Many types of salts have been employed to effect protein separation and purification through salting-out. Of these salts, ammonium sulfate has been the most widely used chemical because it has high solubility and is relatively inexpensive. Because enzymes are proteins, enzyme purification can be carried out by following the same set of procedures as those for protein, except that some attention must be paid to the consideration of permanent loss of activity due to denaturation under adverse conditions.
There are two major salting-out procedures. In the first procedure, either a saturated salt solution or powdered salt crystals are slowly added to the protein mixture to bring up the salt concentration of the mixture. For example, the salt concentration reaches 25% saturation when 1 ml of the saturated salt solution is added to 3 ml of the salt-free protein solution; 50% for 3 ml added; 75% for 9 ml added; and so on. The precipitated protein is collected and categorized according to the concentration of the salt solution at which it is formed. This partial collection of the separated product is called fractionation. For example, the fraction of the precipitated protein collected between 20 and 21% of salt saturation is commonly referred to as the 20-21% fraction. The protein fractions collected during the earlier stages of salt addition are less soluble in the salt solution than the fractions collected later.
Whereas the first method just described uses increasing salt concentrations, the following alternative method uses decreasing salt concentrations. In this alternative method, as much protein as possible is first precipitated with a concentrated salt solution. Then a series of cold (near 0ºC) ammonium sulfate solutions of decreasing concentrations are employed to extract selectively the protein components that are the most soluble at higher ammonium sulfate concentrations. The extracted protein is recrystallized and thus recovered by gradually warming the the cold solution to room temperature. This method has the added advantages that the extraction media may be buffered or stabilizing agents be added to retain the maximum enzyme activity. The efficiency of recovery typically ranges from 30 to 90%, depending on the protein. The recrystallization of protein upon transferring the extract to room temperature may occur immediately or may sometimes take many hours. Nevertheless, very rarely does recrystallization fail to occur. The presence of fine crystals in a solution can be visually detected from the turbidity.
The recovery of protein can have very significant economical implications. Because a fixed fraction of the original protein stays soluble in the solution, the recovery of protein is often not near 100%. Of course, a yield of over 100% indicates that there may be problems associated with the assay method.
In a typical protein preparation or purification step carried out in a laboratory where the aim is to isolate a small quantity of a product for structural or kinetic studies, a saturated ammonium sulfate solution is routinely used. It is also the procedure taken in this experiment. However, in an actual large scale commercial process, it is better to add ammonium sulfate directly into the protein mixture as powdered solids so that the effect of dilution by the salt solution is minimized.