Ammonium sulfate protein fractionation

Beef liver (or lung) was minced and then autolyzed for twenty-four hours before extraction with an alkaline solution saturated with ammonium sulfate. Protein was precipitated by warming the extract, and the heparin-protein complex was precipitated from the supernatant liquor on acidification. Extraction of the complex with ethanol removed fatty material, and tryptic digestion removed most of the protein. The heparin was precipitated with ethanol, redissolved in warm alkaline solution to destroy trypsin, and reprecipitated with acetone. This material, crude heparin, was isolated in a yield of 15-50 g. per 100 lb. of animal tissue. In a later paper," the purification of crude heparin by fractionation successively with Lloyd s reagent, cadmium chloride, and acetone, was described. The purified heparin w-as 100 times as active as the crude material. Scott and Charles" reported the presence of nitrogen... 

The only other comparison that has been made between the azV mutants and the wild type is a very preliminary examination of the disc gel electrophoretic patterns of several ammonium sulfate-precipitated fractions of crude extracts. Thus far, no protein that might be a repressor has been found missing in the azV extracts. We are therefore left with the rather operational interpretation that the azl gene may be a genetic element required for multivalent repression. Whether that element is or is not the structural gene for the postulated aporepressor of the Jacob-Monod model is, of course, impossible to state at this time. 

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. 

Purified LBP is obtained from the crude LBP separated in the gel filtration of the 35 kDa luciferase on Sephadex G-100 (see Fig. 8.2). The fractions of crude LBP are combined and the protein is precipitated with ammonium sulfate (75% saturation). The precipitate is dissolved in a small volume of lOmM Tris-HCl/5 mM 2-mercaptoethanol, pH 8, and a small amount of luciferin is added as a tracer. Then, the crude LBP is purified on a column of Sephadex G-200 (Hastings and Dunlap, 1986). The fractions of LBP are identified by luminescence produced by the addition of luciferase at pH 6.3 the luminescence due to the tracer luciferin is proportional to the amount of LBP in each fraction. 

Cormier and Dure (1963) found another type of luciferin and called it protein-free luciferin. Protein-free luciferin was found in the vapor condensate of freeze-drying whole animals, and also in the 3 5-56 % ammonium sulfate fraction of the crude extract noted above. The protein-free luciferin behaved like an aromatic or heterocyclic compound and it was strongly adsorbed onto Sephadex and other chromatography media, requiring a considerable amount of solvent to elute it. The luminescence reaction of protein-free luciferin in the presence of luciferase required a 500-times higher concentration of H2O2 compared with the standard luciferin preparation. Both types of the luciferin preparation had a strong odor of iodoform. 

A wheat germ, cell-free, translation extract was fractionated into three concentrated parts using ammonium sulfate the 0 - 40 % saturated fraction, the 40 - 60 % saturated fraction, and the ribosome fraction. These fractions were tested for their ability to enhance the translational activity of the wheat germ, cell-free extract for dihydrofolate reductase. The fortified cell-free system supplemented with the 0 - 40 % ammonium sulfate fraction enhanced the efficiency of protein synthesis by 50 %. 

Fig. 1. Reconstruction of the cell-free protein synthesizing system with the partially purified wheat germ extracts. Control normal wheat germ cell-free system, (I) 0 - 40 % ammonium sulfate fraction 3 pi, 40 - 60 % ammonium sulfate fraction 4 pi, and ribosome 3 pi were added to 25 pi reaction mixture, (II) 0-40 % ammonium sulfate fraction 4 pi, 40 - 60 % ammonium sulfate fraction 4 pi, and ribosome 1.5 pi were added to 25 pi reaction mixture.

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. 

The catalytic activities of the fortified wheat germ cell-free systems supplemented with each fraction were investigated (Fig. 2). As shown in Fig. 2, only 0 - 40 % ammonium sulfate fraction showed an enhancement in DHFR protein synthesis. This enhancement of protein experimental results and the fact that the various eukaryotic initiation factors are contained in synthesis was also confirmed by SDS-PAGE and autoradiography (Fig. 3). From the above 0-40 % ammonium sulfate fraction [5, 6], it can be concluded that the amount of initiation factors in a conventionally prepared wheat germ cell-fi extract is deficient for the translation of DHFR with internal ribosome entry site. Therefore, it needs to supplement a wheat germ cell-free extract with the fraction containing the limited initiation factors for the efficient protein translation, and this fortified cell-free system can be easily made by simple... 

Fig. 3. Autoradiograph of SDS-PAGE of in vitro translated dihydrofolate reductase (DHFR) in the wheat germ cell-free protein synthesis systems with (n) 4 pi of ribosome fiaction, (III) 4 pi of 0 -40 % ammonium sulfate fraction, or (IV) 4 pi of 40 - 60% ammonium sulfate fraction, respectively. Lane I is control dihydrofolate reductase produced in the normal wheat germ cell-free protein synthesis system.

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 (- - ).

Fig. 4. — Monitoring of the Multiple Molecular Forms of Tomato Pectinesterase by Starch-gel Electrophoresis.98 [ENZ, detection of pectinesterase activity by paper print with pectin and Bromothymol Blue PROT, protein staining with nigrosin O, origin. Key A, 1 crude tomato extract after ammonium sulfate salting-out, and dialysis 2 pectinesterase fraction from column of DEAE-Sephadex A-50 3 and 4 pectinesterase fractions from column of Sephadex G-75. B, Two parts of the same gel after horizontal slicing 1, 500 fig of the isolated form of pectinesterase from a column of CM-Seph-adex C-50 with 175 mM phosphate-sodium chloride buffer 2, active fraction at 150 mM buffer 4 and 5, 250 fig and 1 mg of the isolated form of pectinesterase, respectively.]...

Solubility curves for different types of Hp have been presented (H5, H7). The higher solubility of type 1-1 than that of the others is in conformity with its lower molecular weight. The irregularity of the solubility curves for Hp of 2-2 and 2-1 type reflects the molecular heterogeneity of these two proteins. It is known from fractionation experiments with ammonium sulfate as well as with ethanol that the slower Hp bands are enriched in those Hp fractions that are precipitated first. So far, we have not been able to separate any of the Hp bands completely from the others, except the 1-1 band in ascitic fluid of type 2-1. 

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. 

Since ammonium sulfate fractionation will also cause precipitation of other proteins, antibody concentrations obtained from absorbance measurements at 280 nm are only estimates. Alternatively, a sample of the dialyzed solution can be resolved on a SDS-polyacrylamide gel alongside a series of known concentrations of IgG. Staining the gel with Coomassie blue can then be used to estimate the amount of immunoglobulin obtained and can also give an estimate of purity. 

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.

Ammonium sulfate is one of the cheapest and smoothest agents for precipitation of proteins. It is suitable for protein concentration as well as for protein fractionation. 

Figure 3-4 Hypothetical behavior of a solution containing three proteins, A, B, and C, upon ammonium sulfate fractionation. The concentration of protein remaining in the solution is plotted against ammonium sulfate concentration (usually expressed as % saturation). Addition of ammonium sulfate to concentration c, will precipitate largely protein B, which can he removed by centrifugation. Addition of additional salt to c2 will precipitate largely protein C, while A remains in solution.

A cell extract was subjected to ammonium sulfate fractionation and the dialyzed protein was then poured through the affinity column which held the cytidine deaminase molecules because of their affinity for the cytidine structures that were bound to the agarose. 

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