Materials Included In Kit

Additional Materials Required

Prelab Preparation

Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron throughout this prelab preparation.

1. Dilute the hydrogen peroxide solution from 3% to 1.5% (0.44 M). Complete the afternoon before or the morning of the lab.
   1. Use a graduated cylinder to measure 473 mL distilled water into a 1-L beaker.
   2. Slowly stir in the 473 mL of 3% hydrogen peroxide.
   3. Mix well.
   4. Decant into the supplied plastic, amber 16-ounce bottles.
   5. Cap using the green caps.
   6. Label bottles with “1.5% hydrogen peroxide” and the preparation date.
2. Prepare the ice bath.
   1. Combine ice and tap water in 1-L beaker
3. Prepare catalase solution, 400 units/mL, within one hour of use.
   1. Multiply 50 mg times the units/mg shown on the catalase label and divide by 400 units/mL to calculate the amount of distilled water needed to make the solution. In the event that the label shows two values—one for units/mg solid and one for units/mg protein—use the smaller of the two numbers in the calculation. The smaller number should refer to units/mg solid. For example:
      {10757_Preparation_Equation_3}
   2. Use a graduated cylinder to measure the calculated amount of distilled water into a 500-mL beaker.
   3. Add the catalase to the distilled water and gently stir the solution using the stir plate and the magnetic stir bar until the solid has dissolved.
   4. Store on ice–water bath to inhibit the decomposition of the catalase enzyme.
4. Prepare a boiling water bath just before the laboratory.
   1. Add 750 mL tap water into a 1-L beaker.
   2. Add 2–3 boiling stones to the water.
   3. Place on hot plate.
   4. Adjust hot plate to keep water at a slow boil.
5. Prepare the buret.
   1. Screw a stopcock onto the Luer-lock of each of the 10-mL syringes supplied with the kit.

Safety Precautions

Hydrogen peroxide is an oxidizer and a skin and eye irritant. Sulfuric acid is extremely corrosive to skin and eyes and other tissues. Potassium permanganate is a skin irritant and dye. The scalpel is a sharp object, care must be taken when cutting the potato or liver with the scalpel. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron throughout this lab. Follow all normal classroom guidelines. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please consult 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. Dispose of excess potassium permanganate using Flinn Suggested Disposal Method #12a. Dispose of excess sulfuric acid solution using Flinn Suggested Disposal Method #24b. Dispose of excess hydrogen peroxide solution using Flinn Suggested Disposal Method #22a. Dispose of excess Catalase power using Flinn Suggested Disposal Method #26a. The experimental byproduct solutions are acidic and contain Mn²⁺ ions. Neutralize the acid with sodium carbonate or sodium bicarbonate according to Flinn Suggested Disposal Method #24b, and add extra hydrogen peroxide (about 5 mL) to the solution to convert the Mn²⁺ ions to MnO₂, a brown solid. Separate the resulting mixture by filtration and dispose of the solid MnO₂ according to Flinn Suggested Disposal Method #26a.

Lab Hints

• Ensure students label all materials with their group number and the activity number or chemical used in the container.
• The baseline titration must be repeated each day that titrations are performed. Hydrogen peroxide decomposes and consequently affects the rate of the catalase reaction.
• Potassium permanganate stains clothing and other materials. Students may be advised to wear old clothing in addition to their chemical apron. Stains may be removed by applying dilute hydrogen peroxide to the area.
• For Activity 3, complete all reactions during one lab period, then cover samples and titrate during the next lab period.
• Do not store potassium permanganate in the burets.|Activity 1 may be completed as a demonstration. Hydrogen peroxide slowly decomposes to H₂O and O₂. Several days of decomposition may be required to show a change during titration. Remind students that Activity 2a demonstrates the body’s need to catalyze the decomposition reaction.

