Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Prepare the catalase stock solution by adding 100 mg (0.10 g) of catalase to 500 mL of distilled water and stirring on a magnetic stirrer until catalase is dissolved. Catalase is only slightly soluble in water and dissolving it completely takes several minutes. To make a 10% solution of this catalase solution, add 1 mL of the catalase solution to 9 mL of distilled water.
2. Before class, make 300 mL of diluted catalase solution for the Introductory Activity. Since catalase activity decreases over time, the amount of catalase stock solution needed will vary based on how long the solid catalase had been stored, particularly if it was previously opened.
   1. Add 0.5 mL of 3% H₂O₂ and 1 mL of distilled water to a reaction plate well.
   2. Soak a filter paper disk in the catalase solution and put it at the bottom of the well containing the H₂O₂.
   3. Observe the amount of time it takes to float and adjust the concentration of the solution as specified in the following table.
      {11315_Preparation_Table_1}
3. Filter paper disks can be punched prior to class to save time and to offset the need for multiple paper punches.

Safety Precautions

The hydrogen peroxide used in this experiment is very dilute but can cause skin and eye irritation upon contact. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash thoroughly before leaving the lab. 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. Dilute peroxides may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method 22a. For any chemical not specifically listed in the materials, but used in the inquiry portion of the lab, 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 students begin experimentation.

Lab Hints

• Enough materials are provided in this kit for five classes of 30 students working in pairs or for 15 student groups per class. Part A and Part B will each require one 50-minute class period with discussion time. Part C may take 1–3 days, depending on whether work is completed in class or as homework.
• The simple assay method utilized in this activity provides excellent opportunities for students to be involved in higher level science practices such as refining a protocol or exploring inherent error. 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. You may wish to provide equipment for a variety of opportunities.
• Using purified catalase allows for investigation of enzyme concentration with far more certainty than using liver or yeast.
• Catalase solutions should be made fresh each day.
• Store solid catalase in a lab refrigerator. It is normal for the activity level to decrease over time, so catalase should be replaced regularly.
• The amount of time the disk soaks in the catalase will impact results. Guide students toward controlling this variable if their results are inconclusive.

Teacher Tips

• Two key discussion times occur during this laboratory. Be sure to discuss observations and results after the initial trial with the assay technique (Part A). 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 catalase 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 C). This discussion could be conducted as a scientific seminar. Each research team can present their design, controls, results and conclusions to drive a class discussion and compare various approaches using the same variables.
• Students may need help with the dilution procedures to be sure they know how the concentrations have changed and how to keep their units comparable when they make graphs.
• Temperature, pH and catalase concentration are the most likely variables students will want to test in Part C. Anticipate these experiments by having water bath materials, thermometers, buffer solutions and pH monitoring equipment available. To prepare catalase solutions at different concentrations, you will need to calculate the concentration of catalase in units of enzyme activity per mL. So, first convert the units from mg/mL to units/mL using the original concentration of the stock solution and the catalase activity units that are printed on the Flinn chemical label (Equation 2).
  {11315_Tips_Equation_2}
  To prepare a dilution of the stock solution, use the concentration of the original stock solution in units/mL and multiply that by the desired dilution factor (Equation 3).
  {11315_Tips_Equation_3}
• Students could also write procedures that use different methods of data collection than the assay method. For example, gas pressure and oxygen sensors can be used to measure oxygen production.
• When testing for pH, students can either add buffer to the hydrogen peroxide or dilute the catalase solution using a buffer solution. Additional catalase solution may be required for students to perform this dilution without compromising their results.

Answers to Prelab Questions

1. Identify the enzyme and the substrate in this experiment.

   The enzyme is catalase and the substrate is hydrogen peroxide.

2. Sketch a model showing how the enzyme-substrate system works to break down toxic hydrogen peroxide.

   Answers will vary. Drawing should show hydrogen peroxide binding to catalase, then water and oxygen gas being produced. Catalase is unchanged.

   {11315_PreLab_Figure_1}
3. Explain how the floating-disk assay measures the rate of reaction.

   Oxygen is a product of the reaction. The gas bubbles stick to the disk, making them float. The time it takes for the disk to float is proportional to the rate of the reaction.

Sample Data

Catalase

Part A. Floating-Disk Assay System

Part B. Effect of Substrate Concentration Guided-Inquiry Experiment
Results from student experiments will likely vary a great deal. The general trends students get are likely to follow “typical” enzyme curves for various variables. See graphs as examples.

Answers to Questions

What is the relationship between the concentration of the substrate and the reaction time?

Possible answers include:
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 how an enzyme affects the decomposition of hydrogen peroxide. Then design additional experiments to further investigate factors affecting the rate of enzyme-catalyzed reactions.

