Enzyme Catalysis Investigation Name ____________________

OBJECTIVES: When you have finished this lab, you will have learned
How to analyze data to identify how molecular interactions affect structure and function.
That change in the structure of a molecular system may result in a change of the function of the system.
That the shape of enzymes, active sites and interaction with specific molecules are essential for basic
functioning of the enzyme.
That the change in function of an enzyme can be interpreted from data regarding the concentrations of
product or substrate as a function of time.
How to pose, refine and evaluate scientific questions.
How to analyze data to identify patterns or relationships.
How to evaluate the evidence provided by a data set related to a particular scientific question.

INTRODUCTION:
Many organisms can decompose hydrogen peroxide (H2O2) enzymatically. Enzymes are globular proteins, responsible
for most of the chemical activities of living organisms. They act as c atalysts, substances that speed up chemical
reactions without being destroyed or altered during the process. Enzymes are extremely efficient and may be used over
and over again. One enzyme may catalyze thousands of reactions every second. Both the temperature and the pH at
which enzymes function are extremely important. Most organisms have a preferred temperature range in which they
survive, and their enzymes most likely function best within that temperature range. If the environment of the enzyme is
too acidic or too basic, the enzyme may irreversibly denature, or unravel, until it no longer has the shape necessary for
proper functioning.
H2O2 is toxic to most living organisms. Many organisms are capable of enzymatically destroying the H2 O2 before it can
do much damage. H2O2 can be converted to oxygen and water, as follows:
2 H2 O2 2 H2 O + O2
Although this reaction occurs spontaneously, enzymes increase the rate considerably. At least two different enzymes are
known to catalyze this reaction: catalase, found in animals and protists, and peroxidase, found in plants. A great deal can
be learned about enzymes by studying the rates of enzyme-catalyzed reactions.
In this experiment, you will measure the rate of enzyme activity under various conditions, such as different enzyme
concentrations, pH values, and temperatures. It is possible to measure the pressure of oxygen gas formed as H2O2 is
destroyed. If a plot is made, it may appear similar to the graph shown.


At the start of the reaction, there is no product, and the pressure
is the same as the atmospheric pressure. After a short time,
oxygen accumulates at a rather constant rate. The slope of the
curve at this initial time is constant and is called the initial rate. As
the peroxide is destroyed, less of it is available to react and the
O2 is produced at lower rates. When no more peroxide is left, O2
is no longer produced.

Purpose: This laboratory demonstrates the action of the enzyme catalase on H202 (hydrogen peroxide). Hydrogen
peroxide is decomposed into water and oxygen gas by catalase activity. The amount of hydrogen peroxide that remains
following various period of catalase action will be measured to determine the rate of catalase activity

Part 1: The Uncatalyzed Rate of H202 Decomposition:

Materials:
- 1 Medicine cup with 1.5% H202 (from Ms. - 10 mL 1.0 H2SO4
Krause) - 10 mL 2% KMnO4
- 2 medicine cups - Marking pen/pencil
- 1 12-mL syringe - Labeling tape
- 1 1mL pipet - Paper, white
- 1 mL Distilled water

Safety warning:
1) Be sure to always wear safety goggles, gloves and a lab apron to protect your eyes
2) Sulfuric acid (H2SO4) is a strong acid and be an irritant if allowed to come in contact with the skin
3) Potassion permanganate (KMnO4) will stain skin, equipment, and clothing if contact occurs
4) Dispose of any waste materials and clean up your work area as directed by your teacher
5) Be sure to always wash your hands before leaving the laboratory
Procedure:
1) Remove 10 mL of the H202 from the cup using the 12 mL syringe and transfer it to another medicine cup labeled
Uncatalyzed H2 02 Activity. Thoroughly rinse the syringe after using it (this means go to the sink and remove
popper from syringe and rinse)
2) Using the 1 mL pipet, add 1 mL of distilled water to the H2 02 in the Uncatalyzed H2 02 Activity cup
3) Using the 12 mL syringe, add 10 mL of 1.0 M H2SO4 to the cup. Thoroughly rinse the syringe after using it (this
means go to the sink and remove popper from syringe and rinse)
4) Swirl gently to mix the contents of the cup
5) Label the remaining medicine cup Uncatalyzed Assay. Using the 12 mL syringe, transfer 5 mL of the
Uncatalyzed H202 Activity mixture to this cup. Thoroughly rinse the syringe after using it
6) Draw 10 mL of 2% KMnO4 into the 12 mL syringe.
7) Place a white sheet of paper on the counter where you will be performing the titration. Place the Uncatalyzed
Assay cup on the white sheet of paper. Read the initial volume of KMnO4 in the syringe and record this is Data
Table 1
8) Holding the 12 mL syringe over the cup, carefully depress the plunger so that one drop of KMnO4 falls into the
cup. Swirl the cup gently and observe the solution for any pink to brown coloration that remains after mixing is
complete
9) Complete the titration by repeating Step 8, drop by drop until a pink to brown coloration of the solution persists
after mixing. Record the final volume of KMnO4 solution in the 12 mL syringe in Data Table 1.
10) DISPOSAL: Rinse out the syringe but DO NOT THROW IT AWAY. Throw away the pipette you were using. Keep
ALL other solutions labeled and put them to the side. You will throw them away when I tell you later on in the lab.
Analysis:
Data Table 1: The Uncatalyzed Rate of H2 02 Decomposition