Sample Data

Activity 2

Graph 1
Title Amount of H₂O₂ Consumed Over Time or Catalase Activity Measured as Rate of H₂O₂ Decomposition Post-Lab Questions

*Reaction rate (mL H₂O₂/sec)

Answers to Questions

Activity 2

Part 1. General Reaction

1. What is the enzyme in this reaction?

   Catalase

2. What is the substrate in this reaction?

   Hydrogen peroxide

3. What are the products in this reaction?

   Water and oxygen

4. How could you show that the gas generated in this reaction is oxygen?

   Capture the gas in a test tube. Insert a glowing splint. If the gas is oxygen the glowing splint will reignite and burst into flame.

Part 2. Boiled Catalase

1. How does the reaction compare to the one using the unboiled catalase? Explain the reason for this difference.

   Very few or no bubbles are being generated. Boiling has denatured the catalase, therefore the reaction is proceeding at the uncatalyzed rate, which is very slow.

Part 3. Living Tissue

1. What do you observe?

   Many bubbles effervesce from the potato or liver sample due to the presence of naturally occurring catalase.

2. Hypothesize what would happen if the living tissue were boiled before being added to the H₂? Support your hypothesis using your observations from Activity 2.

   Very few or no bubbles of gas will be produced. Boiling the sample will denature the catalase.

4. What percent of the H₂O₂ spontaneously decomposes in 24 hours? Calculate the amount by dividing the uncatalyzed H₂O₂ decomposition (line d) by the number of days the beaker was allowed to decompose from Activity 1, then multiply by 100%.

   0%

5. Graph the data for enzyme-catalyzed decomposition on Graph 1. See graph in the Sample Data section.
   1. The independent variable (x-axis): Time
   2. The dependent variable (y-axis): Amount of H₂O₂ used (mL)

Post-Lab Questions

2. When is the rate the highest? Explain why.

   The first or second interval. There are a large number of substrate molecules for the enzyme to catalyze.

3. When is the rate the lowest? Explain why.

   Last interval. Most of the substrate molecules have been decomposed so fewer molecules are available for the enzyme to catalyze.

4. Why was sulfuric acid added to the reaction at specific times during Activity 3? Relate this to enzyme structure and chemistry.

   The sulfuric acid denatures the catalase, inhibiting its ability to bind to the hydrogen peroxide.

5. Catalase was kept on an ice–water bath. Predict the effect lowering the temperature would have on the rate of enzyme activity. Explain your prediction.

   Reducing the temperature of the catalase reduces the kinetic activity of the molecules, reducing the number of successful collisions between the enzyme and its substrate.

6. Design a controlled experiment to test the effect of varying pH, temperature or enzyme concentration on the rate of an enzyme-catalyzed reaction. (Answer on a separate sheet of paper.)

   Answers will vary. Variations in protocol, type of enzyme and procedures to adjust the pH, temperature and dilute the enzyme are all viable answers.

7. A small portion of the human population has genetic disorders linked to malformed enzymes. If you were a carrier for an enzymatic genetic disorder that causes extreme mental retardation and death before the age of three, would you want to know? Explain the reasons behind your choice. (Answer on separate sheet of paper.)

   Answers will vary.

Teacher Handouts

10757_Teacher1.pdf

References

Biology: Lab Manual; College Entrance Examination Board: 2001.

Introduction

In this laboratory, the conversion of hydrogen peroxide (H₂O₂) to water and oxygen gas by the enzyme catalase will be observed and studied. The amount of oxygen generated will be measured and then the rate of this enzyme-catalyzed reaction will be calculated.

Objectives
After completing this laboratory, you should be able to:

• Measure the effects of changes in temperature, pH, enzyme concentration and substrate concentration on reaction rates of an enzyme-catalyzed reaction in a controlled experiment.
• Explain how environmental factors affect the rate of enzyme-catalyzed reactions.

Concepts

• Biological enzyme
• Independent/dependent variables
• Catalyst
• Reaction rate
• Experimental design

Background

A catalyst is any substance that speeds up the rate of a chemical reaction without being permanently altered in the process. Thus a single molecule of catalyst can perform the same reaction thousands of times a second. Catalysts cause slow reactions to occur more quickly by lowering the activation energy necessary for the reaction to occur. A ski lift is an analogy for a catalyst. If the reaction is “skiing,” then the skier must first get to the top of the ski hill. One option is for skiers to climb to the top and once they reach the top, enjoy the potential energy they earned as they ski back down the hill. The ski lift allows many more skiers to reach the top of the hill very quickly without the skiers expending much energy. Once at the top, they still enjoy the same energy release as they ski down the ski hill. The reaction can occur many, many more times and the ski lift (the catalyst) is not changed during the process.

