You are trying to purify the enzyme Rubisco. The SDS- PAGE gel of the different
ID: 102371 • Letter: Y
Question
You are trying to purify the enzyme Rubisco. The SDS- PAGE gel of the different purification steps are shown. Exactly 25 mu g of TOTAL protein was loaded into each lane. Based on this, answer the following questions: a. What is the reaction catalyzed by this enzyme? b. Describe an assay to test whether a particular purification step contains the active enzyme? c. I use 25 mu g of TOTAL protein from each purification step to generate Michaelis-Menten curves for Rubisco. Will all steps show the same V_max? If you do not expect all the steps to show the same V_max, which one do you expect will show the highest V_max? Give reasons for your answer. d. I use 25 mu g of TOTAL protein from each purification step to generate Michaelis-Menten curves for Rubisco. Will all steps show the same K_m? If you do not expect all the steps to show the same K_m, which one do you expect will show the highest K_m? Give reasons for your answer. e. I perform a Western blot on the SDS-PAGE gel shown, using an antibody that specifically binds Rubisco. How many bands would I expect to see in each lane? f. Based on the gel, what do you predict is the approximate molecular weight of Rubisco? g. If Rubisco were made up of two different proteins of identical size, how many bands would I expect to see with purified Rubisco in an SDS-PAGE? In a Western blot? h. Assume Rubisco is made up of two different proteins of identical size, and that I somehow managed to separate these two proteins. What would the specific activity of each of the purified proteins be? What would the specific activity be if I mixed the proteins again?Explanation / Answer
a) The enzyme rubisco is ribulose bisphosphate carboxylase. The reaction catalysed by the enzyme is carboxylase or oxygenase reactions.
b) The type of assay that can be used to determine the presence of active enzyme is ELISA (enzyme linked immuno sorbent assay).
c) Vmax is the maximum rate of enzymatic reaction. The highest Vmax would be expected to occur in the affinity purification step. This is because maximum concentration of enzyme is present in this purification step.
d) Km value of an enzyme represents the substrate concentration at half maximal velocity. Small Km value indicates that the enzyme requires only a small amount of substrate to become saturated. Higher Km value implies that larger substrate concentration is needed. Therefore, the enzyme purified by affinity method has the highest Km value.
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