Enzyme Fractionation By Salting-Out: A Theoretical Note

This chapter explores that for many years salting-out by high concentrations of ammonium sulfate has been one of the classical methods of protein separation. 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. Unlike fractionation with organic solvents, it does not tend to inactivate enzymes by denaturation; on the contrary, ammonium sulfate frequently has a protective action on enzymes, and indeed enzymes are frequently stored in the form of suspensions of precipitates in concentrated ammonium sulfate. This not only stabilizes the enzyme but prevents the growth of bacteria. Unlike chromatographic and absorption methods, it affords a procedure for concentrating as well as purifying the enzyme. The chapter stresses on the importance of pH and temperature as variables that have a great effect on the positions and separation of the precipitation peaks. They must therefore be carefully controlled. It highlights that a single protein may crystallize in several different forms with different solubilities and the solubility, and hence the composition of the precipitate may change in a complicated manner with time.