Enzymes are biochemical catalysts. Enzymes perform very specific functions and therefore they only react with very specific substances. This exclusive nature of enzyme/substrate binding means that living organisms must contain thousands of different enzymes to catalyze all the different biochemical reactions that must occur. One type of enzyme may break a molecule into two or more product molecules (digestion). while a different type of enzyme may combine two or more molecules together to build a more complex product (assimilation).

Enzymes are globular, three-dimensional proteins with specific characteristic shapes that allow only a few specific chemicals to temporarily bond with the enzyme. In particular, enzymes have an active site and an allosteric site. At the active site the chemical or protein (substrate) binds to the enzyme so that it may be reacted. At the allosteric site a biofeedback molecule binds to inhibit the enzyme from reacting with the substrate. This feature allows the cell to control how much reaction product is created.

Digestive enzymes are primarily contained in two cellular organelles—lysosomes and peroxisomes. Lysosome enzymes break down proteins, carbohydrates, nucleic acids and foreign particles. Peroxisomes are most common in liver and kidney cells where the enzyme peroxidase metabolizes long-chain fatty acids into cholesterol, hormones, bile acids and cell membrane components, producing hydrogen peroxide, H₂O₂, as a waste product. Hydrogen peroxide also occurs as a normal byproduct of cellular respiration. Hydrogen peroxide is generally considered to be an oxidizer and a buildup of this chemical would kill the cell. Consequently, peroxisomes contain the enzyme catalase. Catalase catalyzes the oxidation–reduction reaction of toxic hydrogen peroxide into harmless water and oxygen.

2H₂O₂ (aq) → 2H₂O(l) + O₂(g) + heat

The products of the catalase reaction are beneficial to the cell. Consequently, most living organisms contain the enzyme catalase.

Enzymes function best under very specific conditions and any change in these conditions will affect the rate of reaction by denaturing the catalyst. When an enzyme denatures, its shape changes, hindering its ability to bind with reactants. The most common analogy is that of a lock and key. If the lock (enzyme) is deformed, the key (reactant) will no longer fit and the lock will be unusable. There are four main conditions that affect the catalytic activity of an enzyme.

1. Activator and Inhibitor Concentration—Activators bind to the enzyme and increase the rate of reaction. This is an important physiological phenomenon that is used by the cell in times of cellular stress. Inhibitors decrease the rate of reaction by binding to the enzyme and physically blocking the active site, unfolding the protein’s structure or by binding to the allosteric site and turning off the enzyme. Many poisons are enzyme inhibitors that limit or prevent important biological processes and lead to cell and organ damage and possibly death.
2. pH—The cytoplasm of the cell is composed of mostly water and buffers resulting in a pH of approximately 7. Many enzymes function best at a pH of 7. Acidic or basic conditions within the cell can cause the enzyme to denature. Specifically, acidic conditions are characterized by an excess of H⁺ ions. These H⁺ ions bind with the protein’s –COO– and –NH₂ side chains creating a deformed catalyst. Basic conditions cause the protein to lose H⁺ ions creating a different deformation to the catalyst. Pepsin is an enzyme found within the human stomach. Pepsin and a few other enzymes function best in the acidic environment of the stomach. The internal environment of a lysosome is also acidic and lysosomal enzymes, like catalase, function best in an acidic environment. If a lysosome breaks open inside a cell, the digestive enzymes will not function well in the neutral pH environment of cytoplasm, protecting the cell from destruction.
3. Temperature—In general, the rate of a chemical reaction increases as temperature increases because the temperature increases the kinetic energy of the molecules. For enzymes this is true up to a specific optimal temperature. If the temperature increases beyond this optimal value, the protein’s structure will deform due to the increased kinetic energy of the system. This creates a dampening affect on the rate of reaction. Most enzymes function best at 37 °C, which is the normal, internal body temperature. An internal body temperature of 44 °C causes death in part due to permanent denaturing of body proteins.
4. Salt Concentration—Salt concentration also affects the rate of an enzyme’s catalyzed reaction. A high salt concentration means there are many dissociated ions that interfere with the polarity of the protein molecule. A low salt concentration hinders the enzyme because it allows the polar amino acid side chains of the enzyme to attract each other and deform the protein’s shape. In homeostasis, human blood has an intermediate salt concentration of 0.9%. Urine may be up to four times more concentrated than blood because the kidneys act to regulate salt concentration in blood. The kidneys selectively excrete or absorb sodium, chloride, potassium and other ions as needed by the body to maintain homeostasis.

