Investigating Catalase Activity Introduction: Proteins are important organic molecules that serve many purposes. Proteins are made up of chains of amino acids, and the sequence of amino acids helps determine the shape and function of the protein. Proteins have a predictable three-dimensional shape, however if the amino acid chain is disrupted it changes the shape and function of the protein. Denaturation due to high heat or wrong pH environment changes the quaternary and tertiary structure of the protein, and leads to loss of function. Almost all enzymes are proteins; they are organic catalysts that help to increase the rate of a chemical reaction, however they do not cause the reaction and are not consumed by the reaction. Billions of chemical reactions catalyzed by enzymes happen at any given second in a living cell. These enzymes have an optimal pH and temperature that they work the best at, and it is usually the temperature and pH of their natural environment. As mentioned before, most enzymes are proteins and therefore their three-dimensional shape is important. If an enzyme changes shapes it changes its function and it cause it to not catalyze its reaction. One enzyme that is present in liver and potato (Huang and Beevers, 1971) is catalase, which breaks down hydrogen peroxide formed in the lysosomes into molecular oxygen and water. In a test tube, formation of oxygen bubbles can be monitored. Though a sample of liver or potato contains many, many enzymes, it is possible to test the activity of a single enzyme in cellular extracts by providing excess substrate, and monitoring the production of a visible result of the reaction. Potatoes are inexpensive, plentiful, and high in catalase. Today we will use an extract of potato along with hydrogen peroxide to measure catalase activity. In Part A, you will explore experimental design and learn the importance of setting up negative control tubes. You will see that adding more enzyme or more substrate can increase the rate of a reaction. You will take readings at various times as the reaction progresses. In Part B, you will explore the effect of pH on catalase activity. In Part C, you will explore the effect of temperature on catalase activity. 1. What is your hypothesis? Will more potato extract lead to a faster reaction? Will more hydrogen peroxide lead to a faster reaction? Will only potato extract, or only hydrogen peroxide, show a reaction at all? 2. What do you expect to be the optimal pH for potato catalase activity? Write a hypothesis and a prediction. 3. What do you expect to be the optimal temperature for potato catalase activity? Write a hypothesis and a prediction. Materials: test tubes Enzyme extract from potato tubers, buffered at pH 7 water bath at 30° C thermometer pH Buffer 4 pH Buffer 7 pipets 3% Hydrogen peroxide small metric ruler Distilled water water bath at 40° C pH paper pH Buffer 5 pH Buffer 8 water bath at 50° C pH Buffer 3 pH Buffer 6 Part A: Experimental Design Methods, part A: Obtain 7 tubes and label them. Add 4 ml of pH buffer 7 to each of tubes 1 and 2. Add 3 ml of pH buffer 7 to tube 3. Add 2 ml of pH buffer 7 to each of tubes 4 and 6. Add 1 ml of pH buffer 7 to each of tubes 5 and 7. Add 1 ml of potato extract to each of tubes 1, 3, 6, and 7. Add 2 ml of potato extract to tube 4. Add 3 ml of potato extract to tube 5. Measure carefully. Mix well. Prepare a set of tubes labeled 3H, 4H, 5H, 6H, and 7H. Add 1 ml of hydrogen peroxide to each of tubes 2, 3H, 4H, and 5H. Add 2 ml of hydrogen peroxide to tube 6H. Add 3 ml of hydrogen peroxide to tube 7H. Locate a stopwatch or other timekeeping device and be sure everyone in your group is ready to move on. Once you potato extract and hydrogen peroxide come into contact, the reaction begins immediately. Once you are all ready, QUICKLY pour the contents of tube 3H into tube 3, the contents of tube 4H into tube 4, the contents of tube 5H into tube 5, the contents of tube 6H into tube 6, and the contents of tube 7H into tube 7. Mix well, then leave the tubes undisturbed at room temperature and wait 2 minutes. After 2 minutes, measure the height of the bubble column in each tube. After 5 minutes total, measure the height of the bubble column in each tube. After 10 minutes total, measure the height of the bubble column in each tube. Results: Table 1 Tube contents of tube # 1 2 3 4 5 6 7 height of bubble column after 2 minutes (mm) height of bubble column after 5 minutes (mm) height of bubble column after 10 minutes (mm) 1ml potato extract + 4ml buffer 7 4ml buffer 7 + 1ml H2O2 1ml potato + 1ml H2O2 + 3ml buffer 2 ml potato + 1 ml H2O2 + 2 ml buffer 3 ml potato + 1 ml H2O2 + 1 ml buffer 1ml potato + 2ml H2O2 + 2ml buffer 1ml potato + 3ml H2O2 + 1ml buffer Discussion: 1. What is the purpose of setting up a negative control tube that you know will not have a reaction? 2. What is the purpose of having two negative control tubes in this experiment? 3. What should you do if your negative control tube does show a reaction? 4. How much potato extract is optimal (i.e. shows good reaction, but does not make a mess spilling out of the tube)? 5. How much hydrogen peroxide is optimal (i.e. shows good reaction, but does not make a mess spilling out of the tube)? 6. The hydrogen peroxide is always added last, and needs to be added to all tubes quickly. Why is that important? 7. Does the speed of bubble formation change over the course of the 10 minute experiment? Explain. 8. Why should each tube contain the same amount of liquid? Part B: The effect of varying pH Methods, part B: Obtain 7 tubes and label them. Add 2 ml of the indicated pH buffer to each tube. Add 1 ml of potato extract to each tube and wait 5 minutes. Obtain pH paper and determine the pH of each of the tubes and record in Table 2. Quickly add 4 ml of hydrogen peroxide to each tube with the exception of your negative control tube, to which you should add water. This will start the reaction. You may want to use a second set of tubes to measure and hold hydrogen peroxide until you are ready for the reaction to start, as you did in part A. Leave the tubes undisturbed at room temperature and wait 5 minutes. After 5 minutes, measure the height of the bubble column in each tube. Results: Table 2 Tube # pH buffer added 3 4 5 6 7 8 Negative control pH height of bubble column after 5 minutes Figure 2 Discussion: 1. What should you do if your negative control tube does show a reaction? 2. Why should each tube contain the same amount of liquid? 3. What appears to be the optimal pH for catalase? 4. Does the data support your hypothesis? 5. If you design a new experiment, which pH will you test? Part C: The effect of varying temperature Methods, part C: Obtain 6 tubes and label them. Add 2 ml of pH 7 buffer to each of your tubes. If you wish to use a different pH, please record what you do. Add 1 ml of potato extract to each tube. Set up a set of tubes 1H, 2H, etc. and add 4 ml of hydrogen peroxide to each of these tubes with the exception of your negative control tube. Move all tubes to the indicated temperatures and wait 5 minutes so the liquid in the tube reaches the temperature of the environment. Quickly combine hydrogen peroxide with potato extract by pouring contents of tube 1H into tube 1, and so on. This will start the reaction. Leave the tubes undisturbed in the various temperature environments you are testing and wait 5 minutes. After 5 minutes, measure the height of the bubble column in each tube. Results: Table 3 Tube # temperature height of bubble column after 5 minutes (mm) refrigerator (____ ° C) room (____ ° C) 30° C 40° C 50° C Negative control Figure 3 Discussion: 1. What should you do if your negative control tube does show a reaction? 2. What appears to be the optimal temperature for catalase? 3. Why would this be the optimal temperature for potato catalase activity? 4. Does the data support your hypothesis? 5. If you design a new experiment, which temperature will you test next? 6. What other modifications might you make if you were to design your own experiment to test catalase? References: Huang, A. H., & Beevers, H. (1971). Isolation of microbodies from plant tissues. Plant Physiology, 48(5), 637-641. Mader, S.S. Laboratory Manual to accompany Biology, Twelfth Edition. McGraw Hill Education, 2016. Print. Morgan and Carter. Investigating Biology Lab Manual, Fifth Edition. Pearson Education, Inc., 2005. Print. Richardson and Richardson. A Laboratory Guide to the Natural World, Second Edition. Prentice Hall, Inc, 2005. Print Williams, J. (1928). THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE. The Journal Of General Physiology, 11(4), 309-337.