Investigation: Catalase

How do enzymes work in living things?

Objectives:

  • to demonstrate the activity of an enzyme in living tissues
  • to learn how changes in the environment affect the activity of catalase
  • to demonstrate the steps of the scientific method
  • to practice observational skills, communicate in writing and report data orally

Introduction:

What might happen to your cells if they made a poisonous chemical? You might think that they would die. In fact, your cells are always making poisonous chemicals. They do not die because your cells use enzymes to break down these poisonous chemicals into harmless substances. Enzymes are proteins that speed up the rate of reactions that would otherwise happen more slowly. The enzyme is not altered by the reaction. You have hundreds of different enzymes in each of your cells.

In this lab, you will study an enzyme that is found in the cells of many living tissues. The name of this enzyme is catalase; it speeds up a reaction which breaks down hydrogen peroxide, a toxic chemical, into two harmless substances—water and oxygen. The reaction is as follows:

2 H2O2 2 H2O + O2

This reaction is important to cells because hydrogen peroxide (H2O2) is produced as a byproduct of many cellular reactions. If the cells did not break the hydrogen peroxide down, they would be poisoned and die.

In this lab, you will study the catalase found in living cells. It may seem strange to use dead cells to study the functions of enzymes, but the enzymes actually remain intact and active for several weeks.

*Recall that pH is the measure of the acidity or alkalinity of a solution. An acidic solution has many hydrogen ions (H+) and a pH below 7. An alkaline, or basic, solution has very few hydrogen ions and a pH above 7. A neutral solution has a pH of 7. You may review a pH chart in your textbook.

*Review the following terms: enzyme, substrate, active site, activation energy, product, optimum conditions, reactant, product, denature, endothermic, and exothermic.

**Rating Reactions: Use the following scale to rate the reactions in your test tubes.

0= no reaction, 1= very slow, 2= slow, 3= medium, 4= rapid, 5= very rapid bubbling

Safety Precautions:

*Be sure to clean stirring rods and test tubes between steps.

*Handle glassware with care.

Presentations:

*All members must participate (speak)

*Use paper/poster to show the following- problem, hypothesis, data, and conclusion

*Introduce the problem and hypothesis, briefly explain the procedure used, report the data collected, explain the conclusion

Part A. Normal Catalase Activity(ALL Groups)

  1. Place 2 mL of hydrogen peroxide into a clean test tube.
  2. Observe the hydrogen peroxide. Record observations.
  3. Place a small piece of animal/plant/fungus into same tube. Push the animal/plant/fungus down into the hydrogen peroxide with stirring rod.
  4. Observe and record data using scale.
  5. Answer questions.

Part B.

1. Read the problem for the activity you have been assigned.

  1. Read the directions for the activity you have been assigned.
  2. Develop a hypothesis.
  3. Carry out procedure/experiment.
  4. Prepare presentation for class.
  5. Make presentation to class.

Problem 1: Is catalase reusable?

  1. Add 2 mL of hydrogen peroxide into a clean test tube and add a small amount of cells. Push cells to bottom with stirring rod.
  2. Observe and record data using scale.
  3. Pour off the liquid into a second test tube.
  4. Add another 2 mL of hydrogen peroxide to the cells remaining in the first test tube.
  5. Observe and record data using scale.
  6. Repeat steps 3 – 5 two more times.
  7. Analyze data and form a conclusion.
  8. Prepare a presentation of your problem, hypothesis, procedure, data and conclusion.

Problem 2: Do both plants and animals have an enzyme that breaks down hydrogen peroxide?

  1. Label first test tube as 1, second tube as 2, third tube as 3 and fourth tube as 4.
  2. Add 2 mL of hydrogen peroxide to each test tube.
  3. Add a small piece of liver to test tube 1,pushing to bottom of tube with stirring rod.
  4. Observe and record data using scale.
  5. Repeat steps 3 – 4 with potato in test tube 2.
  6. Repeat steps 3 – 4 with chicken in test tube 3.
  7. Repeat steps 3 – 4 with carrot in test tube 4.
  8. Analyze data and form a conclusion.
  9. Prepare a presentation of your problem, hypothesis, procedure, data and conclusion.

Problem 3: What effect does boiling temperatures have on catalase activity?

*Caution: Use tongs when placing tubes into hot water baths.

  1. Label one tube as 1 and second tube as 2.
  2. Add a small piece of liver into the bottom of each test tube and cover each with a small amount of distilled water.
  3. *Use a test tube holder and place tube 1 into a boiling water bath (100oC) for 5 minutes. Leave tube 2 at room temperature.
  4. *Use a test tube holder and remove test tube 1 from boiling water bath and allow it to cool. Pour off water from each tube.
  5. Add 2mL of hydrogen peroxide to each test tube.
  6. Observe and record data using scale.
  7. Analyze data and form a conclusion.
  8. Prepare a presentation of your problem, hypothesis, procedure, data and conclusion.

Problem 4: What effect does freezing, room and body temperatures have on catalase activity?

*Caution: Use tongs when placing tubes into hot water baths.

