Enzyme X is a highly pigmented protein that imparts the characteristic color to certain blue-green algae. It also facilitates a reaction necessary to the survival of this species; we can follow the kinetics of this reaction by measuring the conversion of Substance X to Substance Y at various times during purification.
1. Given a pure preparation of these algae, and the required supplies and equipment, devise and outline an empirical procedure for purifying Enzyme X.
2. What is a good indication of purity in your preparation?
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Do an assay of the supernatant and the precipitate to see which one has the highest specific activity. Keep the one that has the highest.
The specific activity is the ratio of biochemical activity to the mg of total protein present. In this case, the conversion of Substance X to Substance Y is the activity that we are looking for. In each step, barring the last, one of our precipitates will contain no amounts (or very little) of the desired enzyme and therefore show no specific activity of the desired nature. The other substance will be the one we will keep for further fractionation and testing.
We can then "salt out" the solution to begin a rough purification process. Salting out will help separate the organic material from water. If solubility is not known, do another assay by measuring again the specific activity and keeping the highest.
Once this is complete, affinity chromatography can be performed since we are seeking an enzyme. The active enzyme will attach to its substrate, thereby hindering it's flow and impeding the passage of the enzyme.
A disc electrophoresis can also be used to more definitely determine the purity of the compound. This process will sort the remaining substances first by size and charge and then only by charge. My suggestion is to search the remaining substance by noting the color. Since substance X is pigmented, a blue color can indicate purity.
However, a final assay should indicate a much higher specific activity since these group of proteins should be entirely pure.