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 yourpreparation?

What I have:

The first step to determining protein identification andpurification is to isolate the protein of interest from thestarting material. This process is initiated by determining theproper assay to be used. An assay is a termed used to describe thetechnique that one uses in protein purification. It describesmethodology, protocol, and detailed technique unique to certainexperiments. In more detail, unique to certain experiments,describes the chemical properties of the desired protein. Theprotein of interest must be removed from the cell. Its associatedenzyme is a useful tool in determining its specific activity. Thisis a quantitative measurement that can assess the progression ofprotein 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 wouldbegin by
extraction of all proteins via salting out technique. Then removethe supernatant and centrifuge, followed by column chromatographyto separate all the proteins based on size, followed by labelingvials according to size. Next, remove a small amount of what is ineach vial and add to it substance X. Because Y is unknown, I dependon the rate of substance X's depletion to detect the presence ofthe enzyme. After recording the change in concentration of X(through fluorescence change) per certain amount of time that isfixed regardless of what protein is being tested, I then identifythe vial that has the mix of proteins that has the most enzyme X,and run SDS-page electrophoresis. This provides an idea about thesize of the different proteins. The difference in their sizes isassessed via the column chromatography. Finally, I run just thatvial through the column and retest with substance X. This indicatespurity in the preparation.

my teachers reponse: what you have submitted is a start howeverthis is general protocol. The purpose of the assignment is tomake sure you the student understands the various proteinpurification techniques discussed in class and in the textbook andwrite a general protocol. The M1 assignment is being returnedto you again to refer back to topic section 1.2 within the classand see what is discussed. It specifically states in the commentary%u201CTo isolate a compound completely, the above methods, and manyothers not described, are usually employed sequentially, and thespecific activity is measured after each step. Only the fractioncontaining the highest specific activity is then passed on to thenext step.%u201D It is telling you the reader that all discussedneed to be done at a minimum to purify a protein and more steps areneeded that would be determined by first doing the%u2018general%u2019 steps as described. Also note that as writtenthe protocol submitted started purificaiton with whole cells - thatis not possible. The protein of interest is inside the cell - thefirst step is to get the protein of interest out of the cell, howis that done?