Concepts

• Catalyst
• Reaction rate
• Enzyme optimization

Background

Enzymes are biochemical catalysts. A catalyst is a substance that accelerates reaction rate but is not consumed during the reaction. Catalysts work by binding to a reactant, called a substrate. When the catalyst reacts with the substrate, the substrate molecule is broken down and the catalyst is free to bond to another molecule of substrate. The enzyme used in this investigation is catalase, and the substrate is hydrogen peroxide. Catalase is an enzyme that is produced by many types of cells. A purified form of the enzyme will be used in this investigation in order to control the concentration of the enzyme. Catalase catalyzes the decomposition of hydrogen peroxide according to Equation 1.

Catalase happens to be one of the fastest enzymes known. In fact, each molecule of catalase can decompose more than 107 molecules of H₂O₂ per second! The efficient production of catalase evolved as a survival mechanism. This is important because not only is H₂O₂ a product in many biochemical reactions, it is also a toxic chemical to cells.

This oxygen production can be used to measure the relative rate of the reaction by using a floating-disk assay procedure. In a floating-disk assay, disks of filter paper are placed within a reaction vessel. As oxygen is produced, the gas bubbles adhere to the disks and the disks float once enough oxygen is produced. The amount of time it takes for this to occur is proportional to the rate of the reaction and to overall enzyme activity.

Experiment Overview

The purpose of Part A is to manipulate the concentration of the substrate to discover the advantages and disadvantages of the floating-disk assay method. The purpose of Part B is to design a precise procedure for determining relative enzyme activity based on concentration of the substrate. In Part C, research, design and carry out an experiment to determine how one variable affects enzyme activity.

Materials

Prelab Questions

1. Identify the enzyme and the substrate in this experiment.
2. Sketch a model showing how the enzyme–substrate system works to break down toxic hydrogen peroxide.
3. Explain how the floating-disk assay measures the rate of reaction.

Safety Precautions

The hydrogen peroxide used in this experiment is very dilute but can cause skin and eye irritation upon contact. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. All student-produced procedures must be reviewed by an instructor. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part A. Floating-Disk Assay System

1. Use a paper punch and punch 20 disks from a piece of filter paper.
2. Pour 10 mL of catalase solution into a small, plastic cup.
3. Pour 10 mL of H₂O₂ into a separate small plastic cup.
4. Use a pipet to transfer 0.5 mL of H₂O₂ and 1 mL of distilled water into one well in the reaction plate.
5. Soak a filter paper disk in the catalase solution and then place it on the bottom of the well and start the timer. Observe carefully and measure the time it takes for the disk to rise. Note: The time it takes for the disk to rise to the top is dependent on 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.
6. While keeping the total volume of liquid the same as in step 4, use a pipet to transfer different concentrations of H₂O₂ into 3–5 other wells. For example, 0.25 mL H₂O₂ with 1.25 mL of water.
7. Soak filter paper disks in catalase solution and then place one disk in each of the various concentrations of H₂O₂. Remember to start timing after placing the disks on the bottom of the wells.
8. Observe and time the rates of the reactions as carefully as you can. Record all observations and results in the Floating-Disk Assay Data Table on the Catalase Worksheet.
9. After the preliminary experiments, share your results with the class and discuss the floating-disk-assay technique, considering the following questions.
   1. What problems were encountered?
   2. How can these problems be overcome?
   3. How can the technique be improved?
   4. How can more consistent results be obtained?

Part B. Effect of Substrate Concentration

1. Based on the class discussions from Part A, refine the experimental procedure and carry it out 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 and then graph the data on the Catalase Worksheet to determine the reaction rate.
3. Based upon an examination of your graph, state the relationship between the concentration of substrate and the reaction time on the Catalase Worksheet.

Part C. Guided-Inquiry Design

1. Consider the following while reflecting upon your knowledge of enzyme-facilitated reactions.
   1. What else might affect the rate of breakdown of H₂O₂ by catalase?
   2. Consider the environmental conditions catalase is generally exposed to (e.g., pH, temperature)?
2. Choose a variable to explore and then design and carry out an experiment to determine the reaction rate of H₂O₂ under selected conditions.
   1. Develop a testable hypothesis.
   2. Discuss and design a controlled experiment to test the hypothesis. Include a detailed procedure based on the introductory activity that collects data to test your hypothesis.
   3. List any safety concerns or precautions that will be taken to protect yourself, your classmates and your instructor during the experiment. Make changes to increase protection and decrease risk.
   4. Determine how you will collect and record raw data.
   5. Determine how you will analyze the data to test your hypothesis.
   6. Review your hypothesis, safety precautions, procedure, data tables and proposed analysis with your instructor prior to beginning your experiment.
   7. Run your experiment. Once the experiment and analysis are complete, evaluate whether the experimental evidence supports, refutes or provides no information concerning the hypothesis.
   8. Make suggestions for revisions to the experiment or hypothesis.

Student Worksheet PDF

11315_Student1.pdf