Enzyme X is a highly pigmented protein that imparts the characteristic color to certain blue-green algae....

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?

The outline of steps to purify Enzyme X should include detail of what the technique is, why that technique is needed, and what the result will be. The reader needs to have enough information so as to be able to do the experiment. Treat the assignment as one that demonstrates your knowledge of biochemical techniques in protein purification.

As for the length of this assignment, you are describing a protocol; therefore, you need to provide the necessary information for the reader to be able to know how, why, and what, as well as the potential outcomes of the steps and the end result.

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Expert Solution

Do a quantitative 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. You are monitoring the concentration of the enzyme as an indicator. The other substance will be the one we will keep for further fractionation and testing.

Salting out will help to separate the organic material from water by changing the solvent ratio. If the 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 for an enzyme. Generally active enzyme will attach to its substrate. There is a receptor to selectively bind the enzyme and thereby hindering its 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. You will see a separation of bands in the electrophoresis lanes that correspond to the other components. Search the remaining substances 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 groups of proteins should be entirely pure.

queen_honey_blossom answered 1 month ago

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