For fractionation of proteins using ammonium sulfate precipi

For fractionation of proteins using ammonium sulfate precipitations:

How come solid ammonium sulfate is preferred rather than using 100% saturated (NH4)2SO4 solution when preparing the high salt (> 60%) fractions? Why is it essential that one record the volume of solvent into which precipitated protein is dissolved?

Solution

The technique exploits the fact that the solubility of most proteins is lowered at high salt concentrations. As the salt concentration is increased, a point is reached where the protein comes out of solution and precipitates. The concentration required for this salting out effect varies from protein to protein, and thus this procedure can be used to fractionate a mixture of proteins.

Using solid ammonium sulfate avoids the precipitation of salt together with protein. Non-precipitating volume of (NH₄)₂SO₄ solution is generally used to further purify the protein after ammonium sulfate precipitation. If a solution of ammonium sulphate is used, the initial concentration of protein becomes less and more ammonium sulfate is required to precipitate the protein. This ends up in the excess use of ammonium sulfate.

Volume of the solvent

A given protein is normally precipitated over a small range of ammonium sulphate concentrations. This reflects the fact that protein-protein aggregation for a given protein suddenly becomes predominant over protein-water and protein-salt interactions. This is because the addition of the salt removes the layer of water molecules surrounding hydrophobic groups on the protein surface, which allows the hydrophobic groups to cause protein aggregation and hence precipitation. The concentration of the solvent is to be measured at various points of protein precipitation (min to max precipitation) to calculate the amount of ammonium sulfate.

To calculate the amount (g) of ammonium sulfate to add to 1 cm³ of solution to achieve a particular concentration, the following formula is used

g = 533 (S₂-S₁) / 100-0.3S₁

Where S₁ is the starting concentration and S₂ is the final concentration. The formula takes into account the change in volume caused by the addition of the ammonium sulphate.