Enzyme X is a highly pigmented protein that imparts the characteristic color to
ID: 779962 • Letter: E
Question
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?
What I have:
The first step to determining protein identification and purification is to isolate the protein of interest from the starting material. This process is initiated by determining the proper assay to be used. An assay is a termed used to describe the technique that one uses in protein purification. It describes methodology, protocol, and detailed technique unique to certain experiments. In more detail, unique to certain experiments, describes the chemical properties of the desired protein. The protein of interest must be removed from the cell. Its associated enzyme is a useful tool in determining its specific activity. This is a quantitative measurement that can assess the progression of protein purification. Proteins can be purified based on size, charge, solubility, and even binding affinity.
Given a pure preparation of algae and purifying Enzyme X, I would begin by
extraction of all proteins via salting out technique. Then remove the supernatant and centrifuge, followed by column chromatography to separate all the proteins based on size, followed by labeling vials according to size. Next, remove a small amount of what is in each vial and add to it substance X. Because Y is unknown, I depend on the rate of substance X's depletion to detect the presence of the enzyme. After recording the change in concentration of X (through fluorescence change) per certain amount of time that is fixed regardless of what protein is being tested, I then identify the vial that has the mix of proteins that has the most enzyme X, and run SDS-page electrophoresis. This provides an idea about the size of the different proteins. The difference in their sizes is assessed via the column chromatography. Finally, I run just that vial through the column and retest with substance X. This indicates purity in the preparation.
my teachers reponse: what you have submitted is a start however this is general protocol. The purpose of the assignment is to make sure you the student understands the various protein purification techniques discussed in class and in the textbook and write a general protocol. The M1 assignment is being returned to you again to refer back to topic section 1.2 within the class and see what is discussed. It specifically states in the commentary %u201CTo isolate a compound completely, the above methods, and many others not described, are usually employed sequentially, and the specific activity is measured after each step. Only the fraction containing the highest specific activity is then passed on to the next step.%u201D It is telling you the reader that all discussed need to be done at a minimum to purify a protein and more steps are needed that would be determined by first doing the %u2018general%u2019 steps as described. Also note that as written the protocol submitted started purificaiton with whole cells - that is not possible. The protein of interest is inside the cell - the first step is to get the protein of interest out of the cell, how is that done?
Explanation / Answer
This is the review with recommended edits to your enzyme purification description.
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.
We can then %u201Csalt out%u201D the solution to begin a rough purification process. Salting out will help separate the organic material from water by changing the solvent ratio. 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. There is a receptor to selectively bind the enzyme and , thereby hindering it%u2019s 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. 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.
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