Question

Enzyme X is a highly pigmented protein that imparts thecharacteristic color to certain blue-green algae. It alsofacilitates a reaction necessary to the survival of this species;we can follow the kinetics of this reaction by measuring theconversion of Substance X to Substance Y at various times duringpurification.
1. Given a pure preparation of these algae, and the requiredsupplies and equipment, devise and outline an empirical procedurefor purifying Enzyme X.
2. What is a good indication of purity in your preparation?

Can you check what I wrote?:
Do an assay of the supernatant and the precipitate to see which onehas the highest specific activity. Keep the one that has thehighest.

The specific activity is the ratio of biochemical activity to themg of total protein present. In this case, the conversion ofSubstance X to Substance Y is the activity that we are looking for.In each step, barring the last, one of our precipitates willcontain no amounts (or very little) of the desired enzyme andtherefore show no specific activity of the desired nature. Theother substance will be the one we will keep for furtherfractionation and testing.

We can then “salt out” the solution to begin a rough purificationprocess. Salting out will help separate the organic material fromwater. If solubility is not known, do another assay by measuringagain the specific activity and keeping the highest.

Once this is complete, affinity chromatography can be performedsince we are seeking an enzyme. The active enzyme will attach toits substrate, thereby hindering it’s flow and impeding the passageof the enzyme.

A disc electrophoresis can also be used to more definitelydetermine the purity of the compound. This process will sort theremaining substances first by size and charge and then only bycharge. My suggestion is to search the remaining substance bynoting the color. Since substance X is pigmented, a blue color canindicate purity.

However, a final assay should indicate a much higher specificactivity since these group of proteins should be entirely pure.