enzyme action: testing catalase activity · lab #803 enzyme action: testing catalase lq by vernier...

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Lab #803 Enzyme Action: Testing Catalase LQ by Vernier Software and Technology,

Gettysburg College, Gettysburg, PA 17325. www.advancingscience.org A Science in Motion Program

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Enzyme Action: Testing Catalase Activity

Pennsylvania Science Standards: S11.A.1.1.4 S11.A.1.3.1 S11.A.2.2.2.1 S11.A.2.2.2.2 Keystone Eligible Content Bio.B.4.1.1, Bio.B.4.1.2, and Bio.B.4.2.5

Introduction and Background Many organisms can decompose hydrogen peroxide (H2O2) enzymatically. Enzymes are globular proteins, responsible for most of the chemical activities of living organisms. They act as catalysts, as 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.

H2O2 is toxic to most living organisms. Many organisms are capable of enzymatically destroying the H2O2 before it can do much damage. H2O2 can be converted to oxygen and water, as follows:

2 H2O2 → 2 H2O + O2

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 pressure of the product as it appears (in this case, O2) measuring the rate of disappearance of substrate (in this case, H2O2) measuring the rate of appearance of a product (in this case, O2 which is given off as a gas)

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 pressure of oxygen gas formed as H2O2 is destroyed. 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 pressure is the same as the atmospheric pressure. 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 O2 is produced at lower rates. When no more peroxide is left, O2 is no longer produced.

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Guiding Questions: How do changes in concentration, pH, and temperature affect the ability of enzymes to function?

Vocabulary: enzyme, catalase, denature, catalyst

Safety: Wear safety goggles Be cautious of over-pressurized test tubes. Be cautious of hot water.

Materials Needed (per group):

LabQuest pH group only: Vernier Gas Pressure Sensor pH buffers (4, 7, 10) pH group only 1-hole rubber stopper assembly Temperature group only: 10 mL graduated cylinder ice 250 mL beaker of tap water thermometer 3% H2O2 (about 12mL per group) Ring stand with clamp enzyme suspension in small beaker (see teacher notes)

600 ml beaker

four 18 X 150 mm test tubes Test tube rack Dropper pipette

1 2 3 4

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Procedure (Part 1 - Testing the Effect of Enzyme Concentration):

1. Obtain and wear goggles.

2. Connect the plastic tubing to the valve on the Gas Pressure Sensor.

3. Connect the Gas Pressure Sensor to LabQuest and choose New from the File menu. If you have an older sensor that does not auto-ID, manually set up the sensor (“sensor” menu → “sensor setup”)

4. On the Meter screen, tap Rate. Change the data-collection rate to 0.5 samples/second and the data-collection length to 180 seconds.

5. Place four test tubes in a rack and label them 1, 2, 3, and 4.

6. Add 3 mL of 3.0% H2O2 and 3 mL of water to each test tube as shown in Table 1.

Table 1

Test tube label

Volume of 3% H2O2 (ml)

Volume of water (ml)

1 3 3

2 3 3

3 3 3

4 3 3

7. Use a clean dropper pipette to add 1 drop of enzyme suspension to test tube 1. Note: Be sure not to let the enzyme fall against the side of the test tube.

8. Insert the 1-hole stopper assembly into the test tube. Note: Firmly twist the stopper for an airtight fit. Gently swirl the test tube to thoroughly mix the contents. The reaction should begin. The next step should be completed as rapidly as possible.

9. Connect the free-end of the plastic tubing to the connector in the rubber stopper as shown in Figure 2.

10. Start data collection by tapping the green play button in the lower left corner. Data collection will end after 3 minutes.

11. Monitor the pressure readings displayed on the handheld screen. If the pressure exceeds 130 kPa, the pressure inside the tube will be too great and the rubber stopper is likely to pop off. Disconnect the plastic tubing from the Gas Pressure Sensor if the pressure exceeds 130 kPa.

12. When data collection has finished, an auto-scaled graph of pressure vs. time will be displayed. Disconnect the plastic tubing connector from the rubber stopper. Remove the rubber stopper from the test tube and discard the contents in a waste beaker.

13. To examine the data pairs on the displayed graph, select any data point. As you tap on each data point, the pressure and time values are displayed to the right of the graph.

Figure 2 Figure 2

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14. Determine the rate of enzyme activity for the curve of pressure vs. time. To help make comparisons between experimental runs, choose your data points at the same time value, ideally the point where the data values begin to increase and the point where the pressure values no longer increase.

a. Choose Curve Fit from the Analyze menu (found on the graph screen). b. Select Linear for the Fit Equation. The linear-regression statistics for these two data columns

are displayed for the equation in the form

y = mx + b

c. Enter the absolute value of the slope, m, as the reaction rate in Table 4. d. Select OK.

15. Store the data from the first run by tapping the File Cabinet icon.

16. Find the rate of enzyme activity for test tubes 2, 3 and 4:

a. Add 2 drops of the enzyme solution to test tube 2. Repeat Steps 8–15. b. Add 3 drops of the enzyme solution to test tube 3. Repeat Steps 8–15. c. Add 4 drops of the enzyme solution to test tube 4. Repeat Steps 8–15.

