Ammonium sulfate fractionation

The solubility of proteins varies according to the ionic strength and hence according to the salt concentration of the solution. Two distinct effects are observed. At low concentrations of salt, the solubility of the protein increases with salt concentration. This phenomenon is called 'salting-in'. However, as the salt concentration (ionic strength) is increased still further, the solubility of the protein begins to decrease. At sufficiently high ionic strength the protein solubility will have decreased to the point where the protein will be almost completely precipitated from solution - an effect called 'salting-out'. The theoretical basis of salting-out is complex but one factor is probably the competition between the protein and salt ions for available water molecules. At high salt concentrations, insufficient water molecules are available for full solvation of the protein so that protein protein interactions become predominant over protein-water interactions, and precipitation occurs.

Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very useful procedure to assist in the purification of a given protein. Indeed, enzyme purification schemes almost invariably include such a step. In practice, ammonium sulfate is the salt commonly used since it is highly water-soluble, relatively cheap and available at high purity. Furthermore it has no adverse effects upon enzyme activity. Typically a small-scale experiment is conducted initially to determine the optimal ammonium sulfate concentration to use. This procedure is typically carried out at 0 - 4º C to maximize protein stability. Great care must be taken to ensure that the salt concentration of the whole solution increases uniformly without the occurrence of local high concentrations which could precipitate the protein of interest along with the undesired proteins. Therefore the solution is stirred continuously as small aliquots of crushed, solid ammonium sulfate (or preferably saturated ammonium sulfate solution) are added. After each addition, the ammonium sulfate is allowed to disperse fully before the next addition. Once the required ammonium sulfate concentration is reached, incubation at 0 - 4 º C is continued for a brief period to allow protein precipitation to occur, and the precipitated protein is then recovered by centrifugation. The concentration of ammonium sulfate in the solution is thus increased stepwise, recovering the precipitated protein at each stage. Each protein precipitate is dissolved individually in fresh buffer and assayed for both total protein content and the activity of the desired enzyme. Based on these data one can then plan a successful ammonium sulfate fractionation procedure for the bulk purification of the enzyme. An ammonium sulfate concentration is chosen which will precipitate the maximum proportion of undesired protein whilst leaving most of the enzyme still in solution. This precipitated protein is removed by centrifugation and then the ammonium sulfate concentration of the remaining solution is increased to a value that will precipitate most of the enzyme whilst leaving the maximum amount of residual protein contaminants still in solution. The precipitated enzyme is recovered by centrifugation and dissolved in fresh buffer for the next stage of purification. Residual ammonium sulfate will be present in this enzyme solution and may need to be removed by gel filtration or dialysis before the next purification step can be attempted.

In addition to its role as an extremely useful and universally applicable purification step, ammonium sulfate purification is often employed again at later stages of purification simply to concentrate the protein from dilute solution after procedures such as gel filtration.

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