Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Prepare the catalase solution by adding 100 mg (0.10 g) of catalase to 500 mL of distilled water.
2. Stir on a magnetic stirrer until catalase is dissolved. Catalase is only slightly soluble in water and dissolving it completely takes several minutes.
3. Prepare 1.0% and 2.0% H₂O₂ solutions in the provided plastic bottles with vented caps. Once the solutions are made, store in a dark, cool place. Solutions should be made the same week they will be used.
   1. To make 300 mL of 1.0% H₂O₂ solution, add 50 mL of 6% H₂O₂ to 250 mL of distilled water.
   2. To make 300 mL of 2.0% H₂O₂ solution, add 100 mL of 6% H₂O₂ to 200 mL of distilled water

Safety Precautions

Hydrogen peroxide can cause skin and eye irritation upon contact. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash hands 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 before students begin experimentation.

Lab Hints

• Enough materials are provided in this kit for five classes of 30 students working in pairs or 15 groups of students per class. The introductory activity and analysis can be completed in one 50-minute class period. The inquiry portion of the laboratory can be completed in one to two class periods, depending on whether or not planning occurs during class or outside of class.
• This lab kit is compatible with Lab 6 in Investigating Biology through Inquiry from Vernier Software & Technology, with the exception that this lab kit includes purified catalase instead of yeast.
• If using a Gas Pressure Sensor instead of an O₂ Gas Sensor, use 50 mL of H₂O₂ solution and 2 mL of catalase solution in a 125-mL Erlenmeyer flask for the introductory activity.
• Using purified catalase enables students to investigate enzyme concentration with greater certainty than using liver or yeast.
• Catalase solution should be made fresh each day. There is enough catalase included in the kit for five days of experimentation.
• 500 mL of catalase solution is enough for 150 groups to complete the introductory activity. The amount of catalase may be scaled down to suit the number of groups doing the experiment in a single 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.
• Students should be familiar with using the Vernier interface and data-collection software. If they are not, allow some extra time to instruct students on their use.
• The O2 Gas Sensor detects oxygen in the air above the sample. Do not submerge the sensor in the solution.
• The normal operating temperature of the O₂ Gas Sensor is 20–30 °C.

Teacher Tips

• Students should have a working understanding of how enzymes functions.
• Temperature, pH and catalase concentration are the most likely variables students will want to investigate in Part B. Anticipate these experiments by having water bath materials, thermometers, buffer solutions of various pH and pH monitoring equipment available. pH buffer envelopes are provided in this kit. Catalase activity is measured and printed on the Flinn chemical label of the catalase bottle. Determine the concentration of the catalase solution using Equation 2, where x = the number of units printed on the bottle of catalase:
  {11319_Tips_Equation_2}
  {11319_Tips_Example}
• When testing for pH, students can choose to either add buffer to the hydrogen peroxide or dilute the catalase enzyme using buffer solution. Make sure more concentrated enzyme is available for students to perform this dilution without changing the concentration of the enzyme.

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.

   {11319_PreLabAnswers_Figure_1}
3. Predict how substrate concentration is expected to influence the reaction rate.

   To a certain point, increasing substrate concentration will increase reaction rate in a proportional way. Note: At about 2%, the concentration of hydrogen peroxide will exceed the ability of the catalase to bind to the hydrogen peroxide and the reaction rate will level off. At 3% and above, the concentration of oxygen is too high for the catalase to effectively work and the rate of reaction decreases.

Sample Data

Catalase

Answers to Questions

Analysis

1. What is the relationship between reaction rate and concentration of substrate as shown in the graph?

   Answers will vary, accept all reasonable answers that reflect the data collected. The 1% and 2% hydrogen peroxide solutions will show an increasing reaction rate. The 6% hydrogen peroxide solution will have a slower reaction rate.

2. What other factors will impact the catalase-facilitated decomposition rate of H₂O₂?

   Answers will vary, accept all reasonable answers. Some common answers will be temperature of catalase, or substrae, pH of substrate or catalase and the addition of enzyme activators or inhibitors.

References

Melville, J.M., Collins, M., and D. Volz. Investigation 6A, Testing Catalase Activity (O₂) in Investigating Biology through Inquiry. Vernier Software and Technology, 2012.

Introduction

The rate of decomposition of hydrogen peroxide by catalase can be measured with an O₂ Gas Sensor. Additional experiments can be performed to investigate how different factors may affect the rate of enzyme-catalyzed reactions.

Concepts

• Catalyst
• Enzyme optimization
• Reaction rate

Background

Hydrogen peroxide (H₂O₂) is a toxin that readily forms as a byproduct of metabolic reactions in most organisms. In order to survive, organisms have enzymes that catalyze the redox reaction that breaks H₂O₂ into non-toxic molecules. Enzymes are biochemical catalysts. A catalyst is a substance that accelerates the 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 class of enzymes that break down hydrogen peroxide are peroxidases. A peroxidase that does not require a reducing agent is called catalase. Catalase catalyzes the decomposition of H₂O₂ according to Equation 1.

Experiment Overview

In Part A of this experiment, three different concentrations of H₂O₂ are used to determine how the concentration of the substrate affects the production of oxygen gas, and therefore the rate of the reaction between H₂O₂ and catalase. In Part B, design an experiment to explore how changing 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. Predict how substrate concentration is expected to influence the reaction rate.

Safety Precautions

Hydrogen peroxide solutions 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 evaluated by an instructor. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part A. Introductory Activity

1. Connect the sensor to the data-collection interface and start the data collection program.
2. Set the duration of the experiment to 5 minutes or 300 seconds.
3. Use a buret clamp to fasten the O₂ gas sensor to a support stand.
4. Add 10 mL of 1.0% H₂O₂ to the gas sampling chamber.
5. Using a graduated pipet, add 1 mL of catalase solution and swirl to mix.
6. Quickly insert and gently twist the O₂ gas sensor into the bottle to form an airtight seal.
7. Start data collection. Data collection will stop after five minutes.
8. Using the data-collection software, perform a linear fit on the area of the graph that is most linear, generally between 50 and 200 seconds. Record the slope of this line, which is the rate of reaction, on the Catalase Worksheet.
9. Rinse the gas sampling chamber with distilled water and dry the inside.
10. Repeat steps 4–9 two more times using 2.0% and 6.0% H₂O₂.
11. On the Catalase Worksheet, graph the reaction rates as a function of concentration of substrate.
12. Complete the remainder of the Catalase Worksheet before beginning Part B.

1. Consider the following questions while reflecting upon your knowledge of enzyme-facilitated reactions.
   1. What else might affect the rate of reaction between H₂O₂ and catalase?
   2. What environmental conditions (e.g, temperature, pH) is catalase generally exposed to?
2. Choose a variable and then design and carry out an experiment to determine how the reaction rate of H₂O₂ and catalase may be affected if the experimental conditions are altered.
   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

11319_Student1.pdf