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Experiment
6A
Biology with Calculators 6A - 1
Enzyme Action:
Testing Catalase Activity
Many organisms can decompose hydrogen peroxide (H 2 O2) enzymatically. Enzymes are globular proteins, responsible for most of the chemical activities of living organisms. They act as catalysts, substances that speed up chemical reactions without being destroyed or altered during the process. Enzymes are extremely efficient and may be used over and over again. One enzyme may catalyze thousands of reactions every second. Both the temperature and the pH at which enzymes function are extremely important. Most organisms have a preferred temperature range in which they survive, and their enzymes most likely function best within that temperature range. If the environment of the enzyme is too acidic, or too basic, the enzyme may irreversibly denature , or unravel, until it no longer has the shape necessary for proper functioning.
H 2 O 2 is toxic to most living organisms. Many organisms are capable of enzymatically destroying the H2O 2 before it can do much damage. H 2 O 2 can be converted to oxygen and water, as follows:
2 H2O 2 ← → 2 H2O + O 2
Although this reaction occurs spontaneously, enzymes increase the rate considerably. At least two different enzymes are known to catalyze this reaction: catalase, found in animals and protists, and peroxidase , found in plants. A great deal can be learned about enzymes by studying the rates of enzyme-catalyzed reactions. The rate of a chemical reaction may be studied in a number of ways including:
- measuring the rate of appearance of a product (in this case, O 2 , which is given off as a gas)
- measuring the rate of disappearance of substrate (in this case, H2O2)
- measuring the pressure of the product as it appears (in this case, O2).
In this experiment, you will measure the rate of enzyme activity under various conditions, such as different enzyme concentrations, pH values, and temperatures. It is possible to measure the concentration of oxygen gas formed as H2O 2 is destroyed using an O 2 Gas Sensor. If a plot is made, it may appear similar to the graph shown.
At the start of the reaction, there is no product, and the concentration is the same as the atmosphere. After a short time, oxygen accumulates at a rather constant rate. The slope of the curve at this initial time is constant and is called the initial rate. As the peroxide is destroyed, less of it is available to react and the O 2 is produced at lower rates. When no more peroxide is left, O 2 is no longer produced.
OBJECTIVES
In this experiment, you will
- use an Oxygen Gas Sensor to measure the production of oxygen gas as hydrogen peroxide is destroyed by the enzyme catalase or peroxidase at various enzyme concentrations.
- measure and compare the initial rates of reaction for this enzyme when different concentrations of enzyme react with H2O2.
Experiment 6A
6A - 2 (^) Biology with Calculators
- measure the production of oxygen gas as hydrogen peroxide is destroyed by the enzyme catalase or peroxidase at various temperatures.
- measure and compare the initial rates of reaction for the enzyme at each temperature.
- measure the production of oxygen gas as hydrogen peroxide is destroyed by the enzyme catalase or peroxidase at various pH values.
- measure and compare the initial rates of reaction for the enzyme at each pH value.
Figure 1
MATERIALS
LabPro or CBL 2 interface enzyme suspension TI Graphing Calculator three 18 X 150 mm test tubes DataMate program ice Vernier O 2 Gas Sensor pH buffers 400-mL beaker test tube rack 10-mL graduated cylinder thermometer 250-mL Nalgene bottle three dropper pipettes 1.5% H 2 O 2 Graphical Analysis (optional) 3.0% H 2 O 2
PROCEDURE
- Obtain and wear goggles.
- Plug the O 2 Gas Sensor into Channel 1 of the LabPro or CBL 2 interface. Use the link cable to connect the TI Graphing Calculator to the interface. Firmly press in the cable ends.
- Turn on the calculator and start the DATAMATE program. Press CLEAR^ to reset the program.
Experiment 6A
6A - 4 Biology with Calculators
f. Press ENTER^ to view a graph of the data and the regression line. g. Press ENTER^ to return to the ANALYZE menu. h. Select RETURN TO MAIN SCREEN from the ANALYZE menu.
- Store the data from the first run so that it can be used later.
a. Select TOOLS from the main screen. b. Select STORE LATEST RUN from the TOOLS MENU.
- Find the rate of enzyme activity for test tubes 2, and 3:
a. Add 10 drops of the enzyme solution to test tube 2. Repeat Steps 7 – 11. b. Add 20 drops of the enzyme solution to test tube 3. Repeat Steps 7 – 10.
- Graph all three runs of data on a single graph. To do this:
a. Select GRAPH from the main screen, then press ENTER^. b. Select MORE, then select L2, L3 AND L4 VS L1 from the MORE GRAPHS menu. c. All three runs should now be displayed on the same graph. Each point of the 5-drop run is plotted with a cross, each point of the 10-drop run is plotted with a box, and each point of the 20-drop run is plotted with a dot. Use the displayed graph and the data in Table 2 to answer the questions for Part I. d. When finished with the graph, press ENTER^ to exit. e. Select RETURN TO GRAPHS SCREEN from the MORE GRAPHS menu. f. Select MAIN SCREEN from the graph screen.
Part II Testing the Effect of Temperature Your teacher will assign a temperature range for your lab group to test. Depending on your assigned temperature range, set up your water bath as described below. Place a thermometer in your water bath to assist in maintaining the proper temperature.
- 0 – 5°C: 400-mL beaker filled with ice and water.
- 20 – 25°C: No water bath needed to maintain room temperature.
- 30 – 35°C: 400-mL beaker filled very warm water.
- 50 – 55°C: 400-mL beaker filled hot water.
- Rinse the three numbered test tubes used for Part I. Fill each test tube with 10 mL of 1.5% H2O 2 and then place the test tubes in the water bath. The test tubes should be in the water bath for 5 minutes before proceeding to Step 15. Record the temperature of the water bath, as indicated on the thermometer, in the space provided in Table 3.
- Find the rate of enzyme activity for test tubes 1, 2, and 3:
a. Add 10 drops of the enzyme solution to test tube 1. Repeat Steps 7 – 10. Record the reaction rate in Table 3. b. Add 10 drops of the enzyme solution to test tube 2. Repeat Steps 7 – 10. Record the reaction rate in Table 3. c. Add 10 drops of the enzyme solution to test tube 3. Repeat Steps 7 – 10. Record the reaction rate in Table 3.
- Calculate the average rate for the three trials you tested. Record the average in Table 3.
Enzyme Action: Testing Catalase Activity
Biology with Calculators 6A - 5
- Record the average rate and the temperature of your water bath from Table 3 on the class chalkboard. When the entire class has reported their data on the chalkboard, record the class data in Table 4.
Part III Testing the Effect of pH
- Place three clean test tubes in a rack and label them pH 4, pH 7, and pH 10.
- Add 5 mL of 3% H2O 2 and 5 mL of a pH buffer to each test tube, as in Table 1.
Table 1 pH of buffer Volume of 3% H 2 O 2 (mL) Volume of buffer (mL) pH 4 5 5 pH 7 5 5 pH 10 5 5
- Using the test tube labeled pH 4, add 10 drops of enzyme solution and repeat Steps 7 – 11.
- Using the test tube labeled pH 7, add 10 drops of enzyme solution and repeat Steps 7 – 11.
- Using the test tube labeled pH 10, add 10 drops of enzyme solution and repeat Steps 7 – 10.
- Graph all three runs of data on a single graph. To do this:
a. Select GRAPH from the main screen, then press ENTER. b. Select MORE, then select L2, L3 AND L4 VS L1 from the MORE GRAPHS menu. c. All three runs should now be displayed on the same graph. Use the displayed graph and the data in Table 5 to answer the questions for Part III. d. When finished with the graph, press ENTER to exit. e. Select RETURN TO GRAPHS SCREEN from the MORE GRAPHS menu. f. Select MAIN SCREEN from the graph screen.
