AP BIOLOGY NAME_____________________

BIOCHEMISTRY
ACTIVITY #9 DATE___________HOUR_____

CATALASE LAB
INTRODUCTION

Hydrogen peroxide (H2O2 ) is a poisonous byproduct of metabolism that can damage

cells if it is not removed. Catalase is an enzyme that speeds up the breakdown of
hydrogen peroxide into water (H2 O) and oxygen gas (O2 ).

Catalase
2 H2O2 2 H2O + O2
Remember: A catalyst is a substance that lowers the activation energy required for
a chemical reaction, and therefore increases the rate of the reaction without being
used up in the process. CATALASE is an enzyme, a biological (organic) catalyst.
Hydrogen peroxide is the substrate for catalase.

***You w ill be working with hot water, acids and bases in this laboratory***
****Use Extreme Caution!!!****

The general procedure for the lab is outlined below, and specific details for each
variable follow.

GENERAL D IRECTIONS :

The assay system used in this lab consists of a filter paper disc coated with the
enzyme and then dropped into a vial of substrate (hydrogen peroxide). As the
catalase breaks down the hydrogen peroxide into water and oxygen gas, the
bubbles of oxygen collect underneath the filter and make it rise to the surface of
the hydrogen peroxide. The time it takes for the filter paper disc to rise is an
indication of the rate of enzyme activity.

R ATE:

Rate of enzyme activity is equal to the distance (depth of hydrogen peroxide in

mm) divided by the time (in sec) for the disc to rise to the surface. We will assume
that each filter is coated with the same amount of catalase (except in the
investigation of the effect of enzyme concentration on enzyme activity).

The enzyme has been prepared for you as follows: 50g of peeled potato was mixed
with 50 ml cold distilled water and crushed ice and homogenized in a blender for 30
seconds. This extract was filtered through cheesecloth and cold distilled water was
added to a total volume of 100 ml. Extract concentration is arbitrarily set at 100
units/ml. The enzyme should be kept on ice at all times!!

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MATERIALS:

Catalase Ice
Hydrogen peroxide 3% and 1.0% Water baths (0 oC, 37 oC, 100oC)
Forceps Vials
Filter paper discs Marking pencils
Water Stopwatch or timer

PROCEDURE :

Each group will investigate and report on two of the following questions. Suggested
procedures for each question are given below. Every student is responsible for
recording the results of all four experiments on this activity.

1. What is the effect of enzyme concentration on enzyme activity?

2. What is the effect of substrate concentration on enzyme activity?
3. What is the effect of pH on enzyme activity?
4. What is the effect of temperature on enzyme activity?

1. What is the effect of enzyme concentration on enzyme activity?

a. Set up five vials containing 40 mL of 3% hydrogen peroxide each.

Measure and record the depth of the hydrogen peroxide in the vials.

b. Dilute the enzyme as follows. Make each dilution in a separate cup.

100 units/mL = 20 ml 100 units/ml

80 units/mL = 12 ml 100 units/ml + 3 ml cold dH2 O
50 units/mL = 10 ml 100 units/ml + 10 ml cold dH2 O
20 units/mL = 3 ml 100 units/ml + 12 ml cold dH2 O
0 units/mL = 20 ml cold dH2 O

c. Using forceps, dip a filter into the enzyme solution at 100 units/ml, then
remove it and drain it on a paper towel.

d. Drop the disc into the vial of hydrogen peroxide labeled 100 units/ml and
time how long it takes the filter to rise to the surface.

e. Repeat this procedure for each of the other enzyme dilutions, and be
sure to use a FRESH vial of substrate for each filter.

f. Record your results in the appropriate data chart.

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2. What is the effect of substrate concentration on enzyme activity?

a. Obtain 1 vial of catalase at 100 units/mL

b. Dilute the substrate (hydrogen peroxide) as described below. Each

dilution should be made in a separate labeled vial. Measure and record
the depth of the hydrogen peroxide.

3.0% H2O2 : 40 mL 3% H2 O2
1.5% H2O2 : 20 mL 3% H2 O2 + 20 ml distilled water
0.75% H2 O2: 10 mL 3% H2 O2 + 30 ml distilled water
0.38% H2 O2: 5 mL 3% H2O2 + 35 ml distilled water
0.0% H2O2 : 40 mL distilled water

c. Dip a filter into the catalase, drain on a paper towel and then drop the
filter into the 3% H2O2 .

d. Time how long it takes the filter to rise to the top.

e. Repeat this procedure for each of the substrate dilutions. Record your
results in the appropriate data chart.

