IGCSE Coordinated Sciences
B03 – Enzymes
Catalase Investigations
Catalase is found in almost every kind of living cell. It catalyses the reaction:
Hydrogen peroxide (H2O2)
catalase
water (2H2O) + oxygen (O2)
According to p. 28 in your textbook, “Catalase is the fastest enzyme known. Hydrogen
peroxide is often formed as a product of reactions in cells. It can be poisonous if it builds
up.
Why do you think catalase has to work so quickly?”
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In this lab you will be investigating the action catalase on hydrogen peroxide, then seeing
how the reaction changes under various conditions, including a change of pH as well as a
change of temperature.
Skills:
C1 – Using techniques, apparatus and materials
C2 – Observing, measuring and recording
C3 – Interpreting and evaluating
Safety:
- wear eye protection
- hydrogen peroxide is a powerful bleach, wash it off with plenty of water if you get it
on your skin
Materials (per group):
- Fresh potato puree (catalase)
- 3% Hydrogen Peroxide
- plastic pipettes
- 10mL graduated cylinder
- test tube rack
- test tubes (5 large & 5 small)
- Buffer solutions of pH 4, 5, 6, 7, 8
Adapted from Cambridge IGCSE Biology
-
Ruler
Forceps
Filter paper
Scissors
Stopwatch
Beakers (50mL, 400mL)
hot plate
- thermometers (x3)
- gloves
- lab apron
- wooden splint
- safety goggles
- tape
Investigation 1: The Effect of Catalase on Hydrogen Peroxide
1. Read through the instructions and check for safety measures.
2. Measure 10 cm3 (10 mL) of hydrogen peroxide into a test tube using a graduated cylinder.
3. Put the test tubes in a test tube rack.
4. Tape a ruler onto the test tube rack so that the 0mm mark is level with the top of the
hydrogen peroxide in the test tube.
5. To the test tube, add 3mL of catalase and star the stopwatch immediately.
6. After 30 seconds, record the height of the bubbles. Keep measuring at 30 second intervals
for 4 minutes.
7. Record quantitative (numerical) & qualitative (observational) data.
Time
(seconds)
30
60
90
120
150
180
210
240
Height of
bubbles
(mm)
Observations:
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8. Light a wooden splint, then blow it out so that it is glowing. Gently push the glowing splint
down through the bubbles in your tubes.
9. Record your observations and explain them as fully as you can.
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10. If time permits, run a second trial adding a different amount of catalase to 10mL of
hydrogen peroxide. When you finish, clean everything thoroughly and put supplies away.
Adapted from Cambridge IGCSE Biology
Investigation 2: The Effect of pH on the activity of catalase
The rate of reaction can be determined from the rate of oxygen production. One indirect
but simple way to measure the rate of oxygen production is to soak up a catalase solution
onto a little square of filter paper and then drop it into a beaker containing a solution of
H2O2.
The paper sinks at first, but as the reaction proceeds, bubbles of oxygen collect on its
surface and it floats up. The time between placing the paper in the beaker and it floating to
the surface is a measure of the rate of the reaction.
In this investigation, you will test this hypothesis:
Catalase works best at a pH of 7 (neutral)
1. Label five 50 mL beakers pH 4, 5, 6, 7, 8
2. Measure 5 mL of 3% hydrogen peroxide solution to each beaker using a graduated cylinder
or pipette.
3. Add 10 mL of the correct buffer solution to each beaker. (A buffer solution keeps the pH
constant at a particular value)
4. Cut out 20 squares of filter paper exactly 5mm x 5mm or use a hole punch to cut out circles
of filter paper exactly the same size. Avoid handling the paper with your fingers, as you
may get grease onto it. Use forceps instead.
5. Pick up a filter paper square with forceps and dip it into the potato puree (catalase).
6. Make sure you are ready to start timing. Then place the filter paper square at the center
bottom of the beaker containing H2O2 and pH 4 buffer solution. DO NOT let it fall near the
side of the beaker.
7. As you put the square into the beaker, start a stopwatch. Stop the watch when the paper
floats horizontally at the surface.
8. Record the time in your table and repeat steps 8 and 9 twice more.
9. Follow steps 8 – 10 for the other pHs.
10. Calculate the mean (average) time taken at each pH and enter it into your table.
11. Draw a graph to show time taken for flotation plotted against pH.
Adapted from Cambridge IGCSE Biology
Investigation 3: The Effect of temperature on the activity of catalase
In this investigation, you will test this hypothesis:
Catalase works best at a pH of 37oC (body temperature)
1. Label four test tubes 5oC, room temperature, 37oC, and 80oC
2. Measure 5 mL of 3% hydrogen peroxide solution to each test tube using a graduated
cylinder.
3. Measure 1mL of catalase into 5 additional test tubes labeled “catalase.”
4. Put the test tubes in a test tube rack.
5. Tape a ruler onto the test tube rack so that the 0mm mark is level with the top of the
hydrogen peroxide in the test tube.
6. Prepare a water bath at 37oC – 40oC by filling a large beaker with water and heating it on a
hot plate, using a thermometer to check the temperature. Do not let the temperature rise
above 40o.
7. Meanwhile, prepare an ice water bath by filling a beaker half full with ice and then adding
water. Use a thermometer to check the temperature and let it drop between 0oC and 5oC.
8. Put the appropriately labeled test tube into the warm water bath and the ice water bath
along with one of the catalase test tubes. Leave the remaining test tubes in the test tube
rack. Let the test tubes sit in the water baths for 5 minutes.
9. To the tube in the ice water bath, add the 1mL of catalase from the same treatment and
start the stopwatch immediately.
10. After 30 seconds, record the height of the bubbles. Keep measuring at 30 second intervals
for 4 minutes. Remember to record your data.
11. Repeat steps 8 & 9 using the test tube in the warm water bath and the test tube at room
temperature.
12. After you have used the warm water bath, raise the temperature of the hot plate so that the
water bath reaches approximately 80oC. Add the appropriately labeled test tube and a
catalase test tube to that water bath and let it remain there for 5 minutes to reach
equilibrium.
13. Repeat steps 8 & 9 using the test tube in the hat water bath.
14. Draw a line graph with four different lines to show the change in oxygen production over
time for each temperature level.
Adapted from Cambridge IGCSE Biology
Data Table for Investigation 2:
Time taken for paper to float (s)
pH
4
5
6
7
8
Trial 1
Trial 2
Trial 3
Mean
Observations:
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Data Table for Investigation 3:
Time (seconds)
Temperature
30
60
90
120
150
180
210
240
Height of bubbles (mm)
Cold:
________oC
Room Temp:
________oC
Body Temp:
________oC
Hot:
________oC
Observations:
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Adapted from Cambridge IGCSE Biology
Post Lab questions – please answer in your science journal:
1. (Investigation 2) Does the enzyme have an optimum pH? If it does, what do your
results suggest it to be?
2. (Investigation 2): Why do the filter paper squares/circles have to be exactly the
same size?
3. (Investigation 3) What is the effect of heating the catalase?
4. Do your results support the hypothesis you were testing for each investigation, or
do they disprove it? Explain your answers?
a. Investigation 2:
b. Investigation 3:
5. In most experiments in biology, we can never be quite sure that we would get
exactly the same results if we did it again. There are always some limitations on the
reliability of the data that we collect. Can you think of any reasons why the results
you got in your experiment might not be absolutely reliable? For example:
a. Might there have been any variables that were not controlled and that might
have affected the results?
b. Were you able to measure the volumes and times as accurately as you would
have liked?
Adapted from Cambridge IGCSE Biology