  1. Label two test tubes as 1, two test tubes as 2 and two test tubes as 3.
  2. Add a small piece of liver to one of tubes labeled as 1, 2, and 3. Push to bottom of tube with stirring rod. Cover liver with water.
  3. Add 2 mL of hydrogen peroxide to the other tubes labeled as 1, 2, and 3.
  4. Place the tubes labeled as 1 into a 0oC ice bath for 3 minutes.
  5. Place the tubes labeled as 2 into a room temperature bath (22oC) for 3 minutes.
  6. Place the tubes labeled as 3 into a body temperature bath (37oC) for 3 minutes.
  7. After the 3 minutes, pour off water in tubes with liver.
  8. Pour each test tube of hydrogen peroxide into the corresponding tube of liver.
  9. Observe and record data using scale.
  10. Analyze data and form a conclusion.
  11. Prepare a presentation of your problem, hypothesis, procedure, data and conclusion.

Problem 5: What effect does an acid pH have on catalase activity?

*Caution: Do NOT allow acids or bases to come into contact with skin or clothing!

*Swirl test tube after adding each drop and check the pH with pH paper, by dipping stirring rod into the solution and touching the pH paper with solution on the tip of the rod. Clean and dry the rod between tests.

  1. Label one tube as 1 and second as 2.
  2. Add a small piece of liver to each test tube, pushing to bottom of tube with clean stirring rod.
  3. For tube 1, add one drop of 1N HCl (acid) at a time until it results in a pH of 3.
  4. For tube 2, add one drop of buffer (pH of 7).
  5. Record pH of each tube.
  6. Allow 2-3 minutes before proceeding to step 7.
  7. Add 2 mL of hydrogen peroxide to tube 1.
  8. Observe reaction and record data using scale.
  9. Repeat steps 7 – 8 with test tube 2.
  10. Analyze data and form a conclusion.
  11. Prepare a presentation of your problem, hypothesis, procedure, data and conclusion.

Problem 6: What effect do heavy metals (inhibitors) have on catalase?

  1. Add a small piece of liver to tube, pushing to bottom of tube with clean stirring rod.
  2. Add 5 drops of heavy metal (AgNO3) solution to same tube.
  3. Allow 2 – 3 minutes before proceeding to step 4.
  4. Add 2 mL of hydrogen peroxide into test tube.
  5. Observe reaction and record data using scale.
  6. Analyze data and form a conclusion.

7. Prepare a presentation of your problem, hypothesis, procedure, data and conclusion

Problem 7: What effect does a basic pH have on catalase activity?

*Caution: Do NOT allow acids or bases to come into contact with skin or clothing!

*Swirl test tube after adding each drop and check the pH with pH paper, by dipping stirring rod into the solution and touching the pH paper with solution on the tip of the rod. Clean and dry the rod between tests.

  1. Label one tube as 1 and second as 2.
  2. Add a small piece of liver to each test tube, pushing to bottom of tube with clean stirring rod.
  3. For tube 1, add one drop of 1N NaOH (base) at a time until it results in a pH of 10.
  4. For tube 2, add one drop of buffer (pH of 7).
  5. Record pH of each tube.
  6. Allow 2 – 3 minutes before proceeding to step 7.
  7. Add 2 mL of hydrogen peroxide tube 1.
  8. Observe reaction and record data using scale.
  9. Repeat steps 7-8 with test tube 2.
  10. Analyze data and form a conclusion.
  11. Prepare a presentation of your problem, hypothesis, procedure, data and conclusion.

Problem 8: What effect does Ethanol alcohol (solvent) have on catalase activity?

  1. Add a small piece of liver to a test tube, pushing to bottom of tube with clean stirring rod.
  2. Add one drop of alcohol to the liver. Wait for one minute.
  3. Add 2 mL of hydrogen peroxide to the tube.
  4. Observe reaction and record data using scale.
  5. Analyze data and form a conclusion.
  6. Prepare a presentation of your problem, hypothesis, procedure, data and conclusion.

Name ______

Catalase Analysis

  1. In the reaction that you are studying in this lab,

a)what is the enzyme? ______

b)what is the substrate? ______

c)what are the products? ______

  1. Before adding the H2O2 to the liver/chicken/carrot/potato, does it bubble on its own? ______
  1. Observe the bubbling of the H2O2 when added to the control in Part A. What gas is being released? ______
  1. Data Table:Observation for each problem

**Rating Reactions: Use the following scale to rate the reactions in your test tubes.

0= no reaction, 1= very slow, 2= slow, 3= medium, 4= rapid, 5= very rapid bubbling

Catalase Reaction

Observation (rate or description)
C – Control (Part A)
Problem 1 – Is catalase reusable?
Problem 2 – Do plants and animals have enzyme to break down H2O2?
Problem 3 – What effect does boiling have on the break down H2O2?
Problem 4 – What effect do freezing, room, and body temperatures have on the break down H2O2?
Problem 5 – What effect do acids have on catalase activity?
Problem 6 – What effect do heavy metals have on catalase?
Problem 7 – What effect to bases have on catalase activity?
Problem 8- What effect does ethanol alcohol have on catalase activity?