This laboratory will focus on the enzyme catalase. A rate for the decomposition of hydrogen peroxide by catalase will be determined. The rate of reaction can be determined by measuring the amount of substrate used or H₂O₂ consumed from the moment the reactants are brought together until the reaction has stopped. By measuring the amount of product formed at regular intervals, a graph can be plotted for use in determining the rates of reaction. The graph that follows is an example (see Figure 1). Note: The graph is for the artificial environment of a beaker. Graphs for the rate of reaction within a living cell vary because the system varies as directed by the nucleus. In comparing the kinetics of one reaction with another, it is best to compare rates at the beginning of the reaction, before the slope of the line changes (see Figure 1). To compare the effectiveness of catalase obtained from a potato with that of catalase obtained from liver, the comparison should be within this initial velocity stage. In the first few minutes of this enzymatic reaction, the number of substrate molecules is large compared to the number of enzyme molecules. Consequently, the enzyme is consistently having successful collisions with substrate molecules. The enzyme is able to react with the substrate molecules as fast as it can during this initial period. When the amount of product formed or substrate consumed is plotted versus time as a line graph, the initial slope of the line is called the initial velocity of the reaction. The initial velocity is the same for a specific enzyme and substrate only if temperature and pH are constant and the substrate is present in excess.

To determine a reaction rate, pick any two points on the straight-line portion of the curve. Divide the difference in the amount of product formed between these two points by the difference in time between them. The result (the shape of the line) will be the rate of the reaction expressed as μmoles of product/second. This equation is: In the graph shown in Figure 1, the rate between two and three minutes is calculated:

Experiment Overview

Activity 1. The Uncatalyzed Rate of H₂O₂ Decomposition
Activity 1 is used to determine the rate of spontaneous conversion of H₂O₂ to H₂O and O₂ in an uncatalyzed reaction.

Activity 2. Test of Catalase Activity
In Activity 2, the general reaction of catalase on hydrogen peroxide, the effect of boiling the catalase on the reaction and the presence of catalase in living tissue will be observed. 

Activity 3. Enzyme-Catalyzed Rate of H₂O₂ Decomposition Over Time
Activity 3 procedures determine the enzyme-catalyzed rate of H₂O₂ decomposition over time. In this experiment, the disappearance of the substrate, H₂O₂, is measured as follows (see Figure 2):

1. The substrate, H₂O₂, and catalase are mixed in a beaker. The enzyme catalyzes the conversion of H₂O₂ to H₂O and O₂(gas).
2. After a specific amount of time, the reaction is stopped by adding sulfuric acid (H₂SO₄) to the enzymatic reaction. The lower pH, created by adding the acid, denatures the enzyme and stops the reaction.
3. The amount of substrate, H₂O₂, remaining in the beaker is measured by titrating with potassium permanganate, KMnO₄. Potassium permanganate reacts with H₂O₂ and H₂SO₄ as follows:

   5H₂O₂ + 2KMnO₄ + 3H₂SO₄ → K₂SO₄ + 2MnSO₄ + 8H₂O + 5O₂

   Note: After the H₂O₂ is consumed in this reaction, any excess KMnO₄ that is added will not react. The excess potassium permanganate added after this point will remain pink when added to the solution. Therefore, the amount of H₂O₂ remaining is determined by adding KMnO₄ until the whole mixture stays a faint pink or brown, permanently. The amount of KMnO₄ added is proportional to the amount of H₂O₂ remaining in the beaker.

