Lab 1 Enzymatic Analysis (1)

Lab 1 Enzymatic Analysis (1) - Enzymatic Analysis Zachary...

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Enzymatic Analysis Zachary Bergman Partner: Anuj Shah 1/30/09 Biology 210-2
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Introduction The goal of this lab was to evaluate the affect of different factors on the function of the enzyme isocitrate dehygrogenase (IDH). The different factors that were individually tested were the affect of temperature, pH, cofactor requirement, a competative inhibitor, and varying the concentration of both the enzyme and the substrate. These factors were chosen for testing to determine the optimum conditions required for this specific enzyme to properly function while varying those conditions which could change in a biological system. IDH is a coenzyme (NAPD + ) and cofactor dependent (Mg 2+ ) which allows us to easily measure it's rate of reaction, and therefore the affect of changed condition, with NAPD + using a spectrophotometer. Materials and Methods The materials that were used was the enzyme isocitrate dehygrogenase (IDH), the substrate isocitrate (Iso), an assay buffer with ions or an assay buffer without ions depending on what is being tested, and the coenzyme NAPD + . The assay buffer is what was varied when testing different conditions. The rate and success of the reaction was evaluated using a spectrophotometer which measured the amount of light that was able to pass through the sample at the wavelength 340 nm. The less amount of light that is able to pass through, the farther along the reaction has proceeded. To ensure accurate readings, we blanked the instrument between each experiment. In each reaction the NAPD + was added last because as the coenzyme, it facilitates the IDH binding to the substrate isocitrate and this is the first step in the reaction. The first set of reactions we ran were controls which were used to determine which of the
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This note was uploaded on 02/16/2012 for the course BIOL_SCI 210-2 taught by Professor Somebody during the Winter '09 term at Northwestern.

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Lab 1 Enzymatic Analysis (1) - Enzymatic Analysis Zachary...

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