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.

Given a pure preparation of these algae, and the required supplies and equipment, devise and outline an empirical procedure for purifying Enzyme X.
What is a good indication of purity in your preparation?

I'm totally stumped as to how to answer this question

To devise an empirical procedure for purifying Enzyme X, we need to follow a general purification scheme. Here is a step-by-step outline of the process:

1. Harvesting: Obtain a pure preparation of the blue-green algae culture containing Enzyme X. This can be done by growing the algae in suitable growth medium, under appropriate conditions (light, temperature, etc.), until a sufficient biomass is obtained.

2. Cell Disruption: Break open the cells of the algae to release all cellular components, including Enzyme X. This can be achieved through methods like sonication, homogenization, or grinding. The aim is to break the cells without denaturing the enzyme.

3. Cell Debris Removal: Centrifuge the disrupted cell suspension to separate the crude extract containing Enzyme X from the cellular debris, such as membranes, organelles, and other unwanted components.

4. Precipitation: Consider performing an initial precipitation step, such as ammonium sulfate fractionation, to concentrate Enzyme X. This exploits the enzyme's differential solubility under specific salt concentrations. Precipitated proteins can be collected by centrifugation and resuspended in a suitable buffer.

5. Chromatographic Purification: Use different chromatographic techniques, such as ion exchange chromatography, size exclusion chromatography, or affinity chromatography, to further purify Enzyme X. These methods exploit the differences in protein size, charge, or specific binding affinity to separate and isolate the enzyme from other contaminants.

6. Analysis and Confirmation: At each purification step, confirm the presence and purity of Enzyme X by using appropriate analytical techniques. These may include measuring absorbance at specific wavelengths, assessing protein concentration through techniques like Bradford assay or UV spectroscopy, and running SDS-PAGE or gel filtration to visualize the protein bands.

7. Concentration and Storage: After achieving the desired purity, concentrate Enzyme X using techniques like ultrafiltration or dialysis. Store the purified enzyme under suitable conditions (such as low temperature, pH, and with the addition of stabilizing agents) to maintain its stability and activity.

Regarding the indication of purity in your preparation, a good measure would be to perform a specific activity assay. This assay measures the enzyme activity relative to the total protein content. A high specific activity indicates a pure preparation with minimal contamination. Additionally, confirming the presence of a single band on SDS-PAGE or gel filtration analysis, along with a purified sample showing little to no impurities on UV spectroscopy or other analytical methods, would also indicate a high level of purity.