Quiz 12: Activity and Thermal Stability of Gel-Immobilized Peroxidase
In this problem, we are asked why we washing the acrylamide gel several times during the experiment, and how we determine if the wash is complete. The experiment uses horseradish peroxidase, and we want to remove any of this enzyme that is not trapped on the gel. To ensure we've successfully removed all the enzyme, we would analyze the rinse water to ensure it is there.
In this problem, we are asked to design an experiment that uses the peroxidase in this experiment to determine the Michaelis constant ( K M ) and maximum velocity of the reaction ( V max ). We can calculate these values by plotting data for the initial reaction velocity ( v 0 ) versus the concentration of the substrate that gave us the resulting v 0 ( [S] ). From raw date, when we plot v 0 versus v 0 / [S], we will get back a straight line. On this line, the v 0 -intercept will be V max , and the v 0 / [S]-intercept is V max / K M. In this way, we can find both terms from the graph. Using several different concentrations of substrate with constant concentrations of enzyme; measuring different rates of reactions, we can determine the initial velocities (known as v 0 ) of the reactions based on these different substrate concentrations. By graphing the inverse of the initial rates versus the inverse of the substrate concentrations, we can find K m by finding the intercept of the different substrate graphs.
In this problem, we are asked how we determine if the gel is leaking horseradish peroxidase, and how we can change the set-up to prevent this leakage. We wash the acrylamide gel several times during the experiment, to remove any of this enzyme that is not trapped on the gel. To ensure we've successfully removed all the enzyme, we would analyze the rinse water to ensure it is there; if it's there, we have leakage. To stop this leakage, we can change some aspects of the gel, specifically increase the concentration of methylene bisacrylamide (cross-linker).