Catalase Lab - A Bio ENZYME ACTIVITY Investigation
Created by Gen Nelson, modified by Dr G
INTRODUCTION
Hydrogen peroxide (H2O2) is a poisonous byproduct of metabolism that can damage cells if it is not
removed. Catalase, a biological catalyst, is an enzyme that speeds up the breakdown of hydrogen
peroxide into water (H2O) and oxygen gas (O2) or
2H2O2
𝑐𝑎𝑡𝑎𝑙𝑎𝑠𝑒
⇒
2H2O + 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.)
Hydrogen peroxide is the substrate for catalase.
****YOU WILL BE WORKING WITH HOT WATER, ACIDS AND BASES IN THIS LABORATORY. USE EXTREME
CAUTION AND WEAR GOGGLES AT ALL TIMES!!!
The general procedure for the lab is outlined below, and specific details for each variable follow.
GENERAL DIRECTIONS:
In this lab investigation, a filter paper disc is coated with the enzyme and then dropped into a beaker of
substrate (hydrogen peroxide (H2O2)). As the catalyst breaks down the hydrogen peroxide into water
and oxygen gas, bubbles of oxygen collect underneath the filter and make the filter paper disc rise to the
surface of the hydrogen peroxide solution. The time it takes for the filter to rise to the surface is
correlated with the rate of enzyme activity.
The rate of enzyme activity = distance (depth of hydrogen peroxide solution in mm)/rise time (sec) or
𝑆𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝐷𝑒𝑝𝑡ℎ
𝑅𝑎𝑡𝑒 =
𝑅𝑖𝑠𝑒 𝑇𝑖𝑚𝑒
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 PREPARATION SHOULD
BE KEPT ON ICE AT ALL TIMES!!
Each group will investigate and report on two of the following questions. Suggestions for investigating
each question are given below.
EVERY STUDENT IS RESPONSIBLE FOR RECORDING THE RESULTS OF ALL FOUR EXPERIMENTS in his/her
lab report.
 What is the effect of enzyme concentration on enzyme activity?

What is the effect of substrate concentration on enzyme activity?

What is the effect of pH on enzyme activity?
 What is the effect of temperature on enzyme activity?
MATERIALS:
Catalase solution (see above), hydrogen peroxide 3%, forceps, filter paper discs, distilled water, ice,
water baths, test tubes, beakers, flasks, marking pencils, stopwatch or digital timing device
PROCEDURE: (by question being investigated)
A. What is the effect of enzyme concentration on enzyme activity?
1. Obtain a 50 ml sample of the enzyme solution and make 5 dilutions as follows, each dilution in a
separate test tube.
o 100 units/ml = 20 ml of the enzyme solution (we standardize this as 100 units/ml)
o 75 units/ml = 15 ml of enzyme solution + 5 ml cold distilled H2O
o 50 units/ml = 10 ml of enzyme solution + 10 ml cold distilled H2O
o 25 units/ml = 5 ml of enzyme solution + 15 ml cold distilled H2O
o 0 units/ml = 20 ml of just cold distilled H2O
2. Place 10 ml of 3% hydrogen peroxide in a small reaction beaker. Measure and record the depth
of the hydrogen peroxide solution in the beaker.
3. Using forceps, dip a clean unused filter paper disc into one of the enzyme solutions. Remove the
disc and drain it on a paper towel. Drop the enzyme coated disc into the beaker of hydrogen
peroxide and time how long it takes the filter to rise to the surface. Record the depth of the
solution and the time it took the disc to rise in the appropriate data table.
4. Repeat the above procedure two more times with the same enzyme concentration.
5. Rinse out and dry the reaction beaker and refill it with 10 ml of fresh hydrogen peroxide.
6. Repeat the entire procedure (steps 2-5) for each of the remaining enzyme dilutions.
7. Record your results in the appropriate data table.
B. 2. What is the effect of substrate concentration on enzyme activity?
1. Obtain 20ml of enzyme solution (100 units/ml) in small flask
2. Dilute the substrate (hydrogen peroxide) as described below in separate, labeled test tubes.
o 3.0% H2O2: 20 ml 3% H2O2
o 1.5% H2O2: 10 ml 3% H2O2 + 10 ml distilled water
o 0.75% H2O2: 5 ml 3% H2O2 + 15 ml distilled water
o 0.38% H2O2: 2.5 ml 3% H2O2+ 17.5 ml distilled water
o 0.0% H2O2: 20 ml distilled water
3. Add 10 ml of the undiluted substrate (3% H2O2) to the reaction beaker.
4. Measure and record the depth of the substrate solution.
5. Dip a disc of filter paper into the enzyme solution, drain on a paper towel and then drop the disc
into the H2O2 solution.
6. Time how long it takes the filter paper disc to rise to the top.
7. Repeat the procedure two more times using freshly prepared discs but the same substrate
solution.
8. Empty, rinse and dry the reaction flask after the third trial.
9. Repeat the procedure (steps 3-8) 3 times for each of the substrate dilutions, empting, drying
and filling the reaction beaker between different dilutions.
