Testing for Catalase Enzyme Activity with Filter Paper Disks

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 (H2O)
and oxygen gas (O2).
Catalase enzyme
2H202
2H2O + O2
A CATALYST is a substance that 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.

Methods
The assay system used in this lab consists of a filter paper disc which is coated with the enzyme and
then dropped into a vial (microtiter plate*) of substrate (hydrogen peroxide). As the enzyme breaks
down the hydrogen peroxide into water and oxygen gas, the bubbles of oxygen collect underneath the
filter disc and make it rise to the surface of the hydrogen peroxide. The time it takes for the filter disc
to rise is an indication of the rate of enzyme activity. You will be doing 5 repetitions for each
experiment.
Rate:
Rate of enzyme activity = distance (depth of hydrogen peroxide in mm)/time (in sec). 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).

Enzyme:
The catalase enzyme has been prepared for you as follows: 50g of peeled potato was mixed with 50 ml
cold water and crushed ice and homogenized in a blender for 30 seconds. This extract was filtered
through cheesecloth and cold water was added to a total volume of 100 ml. Extract concentration is
arbitrarily set at 100 units/ml. ENZYME SHOULD BE KEPT ON ICE AT ALL TIMES!!
Question #1: What is the effect of enzyme concentration on rate of reaction?
1. Make a large number of filter paper discs using a coffee filter and a hole punch.
2. Measure the depth of one sample vial on the microtiter plate. (This will be needed in order to
determine rate).
3. Obtain approximately 100 ml of 3% hydrogen peroxide in a beaker.
4. Fill 5 vials, on microtiter plate, with 3% hydrogen peroxide (substrate). Set the plate aside.
5. Obtain 5 plastic cups and label with the enzyme dilutions listed in table 1.
Table 1 Enzyme Concentrations
Enzyme Dilutions
Enzyme
100 units/ml
20 ml
75 units/ml
15 ml
50 units/ml
10 ml
25 units/ml
5 ml
0 units/ml
0 ml
6.

Cold dH20
0 ml
5 ml
10 ml
15 ml
20 ml

Set up a cold water bath and obtain about 50 ml of enzyme stock solution in a plastic vial. KEEP
YOUR STOCK SOLUTION IN THE COLD WATER BATH AT ALL TIMES!

7.

8.

9.
10.
11.

Set up your first enzyme concentration (100 units/ml) in the appropriate plastic cup. Only make
one enzyme dilution at a time, this will keep your stock solution in the cold water bath as long as
possible. (Check the temperature and make sure to keep it constant throughout the
investigation.)
Using forceps, dip a filter disc into the enzyme solution at 100 units/ml, remove it and drain it by
touching it to the edge of the cup. 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. Repeat this process five times
for the each individual enzyme dilution. Make sure you keep track of the start and stop times for
EACH vial. Use a FRESH set of vials of substrate for each filter paper disc.
Record your results in an appropriate data table.
Repeat steps 7-9 for each of the other enzyme dilutions, be sure to use a FRESH set of vials of
substrate for each filter paper disc.
Clean your materials including plastic cups for the second half of the lab.

Question #2: What is the effect of substrate concentration on the rate of reaction?
1. Label your plastic cups with the substrate dilutions listed in table 2.
2. Use your remaining hydrogen peroxide (get more if necessary) and set up the five dilutions listed in
table 2. Each dilution should be made in a separate, labeled plastic cup.
Table 2 Substrate concentrations
Hydrogen Peroxide Dilutions
3.0% H2O2
2.25% H2O2
1.5% H2O2
0.75% H2O2
0.0% H2O2

H2O2
20 ml
15 ml
10 ml
5 ml
0 ml

dH20
0 ml
5 ml
10 ml
15 ml
20 ml

3. Check and record the temperature of the remaining stock enzyme solution. (Make sure to keep the
temperature constant throughout the investigation.)
4. Fill 5 vials, on microtiter plate, with the 3.0% dilution of hydrogen peroxide (substrate).
5. Stir or shake your enzyme to mix the contents. Using forceps, dip a filter into the enzyme solution
and drain it by touching it to the edge of the cup. Drop the filter into the vial of H2O2. Time how
long it takes the filter to rise to the surface. Repeat this process five times for each individual
substrate dilution.
6. Repeat step 5 for each of the substrate dilutions, be sure to use a FRESH set of vials of substrate for
each filter paper disc.
7. Record your results in an appropriate data table.
* Microtiter Plate microplate is a flat plate with multiple "vials" used as small test tubes.