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Procedure (Part 2 - Testing the Effect of Temperature):

1. Obtain and wear goggles.



On the Meter screen, tap Rate. Change the data-collection rate to 0.5 samples/second and the data-collection length to 180 seconds.

4. Place four clean test tubes in a rack and label them T 0 – 5, T 20 – 25, T 30 – 35, and T 50 – 55.

5. Add 3 ml of 3% H2O2 and 3 ml of water to each test tube, as shown in Table 2.

Table 2

Test tube label Volume of 3% H2O2 (ml)

Volume of water

T 0 – 5°C 3 3

T 20 – 25°C (room temp)

3 3

T 30 – 35°C 3 3

T 50 – 55°C 3 3

7. Measure the enzyme activity at 0 – 5°C :

a. Prepare a water bath at a temperature in the range of 0 – 5°C by placing ice and water in a 600 ml beaker. Check that the temperature remains in this range throughout this test.

b. Place Test Tube T 0 – 5°C in the cold water bath until the temperature of the mixture reaches a temperature in the 0 – 5°C range. Record the actual temperature of the test-tube contents in the blank in Table 4.

c. Add 2 drops of the enzyme solution to Test Tube T 0 – 5°C.





Figure 2

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12. When data collection has finished, an auto-scaled graph of pressure vs. time will be displayed. Disconnect the plastic tubing connector from the rubber stopper. Remove the

rubber stopper from the test tube and discard the contents in a waste beaker.



a. Choose Curve Fit from the Analyze menu (found on the graph screen). b. Select Linear for the Fit Equation. The linear-regression statistics for these two data columns

are displayed for the equation in the form

y = mx + b

c. Enter the absolute value of the slope, m, as the reaction rate in Table 4. d. Select OK.


16. Measure the enzyme activity at 30 – 35°C:

a. Prepare a water bath at a temperature in the range of 30 – 35°C by placing warm water in a 600 ml beaker. Check that the temperature remains in this range throughout this test.

b. Place Test Tube T 30 – 35°C in the warm water bath until the temperature of the mixture reaches a temperature in the 30 – 35°C range. Record the actual temperature of the test-tube contents in the blank in Table 4.

c. Add 2 drops of the enzyme solution to Test Tube T 30 – 35°C.

d. Repeat Steps 8 – 15.

17. Measure the enzyme activity at 50 – 55°C:

a. Prepare a water bath at a temperature in the range of 50 – 55°C by placing hot water in a 600 ml beaker (hot tap water will probably work fine). Check that the temperature remains in this range throughout this test.

b. Place Test Tube T 50 – 55°C in the warm water bath until the temperature of the mixture reaches a temperature in the 50 – 55°C range. Record the actual temperature of the test-tube contents in the blank in Table 4.

c. Add 2 drops of the enzyme solution to Test Tube T 50 – 55°C. d. Repeat Steps 8 – 15.

18. Measure the enzyme activity at 20 – 25°C (room temperature):

a. Record the temperature of Test Tube T 20 – 25°C in Table 4. b. In the tube labeled T 20 – 25°C, add 2 drops of the enzyme solution. c. Repeat Steps 8-15.

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Procedure (Part 3 - Testing the Effect of pH): 1. Obtain and wear goggles.



4. On the Meter screen, tap Rate. Change the data-collection rate to 0.5 samples/second and the data-collection length to 180 seconds.

5. Place three clean test tubes in a rack and label them pH 4, pH 7, and pH 10.

6. Add 3 ml of 3% H2O2 and 3 ml of each pH buffer to each test tube, as in Table 3.

7. In the tube labeled pH 4, add 2 drops of the enzyme solution.





12. When data collection has finished, an auto-scaled graph of pressure vs. time will be displayed. Disconnect the plastic tubing connector from the rubber stopper. Remove the rubber stopper from the test tube and discard the contents in a waste beaker.

Table 3

Test tube Label Volume of 3% H2O2 (ml)

Volume of buffer (ml)

pH 4 3 3

pH 7 3 3

pH 10 3 3

Figure 2

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a. Choose Curve Fit from the Analyze menu (found on the graph screen). b. Select Linear for the Fit Equation. The linear-regression statistics for these two data

columns are displayed for the equation in the form

y = mx + b

c. Enter the absolute value of the slope, m, as the reaction rate in Table 4.

d. Select OK.


16. In the tube labeled pH 7, add 2 drops of the enzyme solution. Repeat steps 8-15.

17. In the tube labeled pH 10, add 2 drops of the enzyme solution. Repeat steps 8-15.

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NAME___________________________________ DATE_______________________

DATA RECORDING & PROCESSING

Table 4

Test tube label (M) Slope, or rate (kPa/min)

1 Drop

2 Drops

3 Drops

4 Drops

0 – 5 C range: _____ C

20 – 25 C range: _____ C

30 – 35 C range: _____ C

50 – 55 C range: _____ C

pH 4

pH 7

pH 10

Plot your data on the corresponding plot

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Part II Effect of Temperature 2. At what temperature is the rate of enzyme activity the highest? Lowest? Explain.