3. What is the effect of pH on enzyme activity?

a. Obtain 1 vial of catalase at 100 units/mL.

b. Set up 5 vials of 40 mL 1% H2 O2 . Measure and record the depth of the

hydrogen peroxide.

c. Label 5 small vials as follows: pH3, pH5, pH7, pH9, pH11 and dilute
catalase into the appropriate vial as directed below:

pH 3: 5 mL catalase + 5 mL pH 3 Buffer
pH 5: 5 mL catalase + 5 mL pH 5 Buffer
pH 7: 5 mL catalase + 5 mL pH 7 Buffer
pH 9: 5 mL catalase + 5 mL pH 9 Buffer
pH 11: 5 mL catalase + 5 mL pH 11 Buffer

d. Dip a filter into the catalase at pH 3, drain on a paper towel and drop it
into the 1% H2 O2 .

e. Time how long it takes the filter to rise to the top.

f. Remove the filter and repeat this procedure for each pH.

g. Record your results in the appropriate data chart.

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4. What is the effect of temperature on enzyme activity?

a. Obtain 1 vial of catalase at 100 units/mL.

b. Set up an ice bath (0oC), a room temp water bath, a 37oC bath and a
boiling water bath

c. Place 5 ml of catalase at 100 units/ml in each of 4 test tubes. Place 1 test

tube in each of the water baths.

d. Place 40 mL 1% H2O2 in each of 4 vials. Measure and record the depth of

the H2O2 . Place 1 vial in the 0 o C bath and leave 3 at room temperature.
This is necessary because heat will destroy the hydrogen peroxide.

e. Allow the catalase and substrate to incubate at each temperature for

about 5 minutes, then test the reaction time at each temperature by
dipping a filter into enzyme at that temperature, draining it, and then
dropping it into substrate at the same temp.

f. Use the second room temperature vial of hydrogen peroxide for the
boiled catalase. Do not boil hydrogen peroxide!!! Time how long it
takes the filter to rise at each temperature.

g. Record your results in the appropriate data chart.

DATA T ABLES WITH R ESULTS

1. What is the effect of enzyme concentration on enzyme activity?

Enzyme Rate:
H2O2 Depth Time Rate
Conc. Class Ave.

0 units/mL

20 units/mL

50 units/mL

80 units/mL

100 units/mL

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2. What is the effect of substrate concentration on enzyme activity?

Rate:
H2O2 Conc. H2O2 Depth Time Rate
Class Ave.

0%

0.38 %

0.75 %

1.5 %

3.0 %

3. What is the effect of pH on enzyme activity?

Rate:
pH H2O2 Depth Time Rate
Class Ave.

11

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4. What is the effect of temperature on enzyme activity?

Rate:
Temp. H2O2 Depth Time Rate
Class Ave.

22

37

100

ANALYSIS :

For EACH variable, use the class average rates to construct a graph of the
independent variable vs. the dependent variable. This means you will have four
graphs:

Enzyme concentration (x axis) vs. Rate (y axis)

Substrate concentration (x axis) vs. Rate (y axis)
pH (x axis) vs. Rate ( y axis)
Temperature (x axis) vs. Rate (y axis)

Attach your graphs to this lab.

CONCLUSION:

Answer the following questions:

1. What is the effect of enzyme concentration on enzyme activity? Explain how

enzyme activity changes as enzyme concentration decreases, and discuss
why this occurs (on a molecular level).

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2. What is the effect of substrate concentration on enzyme activity? How does
enzyme activity change as substrate concentration decreases? Explain your
observations by discussing this reaction on a molecular level.

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3. How does temperature affect the activity of catalase? Explain your

observations by discussing the effect of temperature on protein structure.
Discuss both high and low temperature effects.

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4. How does pH affect the activity of catalase? Consider both high and low pH,
and explain your observations by discussing the effect of pH on protein
structure.

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5. Ectothermic organisms have body temperatures that vary with the

temperature of their surroundings. Discuss the effect this variation might
have on the functioning of enzymes in these organisms. Suggest some ways
ectothermic organisms might cope with this problem.

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