Initial KMnO4 volume in 12 mL syringe (mL)

Final KMnO4 volume in 12 mL syringe (mL)

KMnO4 used in uncatalyzed titration (initial-final) _______mL (transfer this value to Data table 3)

Part 2: Test of Catalase Activity

Materials:
- 30 mL 1.5%H202 - 12 mL syringe
- 3 medicine cups - 1 mL pipet
- 6 mL Catalase solution in medicine cup - Marking pen/pencil
- 1 test tube - Labeling tape
- 1 test tube holder - Hot plate
- 1 test tube rack - 250 mL beaker with water for boiling catalase
- 1 wood macerating stick - Container with ice for catalase (class set)
- 1 cm2 cube of potato - Knife/scalpel for cutting potato
Procedure:
Part A: Observation of Catalase in Action
1) Using the 12 mL syringe place 1.5% H202 into a medicine cup labeled Catalase Extract Activity. Thoroughly
rinse the syringe after using it
2) Using the 1 mL pipet, add 1 mL catalase solution to the H202 in the medicine cup. Be sure to keep the remainder
of the catalase solution in the ice container whenever it is not being used. Thoroughly rinse the pipet after using
it
3) Gently swirl the cup to mix the contents, and observe the reaction occurring in the cup. Record your
observations in Data Table 2.
Part B: The Effect of Boiling on Catalase Action
1) Using the 12 mL syringe, place 5 mL catalase solution into a test tube. Thoroughly rinse the syringe after using
it.
2) Using the test tube holder, place the test tube containing the catalase solution into the boiling water bath.
3) After 5 minutes, remove the tub from the boiling water bath with the test tube holder and place it in the test tube
rack
4) While the catalase solution is cooling, place 10 mL of 1.5% H202 solution in a medicine cup labeled Boiled
Catalase Activity. Thoroughly rinse the syringe after using it
5) When the catalase solution in the test tube has cooled to room temperature, add 1 mL to the H202 in the
medicine cup
6) Gently swirl the cup to mix the contents, and observe any reaction occurring in the cup. Record your
observations in Data table 2.
Part C: Catalase Action in Living Tissue
1) Obtain a 1 cm2 of potato tissue and place it in a medicine cup labeled Liver Tissue Catalase Activity
2) Use the end of the wood macerating stick to grind the tissue into small pieces
3) Add 10 mL of the 1.5% H2 02 solution to the macerated tissue in the cup. Gently swirl the cup to mix the
contents, and observe any reaction taking place in the cup. Record your observations in Data Table 2.
Thoroughly rinse the syringe after using it.
4) DISPOSAL: Dispose of the pipet. Put ALL medicine cups to the side until the end of the lab.
Analysis:
1) Write the overall reaction for this enzymatic reaction:

Data Table 2:
Experiment Enzyme Substrate Product Observation

Test of Catalase
Activity (Part A)

Effect of Boiling on
Catalase (Part B)

Catalase from Living

Tissue (Part C)
Part 3: Baseline Assay and Enzyme catalyzed Rate of H 202 Decomposition
Materials:
- 70 mL 1.5% H2 02 in 120 mL graduated cup - 70 mL 1.0 M H2SO4 in glass beaker
- 8 medicine cups - 40 mL 2% KMnO4 in 120 mL graduated cup
- 1 12 mL syringe - Marking pen/pencil
- 1 1 mL pipet - Labeling tap
- 1 mL distilled water in medicine cup - Stopwbaatch
- 6 mL catalase solution in medicine cup - Paper, white
Procedure:
1) Label seven of the eight medicine cups as shown in the table below. For cups 1-6 the label indicates the length
of time that the reaction will be allowed to proceed.

Cup Label

1 10 seconds

2 30 seconds

3 60 seconds

4 120 seconds

5 180 seconds

6 360 seconds

7 Baseline

2) Into each of the seven labeled cups add 10 mL of 1.5% H202 solution. Thoroughly rinse the 12 mL syringe after
completing this step
3) Using the 1 mL pipet, add 1 mL of distilled water to the H202 in Cup 7 (Baseline)
4) Using the 12 mL syringe, add 10 mL of 1.0 M H2SO4 to Cup 7 (Baseline)
5) Swirl gently to mix the contents of Cup 7. Set this cup aside for later titration
6) Have the stopwatch or clock with second hand ready to begin timing the reaction. Refill the 12 mL syringe with
10 mL of 1.0 M H2SO4 and have it ready to add to the reaction mixture
7) Using the 1 mL pipet, add 1 mL of catalase extract to Cup 1 and begin timing (10 seconds). Swirl the mixture
gently. At the end of 10 seconds add the 10 mL of H2SO4 and swirl. Set Cup 1 aside with Cup 7 for titration later
8) Repeat steps 6 and 7 with Cups 2-6 timing each for the length indicated on their labels. Set each aside for
titration after the reaction is completed. Thoroughly rinse the pipette and syringe after completing this step.
9) Label the remaining medicine cup Titration Vessel. Using the 12 mL syringe transfer 5 mL of the solution from
Cup 7 to the titration vessel. Save the remainder of each solution until all data are collected. Thoroughly rinse
the syringe after using it
10) Draw 12 mL of 2% KMnO4 into the 12 mL syringe
11) Place a white sheet of paper on the counter where you will be performing the titration. Place the titration vessel
on the white sheet of paper. Read the initial volume of KMnO4 in the syringe and record this in Data Table 3
12) Holding the syringe over the titration vessel, carefully depress the plunger so that one drop of KMnO4 falls into
the cup. Swirl the cup gently and observe the solution for any pink to brown coloration that remains after mixing
is complete
13) Complete the titration by repeating step 12, drop by drop until a pink to brown coloration of the solution persists
after mixing. Record the final volume of KMnO4 solution in the 12 mL syringe in Data table 3.
14) Dispose of the sample in the titration vessel by pouring it down the sink, rinsing it with water and drying it with a
SMALL piece of paper towel. Rinse and dry the titration vessel. Repeat the titration on 5 mL of each of the
remaining reaction mixtures (10, 30, 60, 120, 180, 360 seconds). Be sure to clean and dry the titration vessel in
between each titration. Record your data for each mixture in Data Table 4.
 15) DISPOSAL: Rinse out ALL of the medicine cups and place them on the drying paper towel next to the sink.
Rinse out ALL of the 12 mL syringes and put them on the drying paper towel at the syringe station. Dispose of all
other materials
16) Perform the calculations for the H202 decomposition and catalase reaction rates in Data Tables 3,4, and 5. The
reaction rate data from Data Table 5 will then be used to construct a graph.

Analysis:
Notes: The volume (mL) of KMnO4 used in each activity in this laboratory is proportional in a 1:1 ratio to the volume (mL)
of H202 decomposed
Data Table 3: Calculation of Baseline mL H2 02 and % Spontaneous Decomposition of H202 in 24 hours

Baseline Assay

1. Initial KMnO4 volume in 12 mL syringe

2. Final KMnO3 volume in 12 mL syringe

3. KMnO4 volume used in Baseline titration

4. Baseline mL H202 present in 5 mL sample

Calculation of Spontaneously Decomposed H202

5. KMnO4 used in Uncatalyzed reaction (from Data Table

1)

6. Baseline mL KMnO4 (Line 3)- mL of KMnO4 used in

Uncatalyzed titration (Line 5)

7. Volume (mL) of spontaneously decomposed H202

8. % Spotaneous Decomposition of H202 in 24 hours:

((mL Baseline- mL uncatalyzed/7)/ mL Baseline) x 100

Data Table 4: Results of Timed Catalase Reaction

Time

KMnO4 Volumes (mL) 10 30 60 120 180 360

1. Baseline KMnO4 used

(from Data Table 3)
Note: enter this value in
all columns in this row

2. Initial KMnO4 volume

in 12 mL syringe

3. Final KMnO4 volume

in 12 mL syringe

4. KMnO4 used in each

timed titration (initial-
final)

5. Volume of H202
decomposed at each time
interval (line 1-line 4)

Calculate the rate of the reaction for each of the time increments listed in Data table 5 by dividing the difference in
volume of H202 in one time interval by the number of seconds in that interval. This is the same calculation necessary to
determine the slope of the line.
Data Table 5: Reaction Rates for Catalase Decomposition of H202
TIme Increments

Rate of 0-10s 10-30 s 30-60 s 60-120 s 120-180s 180-360s

Reaction:

(mL H202 /sec)

Graphing the results of the enzyme catalyzed decomposition of H202:

You will construct a graph of the rate of catalase action below. Use the following instructions to construct the
graph: Plot the dependent variable on the Y-axis; plot the independent variable on the x-axis. Dont forget to label each
axis with a descriptive label as well as any appropriate units. Give the graph an appropriate title as well.
The independent variable in a scientific experiment is the condition that the experimenter deliberately varies.
What condition in this experiment did you allow to vary to see if that had any effect on the experiments results?
Independent variable: _______________________________
The dependent variable in a scientific experiment is what the experimenter designed the experiment to
measure. What variable did you measure at the completion of this experiment?
Dependent variable: _________________________________
Graph:

Discussion Questions: Answer these on a separate piece of paper to be attached to your lab report.
1) Why was performing a baseline assay necessary to interpret the results of your experiments?
2) Why was performing an assay on uncatalyzed H202 important? What does this information tell you about the
shelf life of H2 02 , which is sometimes a disinfectant?
3) Explain the results of the exercise that used boiled catalase extract. What would your predictions have been for
an experiment that used cooked potato instead of fresh potato as a catalase source?
4) Why was H2SO4 used in these experiments? How did adding H2SO4 have the desired effect on the reaction
mixture?
5) During which time increment on your graph is the reaction rate the steepest? Why
6) During which time increment(s) on your graph is the reaction rate the slowest? Why?