3. How does changing the temperature affect the rate of enzyme activity? Does this follow a pattern you anticipated?

4. Why might the enzyme activity decrease at very high temperatures?

Part III Effect of pH 5. At what pH is the rate of enzyme activity the highest? Lowest?

6. How does changing the pH affect the rate of enzyme activity? Does this follow a pattern you anticipated?

QUESTIONS Part I Effect of Enzyme Concentration 1. How does changing the concentration

of enzyme affect the rate of decomposition of H2O2?

2. What do you think will happen to the rate of reaction if the concentration of enzyme is increased to five drops? Predict what the rate would be for 5 drops.

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7. List 3 sources of error that may have affected the results of your experiment. List possible

changes in the procedure to reduce the source of the error.

Source of Error Possible improvement to Procedure

EXTENSIONS

1. 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.

2. Presumably, at higher concentrations of H2O2 there is a greater chance that an enzyme molecule might collide with H2O2. If so, the concentration of H2O2 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.

3. Design an experiment to determine the effect of boiling the catalase on the reaction rate.

4. Explain how environmental factors affect the rate of enzyme-catalyzed reactions.

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Enzyme Action: Testing Catalase -- Teacher Notes

Set Up Time: 30 minutes Time needed to complete the lab: 45-50 minutes for one or two of the parts. 70-80 minutes for all three parts. Target Grade Level: 8-12, Biology

Activity 1. Assign each lab group one of the three variables to test. Share the collected data for analysis.

2. Your hot tap water may be in the range of 50-55°C for the hot-water bath. If not, you may want to supply pre-warmed temperature baths for Part 2, Step 17, where students need to maintain very warm water. Warn students not to touch the hot water.

3. Many different organisms may be used as a source of catalase in this experiment. If enzymes from an animal, a protist, and a plant are used by different teams in the same class, it will be possible to compare the similarities and differences among those organisms. Often, either beef liver, beef blood, or living yeast are used.

4. To prepare the yeast solution, dissolve 7 g (1 package) of dried yeast per 100 ml of 2% glucose solution. Incubate the suspension in 37 – 40°C water for at least 10 minutes to activate the yeast. Test the experiment before the students begin. The yeast may need to be diluted if the reaction occurs too rapidly. The reaction in Part 1 with 3 ml of 3% hydrogen peroxide, 3 ml of water, and 2 drops of suspension should produce a pressure of 1.3 atmospheres in 40 to 60 seconds. To prepare a 2% sugar solution, add 20 grams of sugar to make one liter of solution (100 ml per group is needed).

5. To prepare a liver suspension, homogenize 0.5 to 1.5 g of beef liver in 100 ml of cold water. You will need to test the suspension before use, as its activity varies greatly depending on its freshness. Dilute the suspension until the reaction in Step 12, with 3 ml of 3% hydrogen peroxide, 3 ml of water, and 2 drops of suspension produces a pressure of 130 kPa in 40 to 60 seconds. The color of the suspension will be a faint pink. Keep the suspension on ice until used in an experiment.

6. 3% H2O2 may be purchased from any supermarket. If refrigerated, bring it to room temperature before starting the experiment.

7. Emphasize to your students the importance of providing an airtight fit with all plastic-tubing connections and when closing valves or twisting the stopper into a test tube.

8. The length of plastic tubing connecting the rubber stopper assemblies to each gas pressure sensor must be the same for all groups. It is best to keep the length of tubing reasonably small to keep the volume of gas in the test tube low. Note: If pressure changes during data collection are too small, you may need to decrease the total gas volume in the system. Shortening the length of tubing used will help to decrease the volume.

9. You may need to let students know that at pH values above 10 enzymes will become denatured and the rate of activity will drop. If you have pH buffers higher than 10, have students perform an experimental run using them.

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Sample Results:

Table 4

Test tube label Slope, or rate (kPa/min)

1 Drop 10.23

2 Drops 44.98

3 Drops 59.36

4 Drops 98.26

0 – 5 C range: 4C 41.43

20 – 25 C range: 21C 48.02

30 – 35 C range: 34C 73.85

50 – 55 C range: 51C 27.55

pH 4 36.57

pH 7 66.86

pH 10 75.27

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ANSWERS TO QUESTIONS

1. 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 H2O2 molecule.

2. 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 5 drops, the rate in the above experiment should be about 111 kPa/min.

3. 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.

4. The rate increases as the temperature increases, until the temperature reaches about 50°C. Above this temperature, the rate decreases.

5. At high temperatures, enzymes lose activity as they are denatured.

6. Student answers may vary. Activity is usually highest at pH 10 and lowest at pH 4.

7. 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.

Sources of error:

Number of drops, size of drops, temperature not maintaned, snug fit of stopper, improper mixing, did not cap quick enough, improper concentration of hydrogen peroxide to water…

1 kPa is approximately the pressure exerted by a 10-g mass resting on a 1-cm2 area. 1 01.3 kPa = 1 atm. There are 1,000 pascals in 1 kilopascal.

enzyme action: testing catalase activity · lab #803 enzyme action: testing catalase lq by vernier...

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