Enzyme Lab
Enzymes are biological catalysts (molecules that increase the rate of a reaction) that control chemical reactions
that would otherwise virtually never occur at normal body temperature (37°C). Thousands of chemical reactions are
occurring in every organism every moment it is alive, and each of these reactions is controlled by a particular enzyme.
Some of the chemical reactions that take place in cells produce toxic by-products, which must be quickly
degraded or converted. For example, many cells produce hydrogen peroxide (H2O2) during the process of cell
respiration (metabolic energy production). Hydrogen peroxide is extremely poisonous and will damage cells if it is
not removed quickly. Catalase is an enzyme that is able to break down the hydrogen peroxide into harmless water
(H2O) and oxygen gas (O2). This breakdown prevents the peroxide from causing unwanted oxidation (breakdown) of
important biomolecules.
Enzymes are extremely efficient. One molecule of catalase can deal with six million molecules of hydrogen
peroxide in one minute. This same reaction can be catalyzed by iron. However, to achieve the same speed there would
need to be about six tons of iron.
In this lab we will use catalase from the skin of red potatoes to demonstrate how various factors affect the
activity of the enzyme. Enzymes only work within specific temperature and pH ranges. There is also an ideal ratio of
substrate to enzyme. Your task is to identify the ideal ranges of temperature and pH as well as substrate/enzyme
ratios for catalase. The amount of foam produced will indicate the activity level of the enzyme (more foam = more
oxygen gas production = more catalase activity).
Part 1: Control
Procedure:
1. Set up two test tubes, one labeled A and the other labeled B. Fill each
with 3 ml distilled water and 2 ml hydrogen peroxide.
2. Pour about 3 ml of the enzyme solution into test tube B.
3. Observe what happens for 2 minutes, answer analysis question # 1.
Option #1: The Effect of Temperature on Enzyme Activity
1. Pour 3 ml of the enzyme solution into four of the test tubes (labeled A-D).
2. The four test tubes should be placed in water baths at (or within 2*C) the following temperatures:
(Note: temperatures are approximate, you must report the ACTUAL temperatures in your analysis)
A. 5 degrees Celsius
B. 20 degrees Celsius
C. 35 degrees Celsius
D. 50 degrees Celsius
3. To the second set of test tubes (labeled A-D) add 2 ml H2O2.
4. After each of the first four test tubes -- containing the enzyme -- have sat at their temperatures for 4 minutes,
dump their contents into the set of test tubes containing the H2O2. All four of the solutions should be
combined at the same time. Be sure to dump the enzyme from A into the other A, B into B, etc.
5. Allow the enzyme to work for 2 minutes then measure the height of the foam produced. Record the results.
Option #2: The Effect of pH on Enzyme Activity
1. Pour 2 ml of H2O2 into four of the test tubes (labeled A-D). To each test tube, add the following:
A.
B.
C.
D.
1 ml vinegar (pH 2.4)
1 ml tea (pH 5)
1 ml distilled water (pH 7)
1 ml baking soda solution (pH 9)
2. To the second set of test tubes (labeled A-D), add 3 ml of the enzyme solution
3. Pour the enzyme solution into the set of test tubes containing the H2O2 mixture. All four of the solutions
should be combined at the same time. Be sure to dump the enzyme from A into the other A, B into B, etc.
4. Allow the enzyme to work for 2 minutes then measure the height of the foam produced. Record the results.
Option #3: The effect of substrate concentration on enzyme activity
1. Pour 3 ml of the enzyme solution into four of the test tubes (labeled A-D).
2. To the second set of test tubes (labeled A-D), add the following amounts of H2O2:
A. 1 ml
B. 3 ml
C. 5 ml
D. 7 ml
3. Pour the enzyme solution into the set of test tubes containing the H2O2. All four of the solutions should be
combined at the same time. Be sure to dump the enzyme from A into the other A, B into B, etc.
4. Allow the enzyme to work for 2 minutes then measure the height of the foam produced. Record the results.
Option #4: The effect of enzyme concentration on enzyme activity
1. Pour 2 ml of H2O2 into four of the test tubes (labeled A-D).
2. To the second set of test tubes (labeled A-D), add the following amounts of the enzyme solution:
a. 1 ml
b. 2 ml
c. 4 ml
d. 6 ml
3. Pour the enzyme solution into the set of test tubes containing the H2O2. All four of the solutions should be
combined at the same time. Be sure to dump the enzyme from A into the other A, B into B, etc.
4. Allow the enzyme to work for 2 minutes then measure the height of the foam produced. Record the results.
Enzyme Pre-LAb
Answer all question on a separate sheet of paper
Pre-lab Questions
1. What is an enzyme?
2. During what process is hydrogen peroxide commonly produced?
3. Why is it important for H2O2 to be broken down?
4. What enzyme breaks down hydrogen peroxide?
5. What is produced when H2O2 is broken down?
6. Describe the relationship between a substrate and an enzyme.
7. Identify the enzyme and substrate in this experiment.
8. In this lab, what will be used as a source of catalase?
9. Identify the control in this experiment.
10. What four independent variables will be tested in this lab? Briefly describe how each will be tested.
11. What independent variable will your group explore in this lab?
12. What will be the dependent variable in this lab?
13. Create a hypothesis for the variable you will be testing.
14. List 3 of the constants in your experiment.
Post lab questions – this section must be typed!!!
1. Describe the difference between the reactions in test tube A and B. Explain why one of the tubes had no
reaction.
2. Complete the data section of a lab report for this experiment (see lab report instructions). You must include a
graph!
3. Was the hypothesis accepted or rejected? Defend your response using data from the experiment.
4. Based on the data from this experiment (you will need to get data from students who tested the other 3
variables), what are the ideal conditions for catalase?