Ammonium sulfate-precipitated

The gaseous ammonia is passed through electrostatic precipitators for particulate removal and mixed with the cooled gas stream. The combined stream flows to the ammonia absorber where the ammonia is recovered by reaction with a dilute solution of sulfuric acid to form ammonium sulfate. Ammonium sulfate precipitates as small crystals after the solution becomes saturated and is withdrawn as a slurry. The slurry is further processed in centrifuge faciHties for recovery. Crystal size can be increased by employing one of two processes (99), either low differential controUed crystallization or mechanical size enlargement by continuous compacting and granulation. 

The purple protein is reddish in solutions and purple in the ammonium sulfate precipitate. The molecular weight is 39,000 by the sedimentation equilibrium method. The purple protein is brightly red-fluorescent, but the fluorescence characteristics cannot be related to the luminescence of Latia (Fig. 6.1.5 Shimomura and Johnson, 1968c). 

Step 3. The photoprotein fractions are combined and the NaCl concentration of the solution is reduced to 50 mM by gel filtration through a column of Sephadex G-25. Then, the solution is poured onto a column of DEAE cellulose. The photoprotein adsorbed on the column is eluted by a linear increase of NaCl concentration from 50 mM to 0.4 M in the same pH 6.7 buffer. Active fractions are combined and concentrated by ammonium sulfate precipitation. 

Step 4. A repetition of gel filtration on a smaller column of Ultragel AcA 54, followed by the concentration of the eluted photoprotein by ammonium sulfate precipitation. 

Ammonium Sulfate Precipitation. The extract was made up to 40% saturation with the slow addition, with stirring, of ammonium sulfate at 4°C. After several hours, the precipitate was removed by centrifugation at 30,000 g for 30 min and the supernatant retained. It was brought to 100% saturation in similar conditions, the precipitate was collected by centrifugation, dissolved in the minimum of distilled water, dialyzed against water and then against 1% glycine, and lyophilized. 

Figure 2. Gel filtration. The dry residue obtained after ammonium sulfate precipitation was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 M NaCl 0.013 % sodium azide, which was loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fractions were collected. The fractions were assayed for protein content (— ) and PNL activity (- - ).

Figure 5 shows the pattern of lyase isoenzymes along the purification process at first, three bands with lyase activity (pis 9.20, 9.00 and 8.65) were detected in the ammonium sulfate precipitate (B 1) in the peak eluted from the Superdex 75HR1030 column, only one band with lyase activity was detected, that correspond to the PNL with pi 9.20 (B 2), but more proteins were detected by silver staining (A 2). 

Figure 11.9 Production of rhGH in . coli (as an intracellular protein). Subsequent to fermentation, the cells are collected by centrifugation or filtration. After homogenization, nucleic acids and some membrane constituents are precipitated by the addition of polyethyleneimine. Ammonium sulfate precipitation of the supernatant concentrates the crude rhGH preparation. Chromatographic purification follows, as illustrated...

Jayle, Herman-Boussier, and Moretti have shown that different types of human Hp in amounts large enough for analysis can be prepared by fractional ammonium sulfate precipitation combined with a rather conventional, preparative electrophoresis in an acetate buffer of pH 5.8. Schultze and Heide (S2) utilize a more complicated procedure, in which preparative electrophoresis (acetate buffer, pH 4.4) is likewise the final step. 

Ammonium sulfate-precipitated antiphosphotyrosine antibodies (see Chapter 2 hybridomas are available from ATCC). 

Specific Antibody Determination. Serum samples were prepared from each bleed by centrifugation to remove clotted material. 100 ul of the sera was incubated for 30 minutes with sufficient H-STXOL to provide a ca. 20 fold excess of hapten to the anticipated quantity of specific binding sites. The radioactivity of the protein pellet was determined after ammonium sulfate precipitation. After correction for a small amount of non-specific adsorption of label by control sera proteins the mg/ml of specific antibody in the sample was calculated. 

Figure 6. Affinity chromatography of EGD from Clostridium thermocellum. Nucleic acid preparation, heat treatment and ammonium sulfate precipitation (0-70%, 70-100%) were carried out as described (10). The final precipitate ( 50 mg protein), dissolved in 50 mM sodium acetate, pH 5.0, was applied (after centrifugation) on the affinity column (2 x 25 cm) (4 -aminobenzyl l-thio-/ -cellobioside coupled to Sepharose 4B) (11). Protein was monitored at 280 nm and the activity of the fractions (2 ml) determined using 2 -chloro-4 -nitrophenyl / -cellobioside (pH 6.5, 25°C) as described in the text. Elution with 10 mM G2 was started as indicated.

A number of approaches have been used to determine the amount of HA protein in a sample. The most successful of these is based on adding a fixed amount of TA to a sample (Thompson and Forward, 1969) after incubation, the turbidity is measured and the increase in turbidity observed is presumed to be proportional to the amount of HA protein in the sample. This method has the advantage that only substances able to form haze with polyphenols respond. The saturated ammonium sulfate precipitation limit (SAPL) method has also been widely used, but is far inferior in providing useful information (Berg et al., 2007 Siebert et al., 2005). 

In vitro studies were conducted with enzymes extracted from peanut (7), pea (. ), and onion (9). The enzymes were fractionated by ammonium sulfate precipitation, dialyzed, and stored frozen until used. The enzymes were assayed for various activities as described In the Results and Discussion. 

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