Question

In your purification of GAPDH, during the second ammonium sulfate precipitation step, you notice that your resuspended pellet does not have activity. You retrace your steps and see that there is activity in the supernatant. What might have happened to cause this? What can you do to resolve this problem?

Answer 1

pecific activity of the protein represents the purity of the enzyme. In the above isolation and purification of GAPDH, resuspended protein pellet does not have activity initially thereby it is representing the protein is not still purified.

Sulphate series of ammonium has the ability to rise the surface tension of solvent used in the purification finally enable decreasing the solubility of nonpolar products result in salting out phenomemnon by profoundly reinforcing hydrophobic interactions.

The solubility of precipitating protein changes based on the ionic strength (isoelectric point of protein) and salt concentration. Thereby it is illustrated that as the ammonium sulphate concentration increases the protein concentration and solubility decreases and initiate precipitation. But majority of desired protein will be in the solution itself. Another reason is ammonium sulphate usually rise the total volume of the solution.

In order to overcome the problem ammonium sulphate addition should be done in a step wise manner to attain a suitable desired ionic strength (nomogram calculations) to separate GAPDH. As soon as reaching suitable and sufficient high ionic strength, the desired protein will be fully isolated (salting out) the from the solution.

Reasons:

Enzyme specific activity: It is the enzyme peptide activity which denotes the enzyme purity in the given purification mixture. That means it denotes the considerable activity of the enzyme per a milligram of the entire protein thereby it is considered as an indicator of enzyme processivity at a particular concentration of substrates. The MKS units to denote specific activity of enzyme is μmol min−1mg−1.

The specific activity of the enzyme rises at every purification process of the protein mixture. Thereby it is illustrated that higher the specific activity higher the protein purity and specific activity of the enzyme is an extremely perfect indicator of purification process.