DATA
Part I Effect of Enzyme Concentration
Table 2 Test tube label Slope, or rate (%/s) 5 Drops 10 Drops 20 Drops
Enzyme Action: Testing Catalase Activity
Biology with Calculators 6A - 7
- Why might the enzyme activity decrease at very high temperatures?
Part III Effect of pH
- At what pH is the rate of enzyme activity the highest? Lowest?
- How does changing the pH affect the rate of enzyme activity?
EXTENSIONS
- Repeat Step 12a to collect data with 10 drops of enzyme suspension. Using the Graphical Analysis computer software, import your collected data into a computer. In Graphical Analysis, use the mouse to select each of the time intervals from Table 6—calculate the rate using the Regression function found in the Analyze menu.
Table 6 Time intervals (Minutes)
Rates 0-30 s 30-60 s 60-90 s 90-120 s 120-180 s
10 Drops
Questions When is the reaction rate highest? Explain why. When is the reaction rate lowest? Why?
- Different organisms often live in very different habitats. Design a series of experiments to investigate how different types of organisms might affect the rate of enzyme activity. Consider testing a plant, an animal, and a protist.
- Presumably, at higher concentrations of H 2 O2, there is a greater chance that an enzyme molecule might collide with H2O2. If so, the concentration of H2O 2 might alter the rate of oxygen production. Design a series of experiments to investigate how differing concentrations of the substrate hydrogen peroxide might affect the rate of enzyme activity.
- Design an experiment to determine the effect of boiling the catalase on the rate of reaction.
- Explain how environmental factors affect the rate of enzyme-catalyzed reactions.
Experiment 6A
6A - 2 T (^) Biology with Calculators
SAMPLE RESULTS
Sample class data
Test tube label Slope, or rate (%/s)
5 Drops 0.
10 Drops 0.
20 Drops 0.
0 – 5 °C range: 4°C 0.
20 – 25 °C range: 21 °C 0.
30 – 35 °C range: 34 °C 0.
50 – 55 °C range: 51 °C 0.
pH 4 0.
pH 7 0.
pH 10 0.
The effect of H (^) 2O 2 concentration on the rate of enzyme activity
The effect of pH on the rate of enzyme activity
The effect of temperature on the rate of enzyme activity
Teacher Information Enzyme Action: Testing Catalase Activity
Biology with Calculators 6A - 3 T
ANSWERS TO QUESTIONS
- The rate should be highest when the concentration of enzyme is highest. With higher concentration of enzyme, there is a greater chance of an effective collision between the enzyme and H2O 2 molecule.
- Roughly, the rate doubles when the concentration of enzyme doubles. Since the data are somewhat linear, the rate is proportional to the concentration. At a concentration of 30 drops, the rate in the above experiment should be about 0.041 %/s.
- The temperature at which the rate of enzyme activity is the highest should be close to 30°C. The lowest rate of enzyme activity should be at 60°C.
- The rate increases as the temperature increases, until the temperature reaches about 50°C. Above this temperature, the rate decreases.
- At high temperatures, enzymes lose activity as they are denatured.
- Student answers may vary. Activity is usually highest at pH 10 and lowest at pH 4.
- Student answers may vary. Usually, the enzyme activity increases from pH 4 to 10. At low pH values, the protein may denature or change its structure. This may affect the enzyme’s ability to recognize a substrate or it may alter its polarity within a cell.
ANSWERS TO EXTENSION 1
- Student answers vary. Ideal data would have the rate being the highest during the first (and maybe second) interval. This is because there are a large number of substrate molecules in comparison to the number of enzyme molecules and there will be a maximum number of collisions between the enzyme and the substrate
- Student answers vary. Ideal data would have the rate being lowest (the rate would be zero or would approach zero) during the last intervals. As the number of substrate molecules decreases and the number of product molecules increases, the number of collisions between the enzyme and the substrate decreases.
