Factors Affecting Enzyme Activity
Objective: To study several factors that affect the activity of enzymes
Background: Catalase is an enzyme present in most cells and found in high concentration in liver and
blood cells. You will use liver homogenate as the source of catalase. Catalase promotes the decomposition
of hydrogen peroxide (H2O2) in the following reaction:
2 H2O2 2 H2O + O2
Hydrogen peroxide is formed as a by-product of chemical reactions in cells. It is toxic and would soon kill
cells if not immediately removed or broken down.
Materials: (per team of 2)
2 pairs safety goggles
2 lab aprons
50 mL beaker
10 mL and 50 mL graduated cylinder
Test tubes
Fresh 3% H2O2
forceps
Water pan
Catalase solution
Test tube rack
Thermometer
Filter Paper
Paper punches
Catalase
Reaction chambers (Drosophila vials with 1-hole stoppers
Stop watch
Ice
Also needed to prep lab: liver, cheesecloth, blender
Procedure:
In all experiments, make sure that your reaction chamber is scrupulously clean. Catalase is a potent enzyme,
and if the chamber is not washed thoroughly, enough will adhere to the sides to make subsequent tests
inaccurate. Measure all substances carefully. Results depend on comparisons between experiments, so the
amounts measured must be equal or comparisons will be valueless. Before you do the experiment, read
through the instructions 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 his or her assigned function.
Part A The Time Course of Enzyme Activity
1. Prepare a table in your data book similar to Table 1.
2. Obtain a small amount of stock catalase solution in a 50-ml beaker
3. Obtain a reaction chamber and a number of filter-paper disks.
4. Place four catalase-soaked filter paper disks high on one interior sidewall of the reaction chamber. (They
will stick to the sidewall.) Prepare a disk for use in the reaction chamber by holding it by its edge with a
pair of forceps and dipping it into the stock catalase solution for a few seconds. Drain the disk against
the sidewall of the beaker before you transfer it to the reaction chamber. CAUTION: Forceps are
sharp. Handle with care.
5. Stand the reaction chamber upright and carefully add 10 mL of 3% hydrogen peroxide solution. Do not
allow the peroxide to touch the filter paper disks.
6. Tightly stopper the chamber.
7. Fill a pan almost full with water.
8. Lay the 50-mL graduated cylinder on its side in the pan so that it fills with water completely. If any air
bubbles are present, carefully work these out by tilting the cylinder slightly while keeping it underwater.
Turn the cylinder upside down into an upright position keeping its mouth underwater at all times.
9. Carefully place the reaction chamber and its contents on its side in the pan of water. Make certain that the
side with the disks faces upward.
10. Move the graduated cylinder into a position so that its mouth comes to lie directly over the tip of the
dropping pipet. One member of the team should hold it in this position for the duration of the experiment.
11. Rotate the reaction chamber 180o on its side so that the hydrogen peroxide solution comes into contact
with the catalase-soaked disks.
12. Measure the gas levels in the graduated cylinder at 30-second intervals for 10 minutes. Record the levels
in your data table.
TABLE 1: Catalase activity over time
Time
30 seconds
1:00
1:30
2:00
2:30
3:00
3:30
4:00
4:30
5:00
5:30
6:00
mL O2 evolved
6:30
7:00
7:30
8:00
8:30
9:00
9:30
10:00
A Simple Enzyme Lab as Guided Inquiry
Print these and have students select one. Have them brainstorm a procedure, and then explain to you their hypothesis and basic plan before
leaving class. These are very basic ideas which we initially use with 9 th graders. You may research additional ideas online. What is the
optimum temperature? pH? effect of salt? activity of catalase from different sources? temperature optimum for potato vs. chicken liver source?
Effect of Concentration of Enzyme on Catalase Activity
Devise a plan to prepare 4 concentrations of enzyme. Include a control.
Discuss your plan with your teacher.
Hypothesis:
Prepare your data chart.
Effect of Concentration of substrate on Catalase Activity
Devise a plan to prepare 4 concentrations of substrate. Include a control. The stock solution of hydrogen
peroxide is 3%.
Discuss your plan with your teacher.
Hypothesis:
Prepare your data chart.
Effect of Temperature on Catalase Activity
Devise a plan to prepare look at the action of catalase at 4 temperatures. The room temperature reaction will
serve as your control.
Discuss your plan with your teacher.
Hypothesis:
Prepare your data chart.
Effect of pH on Catalase Activity
Devise a plan to prepare look at the action of catalase in acidic, neutral and basic environments. Test the pH
of the original experiment. This will serve as your control.
Discuss your plan with your teacher.
Hypothesis:
Prepare your data chart.