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Cells produce hydrogen peroxide (H2O2) as a toxic by-product of normal cellular reactions. One molecule of catalase enzyme may work on 40 million molecules of hydrogen peroxide per second!
If you have used hydrogen peroxide to clean a cut, you have seen catalase in action. When hydrogen peroxide comes in contact with the cut, it reacts with the catalase enzyme in the damaged cells to produce oxygen foam. When the catalase reaction is conducted in a test-tube, the oxygen gas bubbles. The height of the foam is an indication of the amount of catalase activity present.
Investigation of catalase activity in plant and animal tissues
- 5 test-tubes
- wax pencil
- millimeter ruler
- 4 blended tissue extracts (may include apple, potato, onion, Baker's yeast, beef steak or/and beef liver)
- distilled water
- hydrogen peroxide (H2O2) solution (3%)
State a hypothesis concerning the relative abilities of the various tissue types to catalyze the conversion of hydrogen peroxide to water and oxygen.
- Label tubes 1 – 5. Use a wax pencil to draw a line 1 cm from the bottom of
each tube. Draw an additional line at the 2 cm mark
- Add the appropriate extract to each tube to the level of the 1 cm line.
- Add H2O2 to the 2 cm line in each tube. Do not contaminate the dropper. If extract comes into contact with the dropper, discard the dropper.
- Measure the height of the foam (in millimeters) and record the data.
- Wash the test-tubes. Rinse well with water and drain.
Prepare a bar graph of catalase activity. Include labels.
- Did the results of the experiment support the hypothesis? Explain.
- Write the reaction. Use molecular formulas. Identify substrate, enzyme and products.
- What type of gas is released in the bubbles?
- What happens to catalase itself in the reaction – is it used up? destroyed? unchanged?
- The experimental variable (independent variable) is the aspect that varies between the experimental groups. What is the experimental variable in this experiment?
- Controlled variables are those conditions that are kept constant between experimental groups. The controlled variables insure that only one experimental variable is tested per experiment. What are 3 controlled variables in this experiment?
- The dependent variable changes in response to the experimental variable. The dependent variable is what is physically measured to assess the experimental outcome. What is the dependent variable in this experiment?
The effect of temperature on the activity of catalase
- 3 clean test-tubes
- hydrogen peroxide solution
- wax pencil
- extract of liver or yeast
- millimeter ruler
- crushed ice
- water bath at 70oC
State a hypothesis concerning the relative action of catalase at various temperatures. Refer to section 6.1
- Label tubes 1- 3. Use a wax pencil to draw a line 1cm from the bottom of each tube. Draw another line at the 2 cm mark.
- Add extract to the 1 cm line. Note that extract type is a controlled variable in this experiment. Do not add hydrogen peroxide until AFTER step 3.
- Incubate the tubes at the following temperatures for 5 minutes
Tube 1 on ice (record the temperature of the ice)
Tube 2 at room temperature (record the temperature)
Tube 3 at 70oC
- After 5 minutes, return the tubes to the lab bench. Add H2O2 (hydrogen peroxide) to the 2 cm line. Use ice-cold H2O2 for the tube on ice.
- Measure the height of the foam on each tube. Record.
- Wash the test-tubes with water and drain to remove the excess water
Prepare a line graph of catalase activity as influenced by temperature.
- Explain the results shown in the line graph. Did the results support the hypothesis?
- Identify the experimental variable in the experiment.
- List 2 controlled variables in the experiment.
- What is the dependent variable in the experiment?
6.2: Catalase - Biology
AP Biology Lab #2 - Enzyme Catalysis
Objectives: To study the action of enzymes, the characteristics of an enzyme-mediated reaction, and determine the rate of enzyme-catalyzed reactions.
Activity A: Observing the Reaction
Purpose: Examine the interaction of catalase and hydrogen peroxide in different environments.
Hypothesis: The unaltered catalase will react quickly with H2O2 , the heated catalase will react much more slowly with its H2O2, and the liver containing catalase will react the most rapidly of all with its H2O2.
Procedure: First, the control was prepared. For this, 10mL of H2O2was placed into a 60 ml cup. Using a transfer pipet, 1 mL of catalase solution was added to the cup. The reaction was then observed and recorded. Next, the experimental sets were prepared. For one, a hot plate was used to bring a beaker of H2O to a boil. Next, a test tube with 3 mL of catalase in it was placed into the boiling water for 5 minutes. After 5 minutes, 1 mL of catalase was taken from the heated sample, and added to a different 60 mL cup containing 10 mL of H2O2. The reaction was observed and recorded. For the second, a 1 cm 2 piece of beef liver was placed in a cup and was macerated with a glass rod. After the liver was well macerated, 10 mL of H2O2 was added to the cup. The reaction was observed and recorded.
Results: After the addition of the unaltered catalase to the hydrogen peroxide, visible signs of a reaction occurring began after about 2 seconds. Bubbling and foaming happened in the cup, suggesting gas was being produced (The O2). The liver also reacted extremely quickly in the presence of H2O2, producing visible signs of reaction immediately upon addition of H2O2. When the heated catalase was added to the H2O2, no reaction appeared to ensue. Even after waiting 2+ minutes, no signs of any reaction occurred.
Conclusion: My hypothesis that the liver would react the most quickly, that the unaltered enzyme would react the second quickest, and that the heated catalase would react the slowest, was correct. With the unaltered catalase as a control, we can see that 1 mL of catalase begins to visibly react about 2 seconds after the addition of H2O2 under normal conditions and in normal concentrations. The liver piece reacted the most quickly because the livers of organisms have some of the highest concentrations of catalase, and since there was more catalase present in the liver than in the control group, the H2O2 decomposed more quickly. The heated catalase did not react at all, but this was because the change in temperature denatured the protein, and when a protein become denatured, it become inert.
Activities B: Determining Baseline Amount of H2O2 In Solution
Purpose: Determine the initial amount of H2O2 present in solution (Consisting of H2O2, H2SO4, and distilled water). The data collected here will be used as a baseline for later activities.
Procedure: First, 10 mL of H2O2 was placed in a 60 mL cup. Next, 1 mL of distilled water was added, along with 10 mL of H2SO4. The contents were gently swirled to ensure mixing. After the contents were mixed, 5 mL of the solution were transferred to another 60 mL cup. The 5 mL sample was then titrated with KMnO4. The titration protocol was as follows:
Acquire a titration syringe. Draw in about 0.2 mL of air, and then fill with KMnO4. Record the initial volume. Next, proceed to add one drop of the KMnO4 at a time, swirling the solution gently until the color from the drop disappears. Repeat the last step until the solution becomes a persistent pink or brown. That is the endpoint of the titration. Once you have reached the endpoint, record the final volume of KMnO4.