A hydrogen peroxide baseline is established by performing all the steps of the titration without any catalase present. It is an index of the initial concentration of hydrogen peroxide in the solution. A titration baseline should be established at the beginning of each day that a titration is performed.

Determine the rate at which the hydrogen peroxide decomposes when catalyzed by the enzyme catalase. To do this, determine how much H₂O₂ has been consumed after 10, 30, 60, 120, 180 and 360 seconds.

To use lab time more efficiently, set up all of the trials at the same time. Add the catalase and stop the reaction at the proper time. Titrate after all reactions are complete.

Materials

Safety Precautions

Hydrogen peroxide is an oxidizer and a skin and eye irritant. Sulfuric acid is extremely corrosive to skin and eyes and other tissues. Potassium permanganate is a skin irritant and dye. The scalpel is a sharp object, care must be taken when cutting the potato or liver with the scalpel. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron throughout this lab. Wash hands thoroughly with soap and water before leaving the laboratory. Follow all normal laboratory safety guidelines.

Procedure

Activity 1. The Uncatalyzed Rate of H₂O₂ Decomposition

1. Label a 10-mL serological pipet “H₂O₂.”
2. Label a 120-mL beaker “Uncatalyzed “H₂O₂.”
3. Use the H₂O₂ pipet and a pipet bulb to add 15 mL of 1.5% H₂O₂ to the “Uncatalyzed H₂O₂” beaker.
4. Store it uncovered at room temperature for 24 hours to one week, as directed by instructor.

Activity 2. Test of Catalase Activity

Part 1. General Reaction

1. Keep the catalase solution in an ice–water bath at all times.
2. Use the H₂O₂ pipet to transfer 10 mL of 1.5% H₂O₂ into the beaker.
3. Use the 2-mL pipet to add 1 mL of freshly made catalase solution to the beaker.
4. Observe the reaction and answer the Activity 2, Part 1 questions on the Enzyme Catalysis Worksheet.
5. Discard solution into a waste bottle as directed by your instructor.
6. Rinse the 2-mL pipet and beaker thoroughly with distilled water.

Part 2. Boiled Catalase

1. Label a test tube with your group name or number.
2. Use a disposable pipet to transfer approximately 5 mL of catalase solution to the test tube.
3. Place the test tube in a boiling water bath for five minutes.
4. Use the test tube clamp to remove the test tube from the boiling water and place the test tube into the test tube rack. Relabel the test tube, if needed.
5. Use the H₂O₂ pipet to transfer 10 mL of 1.5% H₂O₂ into a clean 120-mL beaker.
6. Use the 2-mL pipet to add 1 mL of the boiled catalase solution to the beaker.
7. Observe the reaction. Answer the Activity 2, Part 2 questions on the Enzyme Catalysis Worksheet.
8. Discard solution into a waste bottle as directed by your instructor.
9. Rinse the 2-mL pipet and beaker thoroughly with distilled water.

Part 3. Living Tissue

1. Use the scalpel and ruler to cut 1 cm³ of raw potato or liver. Caution: The scalpel is a sharp object, use care when cutting.
2. Use the scalpel to cut the 1 cm³ of raw potato or liver into very small pieces.
3. Transfer the tissue into a clean 120-mL beaker.
4. Use the H₂O₂ pipet to transfer 10 mL of 1.5% H₂O₂ to the beaker.
5. Observe the reaction. Answer the Activity 2, Part 3 questions on the Enzyme Catalysis Worksheet.
6. Discard solution into a waste bottle as directed by the instructor.
7. Rinse beaker thoroughly with distilled water.

