Activity 5

Teacher Notes Catalase Enzyme Activity

PS-2820

Teacher Notes Activity 5: Catalase Enzyme Activity

Time Estimates

Preparation: 30 min

Activity: 50 min

Objectives
Students will be able to

measure the concentration of gaseous oxygen in a container with a mixture of dilute hydrogen
peroxide (H2O2) and catalase extract under a variety of conditions (e.g., change in pH, change in
temperature)

compare the production of gaseous oxygen for each trial

calculate the rate of catalase enzyme activity for each trial

state how the catalase enzyme activity is affected by the various changes in conditions

Notes

Sensor calibration is good laboratory practice. However, this activity deals only with
relative changes in measurements. It is not necessary to calibrate the CO2 Gas Sensor
Sensor.

The materials in this lab present no serious safety hazards. The peroxide mixtures can be flushed
down the drain with a large quantity of water. Run the water for around a minute. For
information about the safe handling and disposal of chemicals, refer to the Flinn Scientific
catalog. Contact Flinn Scientific at (800) 452-1261.

As a pre-lab activity or demonstration, allow students to add hydrogen peroxide to a

variety of organisms or tissues and see the bubbles of oxygen that are formed. Suggested
tissue materials include slices of vegetables such as potatoes, turnips, or other starchbearing vegetables; sliced fruits (e.g., apples, pears, and bananas); and cultures of
organisms (e.g., yeast). Slices of animal liver produce a large reaction and an abundance of
oxygen.

Slice or puree the material (about 25 cm3) and place in a small beaker. Add a small amount
(about 25 mL) of undiluted 3% H2O2 to the beaker and the material will begin to foam up.
Ask the students what the foam is and allow them the opportunity to design a simple
experiment to determine what the evolved gas might be.

Students can demonstrate that the gas evolved is oxygen by placing a glowing wooden
splint into a large bubble or a blob of such bubbles. In the presence of oxygen, the glowing
tip of the splint will flash a bright light or burst into flames. Compare the effect the
glowing wooden splint has on the CO2 bubbles produced by mixing vinegar and baking
soda, for instance.

Follow up the inquiry activity or demonstration with a discussion of the importance of

catalase and then have the students work through the lab.

Chicken livers are readily available and inexpensive. A single, onepint package (less than
1 lb or about 0.5 kg) can supply an entire class with all the liver they need. You can freeze
unused livers for about a year before the enzymatic activity of the catalase begins to

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2005 PASCO

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Activity 5

Teacher Notes Catalase Enzyme Activity

PS-2820

decline. Beef livers and pork livers also have high levels of catalase and make good
substitutes for chicken livers.

You can save time by preparing the crude catalase extract solution for the entire class. For
a class with 6-10 computer stations, place 2 entire chicken livers in a blender with 100 mL
of cold, distilled water. Blend on high with three, 10-second bursts. Filter the material
through 3 or 4 layers of cheesecloth and collect the liquid. Throw away the cheesecloth.
Dispose of any unused catalase extract after the class is over. Prepare a new batch for each
class period. Do not try to save the material overnight.

If you prefer not to use liver, lower but adequate levels of catalase may be obtained from
turnips or even potatoes. To make a crude catalase solution, crush about 25 cm3 of the
turnip or potato in a mortar and pestle with about 20 mL of cold distilled water. Filter the
resulting mash through 2 or 3 layers of cheesecloth, squeeze the cheesecloth gently and
collect the liquid. Throw the mash and cheesecloth away. Results will show a lower rate of
oxygen production with this catalase solution than with the animal liver tissue.

If turnips or potatoes are used as a source of catalase, use about 100 grams of the material
with 100 mL of cold, distilled water. Blend and filter as described above.

More Information
Sodium fluoride inhibits a number of enzymatic reactions, so it will be taken up by other
enzymes in the crude catalase material that was extracted from the chicken liver. Because of this,
the inhibitor will not inactivate all of the catalase. If a greater amount of sodium fluoride is
introduced or if pure catalase material is used, the inhibition effect will be more dramatic.
While drastically lowering the pH of the system will degrade the catalase enzyme, it takes time
for the enzyme to be denatured. The pH level will be raised from around 7 to as high as 12,
depending on the initial pH of the distilled water. The natural alkaline buffering capability of the
chicken tissue will prevent the immediate denaturing of the enzyme.
Many cells regularly experience shifts toward acidic conditions as a part of cellular respiration
and photosynthesis. Consequently, many cells have only marginal abilities to buffer shifts that
dramatically lower the pH. Adding the HCl will lower the pH from around 7 to as low as 2 or 3,
depending on the initial pH of the distilled water. This effectively denatured the enzyme.
Cooling the enzyme does not damage or denature the enzyme. In fact, researchers store many
enzymes for long periods of time by freezing them. One of the important reasons that
poikilotherms (so-called cold-blooded) organisms are limited to warmer climes or warm
seasons is because temperatures below around 4C (40F) limit the effectiveness of many
enzyme-moderated metabolic reactions.
An important parallel pertains to the effect that fevers may have on human enzymes. Denaturing
of important enzymes is one of the dangers of high fevers, especially in children. Many enzymes
in humans begin to become inactive (denatured) at temperatures above 42C (106107F).

