 4 C. The second peak was applied to a Sephadex G-200 column equilibrated with 0.01 M... 

It is also to be noted that the usual procedure of expressing concentrations in terms of the fractional saturation of the solution with ammonium sulfate is sometimes ambiguous. The temperature coefficient of solubility is considerable for these solutions, and unless the temperature of the measurements is specified, the given conditions may be difficult for other investigators to reproduce exactly. For temperature values in the neighborhood of 20 to 25 °C. half saturated ammonium sulfate may be taken as equivalent to a 2.05 molal solution. Other fractional saturations may be calculated by proportionality (see also 43). 

The macerated organ is autolyzed for 24 hours at 32° C. and at pH 8.0. Unrelated components are precipitated at pH 7.0 by 40 % saturated ammonium sulfate. When the ammonium sulfate concentration is increased to 80 % of saturation, a precipitate forms which contains 75 % of the enzyme. After solution and dialysis, the enzyme is leprecipitated by addition of 95 % ethanol, all preparations being carried out in the cold. The fractionation with ammonium sulfate is repeated as above. 

The system in rat liver has been partially purified (557,768). The supernatant fraction remaining after homogenization and centrifugation contains the activity. Fractionation of this solution by precipitation with neutralized ammonium sulfate reveals that the enzyme system contains two protein components Fraction I (precipitated at 45% saturation and reprecipitated at 25-40% saturation to... 

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. 

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