10. Record your results in the appropriate data table.
C. What is the effect of pH on enzyme activity?
1. Obtain 1 flask of catalase at 100 units/ml (undiluted enzyme solution)
2. Label 5 test tubes as follows: pH3, pH5, pH7, pH9, pH11.
3. Add the appropriate amount of enzyme solution to each test tube as indicated below:
o pH 3: 5 mL catalase + 5 mL pH 3 Buffer solution
o pH 5: 5 mL catalase + 5 mL pH 5 Buffer solution
o pH 7: 5 mL catalase + 5 mL pH 7 Buffer solution
o pH 9: 5 mL catalase + 5 mL pH 9 Buffer solution
o pH 11: 5 mL catalase + 5 mL pH 11 Buffer solution
4. Fill the reaction beaker with 10 ml 1% H2O2.
5. Measure and record the depth of the hydrogen peroxide solution.
6. Dip a filter paper disc into the catalase solution at pH 3, drain on a paper towel and drop it into
the 1%H2O2 solution.
7. Time how long it takes the filter paper to rise to the top.
8. Remove the filter and repeat this procedure 2 more times and then 3 times for each of the
remaining pH' buffer solution with enzyme added.
9. Record your results in the appropriate data table.
D.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
What is the effect of temperature on enzyme activity?
Set up an ice bath (0oC), a room temp water bath, a 37 oC bath and a boiling water bath
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.
Place 10 ml 3% H2O2 in each of 4 beakers.
Measure and record the depth of the H2O2 solution.
Place 1 beaker in the 0oC bath and leave 3 at room temperature. (This is necessary because heat
will destroy the hydrogen peroxide.)
Allow the catalase and substrate to incubate in the 0oC bath for about 5 minutes
Dip a filter disc into the enzyme incubating at that temperature, drain it, and then drop it into
substrate solution in the reaction beaker at that same temperature.
Time how long it takes the filter paper to rise to the top.
Repeat the above procedure (steps 3-9) for room temperature bath and the 37oC bath.
Time how long it takes the filter to rise at each temperature. Record your results in the
appropriate data table.
Use the second room temp. beaker of hydrogen peroxide for the boiled catalase trial. DO NOT
BOIL HYDROGEN PEROXIDE!!!
Place 10 ml of H2O2 in a reaction beaker , measure the depth of the solution and leave it at room
temperature.
Place the final test tube containing the catalase solution in a boiling hot water bath for 5
minutes. Remove the test tube and quickly transfer the enzyme solution into the reaction
beaker.
Time how long it takes the filter to rise at each temperature. Record your results in the
appropriate data table.
DATA
A. Tables:
1. The effect of enzyme concentration on enzyme activity
Enzyme Conc
H2O2 Depth
Time to Rise
Data Rate
0 units/ml
1.
2.
3.
25 units/ml
1.
2.
3.
50 units/ml
1.
2.
3.
Ave Data Rate
Class Data Rate
75 units/ml
1.
2.
3.
100 units/ml
1.
2.
3.
2. The effect of substrate concentration on enzyme activity
H2O2 Concentration H2O2 Depth
Time to Rise
0%
1.
2.
3.
0.38%
1.
2.
3.
0.75%
1.
2.
3.
1.5%
1.
2.
3
3.0%
1.
2.
3.
Data Rate
Class Data Rate
pH
3
1.
2.
3.
5
1.
2.
3.
7
1.
2.
3.
9
1.
2.
3.
11
1.
2.
3.
8. The effect of pH on enzyme activity
H2O2 Depth
Time to Rise
4. The effect of temperature on enzyme activity
Temperature
H2O2 Depth
Time to Rise
0 oC
1.
2.
3.
22 oC
1.
2.
3.
37 oC
1.
2.
3.
100oC
1.
2.
3.
Data Rate
Class Data Rate
Data Rate
Class Data Rate
B. Graphs
For EACH variable, use the AVERAGE rates to construct a graph of the independent variable vs. the
dependent variable. Make sure each graph has an appropriate title, correctly labeled axes and a bes fit
curve. The four graphs to include are:
1. Enzyme concentration (x axis) vs. Rate (y axis)
2. Substrate concentration (x axis) vs. Rate (y axis)
3. pH (x axis) vs. Rate ( y axis)
4. Temperature (x axis) vs. Rate (y axis)
Useful ANALYSIS 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).
2. What is the effect of substrate concentration on enzyme activity? How does enzyme activity
change as substrate concentration decreases? Explain your observations in terms of the
enzyme’s reaction rate.
3. 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.
4. 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.
5. For each variable you investigated, compare what you expected to happen against what actually
happened. Offer explanations for any “unexpected” results.
6. What possible sources of error may have contributed to your results and how might you
redesign the experiment to minimize them? (Don’t go with human error.)
As you write your lab report, review the lab report guidelines and grading rubric you received earlier.
These same documents can be found on the class wiki.