Activity 3. Enzyme-Catalyzed Rate of H₂O₂ Decomposition Over Time

1. Label each of 8 120-mL beakers with “titration,” “baseline” or “time” where time = 10, 30, 60, 120, 180 or 360 seconds, respectively.
2. Label 10-mL syringe “H₂SO₄.”
3. Use the H₂O₂ pipet to transfer 10 mL of 1.5% H₂O₂ into each beaker.
4. Fill the H₂SO₄ syringe with 10 mL of 2 M H₂SO₄. (Use extreme care in handling acids. Sulfuric acid is corrosive and can cause chemical burns.)
5. Use the 2-mL pipet to add 1 mL of distilled H₂O (instead of catalase solution) to the baseline-beaker.
6. Use the H₂SO₄ syringe to add the 10 mL of 2 M H₂SO₄ to the baseline-beaker.
7. Gently swirl the solution for 30 seconds and then set the baseline-beaker aside.
8. Fill the H₂SO₄ syringe with 10 mL of 2 M H₂SO₄.
9. Use the 2-mL pipet to add 1 mL of catalase extract to the 360-second-beaker. Immediately start timing.
10. Gently swirl for 360 seconds. Note: Swirl for the entire 360 seconds.
11. After 360 seconds, use the H₂SO₄ syringe to add 10 mL of 2 M H₂SO₄ to stop the reaction.
12. Gently swirl the solution to mix the reactants and completely stop the reaction before setting the 360-second-beaker aside.
13. Repeat steps 8 through 12 adding the 2 M H₂SO₄ after 180, 120, 60, 30 and 10 seconds, to each respective beaker.
14. Assemble the titration apparatus as shown in Figure 3.
    {10757_Procedure_Figure_3}
15. Titrate the H₂O₂ remaining in each beaker as follows:
    1. Adjust buret clamp so the buret tip is 1 cm above the top of the “titration” beaker.
    2. Fill the buret with 10 mL of potassium permanganate using a clean disposable pipet. Caution: Potassium permanganate will stain clothes, skin and equipment.
    3. Place waste cup under the buret tip and slowly allow a few drops of potassium permanganate to drip into a waste cup. This removes any air bubbles in the buret tip.
    4. Record the initial volume of KMnO₄ in the buret in Data Table 1. Read from the top of the meniscus each time because the potassium permanganate is too dark to read the bottom of the meniscus as is proper.
    5. Use a clean 10-mL serological pipet to remove a 5-mL subsample from the baseline-beaker. Place this 5-mL sample into the titration-beaker.
    6. Place the titration-beaker on a piece of white paper under the buret on the support stand.
    7. Use the buret to add KMnO₄ one drop at a time to the solution. Note: If the solution turns pink immediately, note the buret reading then add three more drops and see if the solution turns clear again. If it does, disregard the previous final reading and titrate to the true end point.
    8. Gently swirl the solution in the titration-cup after adding each drop.
    9. Continue to add one drop at a time until a pale pink or brown color is obtained and persists for 30 seconds (indicating excess KMnO₄).
    10. Record the final buret reading (volume of KMnO₄) in Data Table 1 on the Enzyme Catalysis Student Worksheet.
    11. Discard the titrated solution into a waste bottle as directed by your instructor.
    12. Rinse the 10-mL pipet and the titration beaker thoroughly with distilled water.
    13. Use the 10-mL pipet to remove a 5-mL subsample from the 360-second-beaker and transfer the sample into the titration-beaker.
    14. Repeat steps a–k for each time trial. Should the end point be overshot, discard sample, rinse the titration beaker and repeat the assay using a new subsample. Do not discard any solutions until the entire lab is completed.
16. Answer the questions for Activity 3 on the Enzyme Catalysis Worksheet.
17. Repeat steps 16 d–n from Activity 3 using the uncatalyzed H₂O₂ from Activity 1 to determine the amount of H₂O₂ remaining. Note: For ease of calculation, assume that 1 mL of KMnO₄ used in the titration represents the presence of 1 mL of H₂O₂ in the solution.
18. Record the measurements in Data Table 1 on the Enzyme Catalyst Worksheet.
19. Complete the table and answer the Activity 3 questions on the Enzyme Catalysis Worksheet.
20. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10757_Student1.pdf