Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Prepare the yeast suspension by adding a ½ package of dry yeast to 150 mL of warm water containing a teaspoon of sugar. Allow the mixture to stand for at least 30 minutes before use. Stir occasionally.
2. Filter paper disks can be punched prior to class to save time and to offset the need for many paper punches. Student volunteers can be enlisted to do the punching prior to class time.

Safety Precautions

The hydrogen peroxide used in this experiment is very dilute but will cause skin and eye irritation upon contact. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Peroxides should be disposed of according to Flinn Suggested Disposal Method #22a.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. Part I and Part II will each require one class period with discussion time. Your teaching schedule, course objectives, availability of equipment and other considerations will determine how much time you allow for Part III of the laboratory.

• The simple assay method utilized in this activity provides excellent opportunities for students to be involved in higher level science processing skills outlined in recent science standards. Let students struggle and explore with their experimental designs. They will eventually realize the need for controls, careful refinement of techniques, and careful recording of data. Do not make everyone do the same experiment. Provide equipment for a variety of opportunities.
• Two key discussion times occur during this laboratory. Be sure to discuss observations and results after the initial trial with the assay technique (Part I). Students will quickly identify key variables affecting results if given “think” time during the open-ended discussion. Consider questions, such as How does the amount of yeast on the disk affect the rate? Can they be dried uniformly? How are the disks dropped into the wells? What if the disks stick to the sides? When does timing start? When does timing end? Are all the disks uniform?
• The second important discussion time is after students complete the experiments they designed (Part II). Conduct a scientific seminar. Have each research team present their entire design, controls, results and conclusions. Discuss each experiment as a class and compare various approaches for the same variables.
• Help students with dilution procedures to be sure they know how the concentrations have declined and how to keep their units comparable when they make graphs.
• Temperature, pH and yeast concentration are the most likely variables students will want to test in Part III. Anticipate these experiments by having water bath materials, thermometers, buffer solutions, acids, bases and pH monitoring equipment ready behind the scenes. Let students utilize other equipment such as pH meters, CBLs or computer probes as available in your science department.

Sample Data

Results from student experiments will likely vary a great deal. Encourage careful analysis based upon the data students collect. The general trends students get are likely to follow “typical” enzyme curves for various variables (see Figure 1). General statements are likely as follows:

“The catalase breakdown of H₂O₂ is decreased as the concentration of H₂O₂ is decreased.”

“The catalase breakdown of H₂O₂ is increased as temperature increases until a critical temperature is reached.”

“There is an optimum pH range for catalase activity.”

Introduction

Explore a floating-disk assay system to study the effect that an enzyme has on the decomposition of hydrogen peroxide. Then design additional experiments to further investigate factors affecting the rate of enzyme-catalyzed reactions.

Concepts

• Catalyst

• Reaction rate
• Dependent/Independent variables
• Enzyme/Substrate
• Experimental design/control

Background

Enzymes are biochemical catalysts. A “catalyst” is a substance that accelerates the reaction rate but is not consumed during the reaction. The enzyme used in this investigation is catalase, an enzyme produced by living cells. Yeast cells are the living cells used in this experiment. Catalase catalyzes the decomposition of hydrogen peroxide according to Equation 1:

In fact, each molecule of catalase can decompose more than 10⁷ molecules of H₂O₂ per second! Catalase happens to be among the fastest enzymes known. Since H₂O₂ is a toxic chemical to cells, the production of catalase and its speed of reaction is a protective device for living cells.

Materials

Safety Precautions

The hydrogen peroxide used in this experiment is very dilute but will cause skin and eye irritation upon contact. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part I. Floating-Disk Assay System

1. Use a paper punch and punch 20 disks from a piece of filter paper.
2. Use three plastic cups to secure:

10 mL of yeast suspension
10 mL of distilled water
10 mL of H₂O₂

3. Fill a reaction plate well one-half full with H₂O₂ using a Beral-type pipet.
4. Soak a filter paper disk in the yeast suspension and put it at the bottom of the well containing the H₂O₂. Observe carefully and measure the time it takes for the disk to rise.

(The time it takes the disk to rise to the top is dependent upon the rate of the reaction shown in Equation 1. The oxygen gas bubbles produced in the reaction adhere to the surface of the filter paper and “float” the disk to the surface. What factors can affect the speed of this enzyme reaction?)

5. Experiment with different concentrations of H₂O₂ in various wells. Use the distilled water and graduated pipet to see if the rate of reaction is affected by the concentration of the substrate (H₂O₂).
6. Observe and time the rate of the reactions as carefully as you can. Record all observations and results on the Catalase Worksheet Part I.
7. After the preliminary experiments, share the results with the entire class and discuss the floating-disk-assay technique. Consider these questions as a class:

• • How can the technique be improved?
  • What problems were encountered?
  • How can these problems be overcome?
  • How can more consistent results be obtained?

Part II. Effect of Substrate Concentration

1. Based upon class discussions after Part I, develop a refined experimental procedure and conduct experiments to determine the relationship between the concentration of the substrate (H₂O₂) and the reaction rate of the catalase reaction.
2. Collect quantifiable data and be as precise as possible. Record the data on the Catalase Worksheet Part II.
3. Use graph paper to graph the results. The graph should depict substrate concentration versus reaction rate.
4. Based upon an examination of your graph, state the relationship between the concentration of substrate and the reaction time. Use the space provided on the bottom of the Catalase Worksheet.

Part III. Test Other Variables

1. What else might affect the rate of reaction of the breakdown of H₂O₂ by catalase? Form a research group as directed by your teacher. Brainstorm hypotheses about various variables. Review the list of hypotheses. Discuss possible experiments that might test each hypothesis. Be sure to discuss any necessary “controls.”
2. Choose the hypothesis that seems to be the most viable and interesting to your group. Design an experiment to test the hypothesis. Write up the experimental design in detail and discuss it with your teacher.
3. Secure any additional materials from your teacher.
4. Conduct your experiment. Create appropriate data tables and record the results from your experimental work.
5. Write a complete laboratory report for your experiment. Be sure to include:

1. A statement of your hypothesis.
2. Describe your experimental design. Include the controls used in the experiment.
3. Document the results of your experiment.
4. Write an interpretation of the experimental results.
5. Was the original hypothesis rejected or accepted based upon the experimental results?
6. What further experiments are suggested by your result?

6. Conduct a class seminar and share all of the experimental designs and results.
7. Consult your instructor for proper disposal of your test solutions.

Student Worksheet PDF

10251_Student1.pdf