BL4820 Lecture 5 - Glutamate Oxaloacetic Acid Transaminase (GOT) Purification -- Expt 3 Part B
1. Ammonium Sulfate Fraction of Protein Mixtures
Increasing the salt concentration to a very high level will cause proteins to precipitate from solution without denaturation if done in a gentle manner. First, we want to understand why the protein precipitates. A protein in a buffer solution is very highly hydrated, in other words, the ionic groups on the surface of the protein attract and bind many water molecules very tightly:
This graphic illustrates how proteins in solution are hydrated by water molecules. When a lot of salt, such as ammonium sulfate, is added to the protein solution, the salt ions attract the water molecules away from the protein. This is partly since the salt ions have a much greater charge density than the proteins. So as the salt is added and these small ions bind water molecules, the protein molecules are forced to interact with themselves and begin to aggregate:
So when enough salt has been added, the proteins will be begin to precipitate. If this is carried out at a cold temperature like in ice, the proteins will precipitate without denaturation. Thus, the proteins can be collected by centrifugation and then redissolved in solution using a buffer with low salt content.
This process is called "Salting Out" and works best with divalent anions like sulfate, especially ammonium sulfate which is highly soluble at ice temperatures (see Table 10-1 on p. 114 of your text where 100% saturation of ammonium sulfate is equivalent to a solution of 3.3 M ammonium sulfate at 0°C).
Salting out or ammonium sulfate precipitation is useful for concentrating dilute solutions of proteins. It is also useful for fractionating a mixture of proteins. Since large proteins tend to precipitate first, smaller ones will stay in solution. Thus, by analyzing various salt fractions, one can find conditions where the protein you are studying precipitates and many of the other proteins in the mixture stay in solution. The end result is that you are also able to increase the purity of your protein of interest while you concentrate it using an ammonium sulfate fractionation procedure.
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