Cellular Metabolism Lab Report

The purpose of the cellular metabolism lab is to determine the effects of inhibitors and other variables on the rate of metabolism in the Krebs Cycle. Inhibitors compete with the substrate to bind to the enzyme, thus slowing down the production of FADH2. When the production of FADH2 is slowed down, the reaction will proceed too slowly for metabolic purposes. In this lab, the enzyme that was catalyzing the reaction was the bean extract. To mimic the FAD that is used in the Krebs cycle, we used 2,6-dichlorophenolindophenol (DPIP). When DPIP is chemically reduced (like the FAD would be), it changes color from blue to clear. The carrier was the phosphate buffer. The inhibitor that was used to test/slow the reaction was Malonate and the substrate used was succinate. In order to view the rate of metabolism that was occurring, we used a spectrophotometer to measure the color absorbance. When DPIP is reduced during the Krebs cycle, the color changes from blue to clear. The …show more content…
We first set up a blank to zero out the spectrophotometer. In tubes 1-4 we added 200 ul of DPIP and 200 ul of the bean extract enzyme, but in tube two, the bean extract was boiled. We then added the appropriated amount of phosphate buffer to the tubes. 200 ul of the inhibitor, malonate, was added to tube three and four. 66 ul of the substrate, succinate, was added to test tube one, two, and three but 200 ul of succinate was added to tube four. Tube one was our positive control. The enzyme was fully efficient and there was no inhibitor. It is expected that tube one will be catalyzed the quickest. Tube two was our negative control. We needed to observe what would occur in the spectrophotometer if the enzyme couldn’t catalyze the reaction and so we added a denatured enzyme to view the effects of no reaction. I expect that the reaction will occur the quickest for tube one, the slowest for tube two, and I expect that tube four will be quicker than tube