Ammonium sulfate concentration

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.

The neutral FDPase and SDPase activities, which were present in the crude spinach extracts, were precipitated at lower ammonium sulfate concentration and could thus be separated from the specific alkaline FDPase. These activities appeared to be associated with the chloroplast fraction and did not require the presence of a divalent cation for activity. In crude extracts only the alkaline FDPase activity was inhibited by antiserum prepared by immunizing rabbits with the purified alkaline FDPase. The neutral FDPase was also active with ribulose diphosphate (RuDP) (98). 

To summarize this set of observations, halophilic proteins adsorb to polysaccharide matrices at high ammonium sulfate concentration. When the matrix is charged the adsorption of proteins having the same charge on the matrix is reduced whereas the adsorption of proteins having opposite charge is greatly facilitated. These observations enabled the development of several powerful purification... 

Ammonium sulfate concentration at which the enzymes eluted from various solid supports. From Mevarech et al. (1976), with permission. 

Ammonium sulfate concentration at which 50% of the activity was found in the supernatant. 

Fig. 6.6. Effect of ammonium sulfate concentration in the polymerization mixture on morphology of a monolithic CEC column prepared from a solution of piperazine diacrylate, N,N-dimethylacrylamide, and vinyl sulfonic acid (52% C, 29% T) in 20 mmol/L phosphate buffer pH 7. Ammonium sulfate concentration 0 (1), 20 (2), and 120 mg/mL (3). (Reprinted with permission of authors from [32]).

In 1M (or greater) ammonium sulfate solutions,actinide(III) ions precipitate as double sulfates. Simultaneously hexamminecobalt (III) sulfate also precipitates. Accordingly, attention has to be paid to the adjustment of the ammonium sulfate concentration. 

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. 

Fig. 6. Rate of change of solubility with ammonium sulfate concentration, obtained by differentiating curves of Fig. 5.

In other words, contrary to widespread and long-standing belief, proteins do not appear in fractions between fixed characteristic limits of ammonium sulfate concentration, but on the contrary the fractionation limits vary with the concentration of the particular protein. 

Fig. 9. Solubility curves for pure crystalline serum albumin, for hemoglobin, and for a mixture of the two, shown as a function of ammonium sulfate concentration. From Roche and Derrien (1946). O = hemoglobin (dog) 9 = serum albumin (horse) X = mixture of both. The curve for hemoglobin has been displaced upwards (scale on right) to show the close correspondence with the curve for the mixture.

All of the enzymes with substitutions at His-357 and His-391 precipitated at the same ammonium sulfate concentrations, and eluted from the ion exchange and gel nitration columns in similar volumes as wild type. This indicated that the physical properties associated with purification (aggregation, quaternary structure, charge, etc.) were not affected by the mutations. The enzymes were greater than 95% pure as analyzed by SDS PAGE. H357F was stable for at... 

A buffer mixture of ammonium sulfate and sulfuric acid can also be employed for the elution of aluminum. Its specific detection is carried out via derivatization with Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt) with subsequent photometry of the complex formed at a wavelength of 313 nm (Fig. 3-160). In an acidic sulfate medium aluminum ions exist as A1S04+ ions depending on the hydrogen ion and sulfate ion concentration. The parameter determining retention is, in this case, the ammonium sulfate concentration. 

Sol in pure water coagulated (denatured) by beat. Precipitate from solution only at high ammonium sulfate concentrations (70-100% of saturation). 

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 ... 

Add (NH4)2S04 at 4 °C slowly to the culture supernatant with stirring until a 35-405K ammonium sulfate concentration is reached. 

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