Protein separation with ammonium sulfat

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. 

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. 

The site of formation of renin is not known, although the indirect and circumstantial evidence favors slightly the juxtaglomerular apparatus rather than the tubules as a source (18). Crude renin, however, is extracted readily from renal cortex by saline extraction, acidification, and precipitation with ammonium sulfate and sodium chloride. Other active protein substances are likewise extracted, and their separation from renin is often a matter of considerable difficulty. The renin substrate, an arglobulin, is found in blood serum and is probably formed by the liver. It can be easily salted out of beef serum as a crude preparation. 

After homogenization, it is often advantageous to perform a salt precipitation, most commonly with ammonium sulfate. The purpose of this precipitation is the separation of cell debris and nucleic acids rather than the purification of the target protein from impurities. Whereas the purification factor of ammonium sulfate precipitation is usually around only 1.5 to 2, the separation of non-proteineous impurities and stabilization of the target protein in ammonium sulfate usually provide sufficient benefit to include this step in any purification protocol. 

With these newer methods of protein separation and amino acid analysis he prepared serum protein fractions by serial salting out with ammonium sulfate and by the Sober and Peterson DEAE cellulose columns (42), using the sera of reptile, fowl, and mammalian blood. Some of the amino acid analyses were carried out by the automatic amino acid methods of Hirs, Moore, and Stein (18). Fortified with this plethora of data, Block now had the opportunity to re-examine not only the ratio of the basic amino acids, but at least 12 amino acids in a variety of protein fractions prepared by at least two different procedures. With the aid of a statistician he determined the significance of the constancy of the molar ratios of pairs of amino acids and found that in spite of the marked variation of the absolute amounts of an amino acid, the molar ratios of certain pairs remain relatively constant among the numerous protein components of animal sera. 

FIGURE 8 Separation of rabbit polyclonal antibodies by ion-exchange chromatography on DEAE Trisacryl M. Column dimensions 16 mm i.d.X 100 mm initial buffer 50 mM Tris-HCI, 0.035 M sodium chloride, pH 8.8 load 5 mL of rabbit serum previously precipitated with ammonium sulfate at 50% saturation and redissolved in column buffer flow rate 50 mL/hr elution of adsorbed protein performed using I M sodium chloride solution in the initial Tris buffer. The first peak represents IgG the second peak is composed of all other serum proteins precipitated by ammonium sulfate. The straight line is absorbance at 280 nm, and the broken line represents the variation of ionic strength of the buffer. The purity of IgG estimated by gel electrophoresis was over 98% and the calculated yield was over 90%. 

The resolved complex is composed of two fractions, a soluble part, which comprises about 15% of complex I proteins, and a water-insoluble part consisting of the rest of the protein and the bulk of complex I lipids. The soluble fraction is easily separated from the insoluble material by centrifugation. Upon fractionation with ammonium sulfate, it yields a soluble flavoprotein containing iron and labile sulfide and a dark brown protein, which contains large amounts of iron and labile sulfide but no flavin. The latter appears to be an iron-sulfur protein and exhibits an EPR signal which is characteristic of iron-sulfur center 2 of intact complex I (46). Its absorption spectrum is shown in Fig. 8. The insoluble fraction also contains equimolar amounts of iron and labile sulfide and little or no flavin. 

For many years salting-out by high concentrations of ammoniiun sulfate has been one of the classical methods of protein separation. There is very little literature on the theoretical basis of the method, particularly as applied to the isolation of enzymes, where it has mainly been used quite empirically. The underlying assumption in most cases seems to have been that the different proteins are precipitated at different fixed ammonium sulfate concentrations, provided the pH and temperature are fixed. For example one may commonly read in instructions for the piuification of an enzyme that the enzyme is precipitated at 65% saturation with ammonium sulfate or that the fraction precipitating between 0.62 and 0.68 saturation should be taken. It is, however, a fairly common experience that when one repeats a published method the enzyme fails to precipitate within the limits given. Furthermore, where the purification of a protein involves more than one salt-fractionation stage, the limits are usually found to be different for the different stages. 

On the addition of oxalate, blood separates into two layers. A dark red layer contains the blood corpuscles and a yellowish layer contains the plasma and plasma proteins. The plasma proteins can then be separated by fractional precipitation with ammonium sulfate solution. A 20%-25% ammonium sulfate solution will precipitate fibrinogens a 33% solution, globulin and a 50% solution, pseudoglobulin while albumins only precipitate at very high ammonium sulfate concentration. Ultracentrifuge measurements enable four components (X, A, G, M) with different sedimentation coefficients to be differentiated and isolated ... 

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