Biology with Xplorer GLX Teacher Notes

2005 PASCO

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Activity 5

Teacher Notes Catalase Enzyme Activity

PS-2820

Sample Data
The first screen shot shows gaseous oxygen level versus time for the catalase and hydrogen
peroxide alone and at room temperature. The second screen shot shows data for the mixture
when the catalase was chilled and the third screen shot shows data for the mixture when the
catalase was warmed in boiling water.

Chilled catalase
Catalase
+ hydrogen
+ H2Operoxide
2

Heated catalase + H2O2

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2005 PASCO

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Activity 5

Teacher Notes Catalase Enzyme Activity

PS-2820

Lab Report - Activity 5: Catalase Enzyme Activity

Answers and Sample Data
Pre-Lab Questions
The catalase enzyme breaks down hydrogen peroxide and releases gaseous oxygen. Measure the
gaseous oxygen level (ppm) in a solution of liver extract and hydrogen peroxide when different
substances are added to the solution.
1.

What effect do you think adding an inhibitor to the hydrogen peroxide will have on the
enzymes ability to catalyze the breakdown of the peroxide?

2.

What effect do you think adding a base (high pH solution) to the hydrogen peroxide will
have on the enzymes ability to catalyze the breakdown of the peroxide?

3.

What effect do you think adding acid (low pH) to the hydrogen peroxide will have on the
enzymes ability to catalyze the breakdown of the peroxide?

4.

What effect do you think decreasing the temperature of the catalase will have on the
enzymes ability to catalyze the breakdown of the peroxide?

5.

What effect do you think boiling the catalase will have on the enzymes ability to catalyze
the breakdown of the peroxide?

Student answers will vary depending on their experience. They may predict that the chemical
inhibitor will stop the enzymes ability to catalyze the breakdown of the peroxide. They may
predict that high and low pH solutions will do the same stop the ability of the enzyme to break
down the peroxide. They may predict that the cold temperature will slow the rate of catalysis,
and that the high temperature will destroy the enzyme.

Data
Make a sketch of one run of O2 concentration versus time, including labels for the y- and x-axes.

O2 Concentration

See Sample Data in

Teacher Notes

Time

Biology with Xplorer GLX Teacher Notes

2005 PASCO

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Activity 5

Teacher Notes Catalase Enzyme Activity

PS-2820

Data Table
Item

Part A

Part B
Inhibitor

Part C
High pH

Part D
Low pH

Part E
Chilled

Part F
Heated

Starting O2

21%

21%

21%

Ending O2

86%

79%

21%

O2 Difference

65%

58%

0%

13%/min

11.6%/min

0%/min

Activity (%/min)

Questions
1.

What does the graph of the reaction between hydrogen peroxide and catalase tell you about
enzyme activity?

The graph of the reaction for the production of oxygen from hydrogen peroxide by catalase
shows that the rate of the reaction is not constant. The reaction rate constantly changes as the
reaction progresses. Initially the reaction is very fast but then the rate slows.
2.

Describe the effect of adding the inhibitor (sodium fluoride) to the peroxide before you add
the catalase to the solution of peroxide. What explanation can you give for the results?

Adding the inhibitor slows the rate at which the enzyme catalyzes the decomposition of hydrogen
peroxide. The process requires over twice as much time.
3.

Describe the effect of adding the base (sodium hydroxide) to the solution of peroxide?
What did the sodium hydroxide do to the pH of the solution in the flask? What does this
tell you about the range of conditions in which catalase may be effective?

The base raised the pH of the solution and slowed the rate at which the enzyme catalyzed the
decomposition of hydrogen peroxide. Eventually, the process was brought to a halt. The change
is approximately half as great as when no base is added. Catalase can apparently tolerate a
temporary rise in pH, but after a time, the elevated pH makes it become inactive.
4.

Describe the effect of adding the acid (hydrochloric acid) to the solution of peroxide? What
did the hydrochloric acid do to the pH of the solution in the flask? What does this tell you
about the range of conditions in which catalase may be effective?

The acid lowered the pH of the solution and denatured or deactivated the enzyme so it could not
catalyze the decomposition of hydrogen peroxide. Catalase cannot tolerate low pH.
5.

Describe the effect of cooling the catalase before adding it to the solution of peroxide.

Cooling the crude catalase before adding it slows the rate at which the enzyme catalyzes the
decomposition of hydrogen peroxide. The process requires over twice as much time.
6.

Describe the effect of heating the catalase to boiling before adding it to the solution of
peroxide. How did the effect of cooling compare to the effect of boiling the catalase? How
can you explain the difference between these two trials?

Heating the crude catalase before adding it effectively denatures or deactivates the enzyme so it
cannot catalyze the decomposition of hydrogen peroxide. Cooling did not deactivate the enzyme;
it merely slowed down its ability to catalyze the breakdown of the hydrogen peroxide.
Biology with Xplorer GLX Teacher Notes

2005 PASCO

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Activity 5

Teacher Notes Catalase Enzyme Activity

PS-2820

Catalase + hydrogen peroxide

Boiled catalase + H2O2

Biology with Xplorer GLX Teacher Notes

2005 PASCO

p. 158