Enzyme Catalysis Lab
AP Biology - Energy
Part 1: Learning the Technique
Background​
:
Catalase​
is an enzyme present in most cells and found in high concentration in liver and blood
cells. Hydrogen peroxide on its own is incredibly toxic to your body, and can cause severe damage
if not disposed of. It is formed as a byproduct of chemical reactions in cells. The breaking part of
hydrogen peroxide into water and oxygen gas (two non-harmful substances) is the job of catalase.
Catalase promotes the decomposition of hydrogen peroxide (H2​
O2​
) in the following reaction:
​
​
2 H​
→ 2 H​
2O​
​ 2​
2O
​ + O​
2
You will measure enzyme activity by measuring the generation of oxygen gas – a product in the
reaction. Our source of catalase will come from yeast cells.
Question​
:
How can we measure the rate of the reaction between the enzyme catalase and its substrate
hydrogen peroxide?
Materials​
(per group):
​
100 mL beaker
10 mL graduated cylinder
50 mL gas measuring tube
Eppendorf pipette
Eppendorf pipette tip
3% hydrogen peroxide (H​
2O
​​
2)​
Plastic container
Room temperature water
Reaction chamber
Ring stand and buret clamp
Catalase solution
In all experiments, make sure that your reaction chamber is ​
very clean​
. Catalase could adhere to
the sides of the chamber if not washed thoroughly introducing inaccuracies into your data.
Measure all substances carefully. Results depend on comparisons between experiments, so the
amounts must be kept ​
constant. ​
Before you do the experiment, ​
read the procedure completely.
Make sure that you have all the required materials on hand, that you understand the sequence of
steps, and that each member of your team knows their role.
Part 1: General Procedure and Set Up
1.
All ​
lab groups will complete Part 1 of the lab.
​
2. Part 2 is open inquiry in which teams investigate different factors affecting enzymes.
3. Set up the equipment as demonstrated by Mr. Erlenbeck.
4. Fill the gas collection tube with water and invert it being sure to plug the opening with your
index finger. A small bubble will make its way to the top of your collection tube. Submerge
the opening of the tube below the surface of the water in your plastic container and clamp
it to the buret clamp leaving room between the bottom of the plastic container and the
opening of the gas collection tube.
5. Have Mr. Erlenbeck inspect your set-up.
6. Obtain a bottle of 3% hydrogen peroxide solution and a 10 mL graduated cylinder.
7. Obtain some stock catalase (yeast) solution from the front of the room. Fill your 100 mL
beaker about half-way with this solution.
8. Pour 10 mL of hydrogen peroxide into the reaction chamber.
9. Pipette 1 mL of of catalase solution using your eppendorf pipette. Be sure the device is set
to 1000 µL (1 mL).
10. Pipette the 1 mL of stock catalase (yeast) solution into the reaction chamber and
IMMEDIATELY ​
stopper the reaction chamber tightly, submerge it in the water bath, and
place the plastic tip of the reaction chamber directly underneath the opening of the gas
collection tube so the bubbles are feeding up into the tube.
11. Be sure to press the reaction chamber to the bottom of the plastic bin. Keep the reaction
chamber level as to not let any of the solution reacting inside to escape into the water in
the bin.
12. Measure the cumulative volume of gas being produced in 30 second intervals for 5
minutes. Record the levels in the provided data tables.
13. Repeat steps 8-12 with 1 mL of water instead of catalase solution to serve as your
experimental control. Be sure to clean out your reaction chamber thoroughly before
running your control.
14. Answer the Analysis Question and begin preparations for Part 2.
Data Table for Oxygen Gas Production by the Enzyme Catalase
Trial One
Time (seconds)
O​
produced (mL)
2​
Control Trial
Time (seconds)
30
30
60
60
90
90
120
120
150
150
180
180
210
210
240
240
270
270
300
300
O​
produced (mL)
2​
Questions: ​
Answer as a group to guide your thinking for Part 2.
1. How can we use the air bubbles in the graduated cylinder to show enzyme activity?
2. What served as the control? What is the independent variable? What’s the
dependent variable?
3. What other factors may affect the rate of enzyme activity? How would you test one
of these factors?
Part 2: Open Inquiry
In Part 2 you’ll investigate the effect of one of the five variables below. You’ll need to
develop a procedure, discuss your plan with Mr. Erlenbeck, prepare data charts, and develop
a hypothesis before beginning your experiment.
Effect of Concentration of Enzyme on Catalase Activity
Devise a plan to prepare 4 concentrations of enzyme. Include a control.
Discuss your plan with Mr. Erlenbeck.
Prepare a data chart.
Hypothesis:
Effect of Concentration of substrate on Catalase Acitivty
Devise a plan to prepare 4 concentrations substrate. Include a control.
Discuss your plan with Mr. Erlenbeck.
Prepare a data chart.
Hypothesis:
Effect of Temperature on Catalase Activity
Devise a plan to observe the action of catalase at 4 different temperatures. The room
temperature reaction will serve as your control.
Discuss your plan with Mr. Erlenbeck.
Prepare a data chart.
Hypothesis:
Effect of pH on Catalase Activity
Devise a plan to observe the action of catalase at pH 6, 7, 8, and 12. Test the pH of the
original experiment. This will serve as your control.
Discuss your plan with Mr. Erlenbeck.
Prepare a data chart.
Hypothesis:
Effect of Salinity on Catalase Activity
Devise a plan to prepare 4 concentrations of salt solution to observe their affect on the
enzyme catalase. The original experiment will serve as your control.
Discuss your plan with Mr. Erlenbeck.
Prepare a data chart.
Hypothesi​
s: