Peroxidase Lab Report PDF

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Alexis Rodriguez Bio 101 Lab Write Up Different Factors That Affect the Rate of Reaction of Peroxidase Abstract: In this lab we have conducted five different experiments, testing the effects of different factors to an enzyme called Peroxidase. First, we tested how the amount of peroxidase would affect the rate of reaction. Then, we tested how temperature, pH, use of boiled enzyme, and presence of an inhibitor (hydroxylamine) would affect the rate of the reaction of the peroxidase. Our hypothesis for the first experiment: The amount of enzyme added to the reaction will increase the rate of reaction. The second experiment: As the temperature increases, the rate of reaction will decrease. The third experiment: As the pH increases, the rate of reaction will decrease. The fourth experiment: If we boil the peroxidase enzyme, the rate of reaction will decrease. Lastly, the fifth experiment: The presence of an inhibitor, hydroxylamine, will decrease the rate of reaction. The results show that these factors have a major affect on the peroxidase. Adding more peroxidase increased the rate of reaction, and the peroxidase at 30.2*C reacted the quickest out of all the other temperatures. As we increased the pH the rate of the reaction increased and when adding an enzyme inhibitor (hydroxylamine) there was little to no reaction.

Introduction: According to Dolphin, Warren (2015) Determining the properties of an enzyme, Biological Investigations: Lab Topic 6 pp. 69-78 enzymes are large proteins that catalyze chemical reactions. Temperature can affect enzyme activity because if the temperature rises , the proteins molecular structure will become more unstable until it denatures and breaks apart. Along with pH, if the environment is too acidic or too basic the enzyme will denature. Enzyme inhibitors are molecules that interact in some way with the enzyme to prevent it from working in the normal manner. Our hypothesis for the first experiment: The amount of enzyme added to the reaction will increase the rate of reaction. The second experiment: As the temperature increases, the rate of reaction will decrease. The third experiment: As the pH increases, the rate of reaction will decrease. The fourth experiment: If we boil the peroxidase enzyme, the rate of reaction will decrease. Lastly, the fifth experiment: The presence of an inhibitor, hydroxylamine, will decrease the rate of reaction. Methods: In order to test our first hypothesis, we labeled 7 tubes 1-7. Next we filled each tube with the exact quantities listed on table 6.1. After each tube has the exact amount, we start by mixing tubes 2 and 3 to start the reaction. Then, we place the reaction quickly into the spectrophotometer set at 470nm and recorded the absorbance in 20 second intervals for 2 minutes. We repeat these steps for tubes 4 and 5, and tubes

5and6. In order to test our second hypothesis, we labeled 9 tubes 1-9. We looked at table 6.3 to get the exact amounts of buffer (ph5), H2O2, peroxidase, and guaiacol in each tube. We placed tubes 2 &3 in an ice bath at 7*C, tubes 4&5 at room temperature (22*C), tubes 6&7 in a heated water bath at 30.2*C, and tubes 8&9 in a heated water bath at 47.2*C for 15 minutes. After the 15 minutes, we took each pair of tubes and mixed them together to start the reaction. Placing the reaction quickly into the spectrophotometer set at 470nm and recording the absorbance in 20 second intervals for 2 minutes. These steps will be repeated for tubes 4&5, 6&7, and 8&9. In order to test our third hypothesis, we labeled 9 tubes 1-9. We looked at table 6.5 to get the exact amounts of buffer (either pH 3, pH 5, pH 7, pH 9), H2O2, peroxidase and guaiacol. Then, we mixed tubes 2&3, put the reaction quickly into the spectrophotometer at 470 nm the recorded the absorbance in 20 second intervals for 2 minutes. These steps were repeated for tubes 4&5, 6&7, and 8&9. In order to test out fourth hypothesis, we added 3 ml of extract to a test tube and place into a boiling water bath for 10 minutes. During the 10 minutes we labeled 5 tubes 1-5 and looked at table 6.7 to add the reagents as called for. After, we remove the extract and let it cool to room temperature. We mix tubes 2&3, put the reaction in the spectrophotometer at 470nm and recorded its absorbance in 20 second intervals for 2 minutes. Then, repeat these steps for tubes 4&5. Results:

In the first experiment, graph 6.1 shows that a greater amount of peroxidase results in a quicker absorbance. In the second experiment, different temperatures had different results to the activity of the peroxidase. According to graph 6.3, the reaction at 30.2*C had the quickest absorbance while the boiled extract had the slowest absorbance causing the denaturing of the enzyme. In the third experiment, the pH of 3 had the slowest reaction but as we increased the pH the absorbance time increased. pH of 5 had the quickest absorbance time while the pH of 7 and 9 were a little slower. Finally, the last experiment resulted in little to no activity of the hydroxylamine treated extract shown in graph 6.9. Results: Graph 6.1

Graph 6.3

Graph 6.5

Graph 6.9

Discussion: All of the hypotheses were supported. Temperature affected the enzyme activity because the increase in temperature , caused the molecular structure to become unstable and denature. The increase in pH, caused the enzyme to denature. The enzyme inhibitor, hydroxylamine, prevented the enzyme from working properly thus slowing down the reaction.

Literature Cited Dolphin, Warren (2015) Determining the properties of an enzyme, Biological Investigations: Lab Topic 6 pp. 69-78...