Enzyme Catalysis Lab Substrate Concentration

Names: Claire Fellbaum, Kiera Bae, Dhanya Rodrigo, Sara Molidor Date: 10/12/2016 Per: 4
AP Inquiry Lab: Catalytic Activity of Enzymes

Courtesy of CIBT (Cornell Institute of Biology Teachers)
Part 1: Introduction
Enzymes are biological molecules that catalyze (speed up) chemical reactions. You could call enzymes
the ‘‘Builders and Do-ers” in the cell; without them, life could not occur. Every cell makes hundreds of
different enzymes to carry out the reactions necessary for life. Fortunately for the cell, enzymes are not
used up when they catalyze a reaction, but can be used over and
over.
The DNA in each cell encodes all the information needed to make its many different enzymes. Enzymes
are relatively large molecules of protein. They are produced whenever the cell ‘‘senses’’ a need for that
particular enzyme; that is, whenever a job needs to be done in the cell which only that enzyme can do it.
The molecule (or molecules) on which an enzyme acts is called its substrate. Enzymes are said to be
very ‘‘specific,’’ meaning that they recognize only one substrate (or a few closely related substrates) and
convert it into a specific product. You could say that each enzyme can do only one type of job. Each
enzyme is specific because it is folded into a particular three-dimensional shape. Within the folds of each
enzyme is the active site, the place where the substrate fits and where the chemical reaction takes place.
Enzymes work very quickly, often catalyzing thousands of reactions per minute. The rate at which an
enzyme works is influenced by many factors including temperature and pH. Enzymes have a temperature
and pH at which they work best, and if an enzyme is exposed to extremes of heat or pH it won’t work at
all! The interactions that hold the protein in its particular shape become disrupted under these conditions,
and the 3- dimensional structure unfolds. In this case, the enzyme is said to be denatured. Other
important factors that influence enzyme activity are the concentration of substrate and the concentration
of enzyme. Up to a point, the more substrate that is present, the faster the reaction. However, when the
substrate concentration is so high that an enzyme is working as fast as it can, further increases of
substrate concentration will have no effect on the rate of product formation.
Background for this Lab

The enzyme that you will study in this experiment is called ‘‘catalase.’’ Its job is to break down its
substrate hydrogen peroxide (H2O2), which is a naturally occurring poison. Without catalase, H 2O2 could
kill the cell. The reaction catalyzed by catalase is:
2H2O2 --> 2H2O + O2
The products remaining after catalase does its job are oxygen gas and water; two very non-poisonous
molecules.
In the home and hospital, hydrogen peroxide is used as an antiseptic to clean out wounds. Have you
ever noticed that when hydrogen peroxide is swabbed on a cut it bubbles? This is because enzymes in
the cut from your body and from infecting bacteria catalyze the rapid degradation of hydrogen peroxide
into water and oxygen. The bubbles are oxygen.
Catalases are very common. They are found in almost all cells that grow in oxygen, including potato
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tubers. In this experiment, a blender is used to grind up a potato in water to release the catalase from the
potato cells. The ground-up potato is filtered through cheesecloth to separate potato skin and cell debris
from the liquid which contains most of the cell’s enzymes, including catalase. To actually measure the
catalase activity, small disks are dipped into the potato cell extract. When this enzyme-containing disk is
placed in a solution of hydrogen peroxide, the enzyme begins to work. As the catalysis occurs, oxygen is
produced, and bubbles of the gas become trapped in the fibers of the disk. When there are enough O 2
bubbles, they lift the filter to the surface. The speed with which the O 2 is produced depends both upon
how much enzyme is present and on the concentration of the hydrogen peroxide. The more enzyme
available, the faster the product (O2) is made. Similarly, the higher the concentration of the substrate,
hydrogen peroxide, the faster the product is made. You can see what happens when you vary either the
concentration of enzyme or the concentration of the hydrogen peroxide.
Part 2: pH Influence on Enzyme Activity (Teacher Demo & Warm Up)
Materials
Five test tubes Test tube rack 3% hydrogen
pH solutions of 3-5-7-9-11 peroxide 100% solution catalase
Metric ruler Graph paper
Procedure
1. Obtain five test tubes and a test tube rack. Label them pH 3, pH 5, pH 7, pH 9, pH 11.
2. Using a pipettor and a 5-ml pipette, measure 4 ml of each of the pH solutions provided into the
correct test tube. Replace the pipette and pipettor into the correct solution. Do not mix them or
lay them down.
3. To each of the test tubes, add 2 ml of 60% catalase solution, using the 5-ml pipette and pipettor.
Swirl the test tube to help mix the pH solution with the catalase. Allow to sit for 5 minutes.
4. With a 1 ml pipette and the pipette, measure 1 ml of 3% hydrogen peroxide into each of the test
tubes. Allow to react for 5 minutes. Foam should form on the top of the solutions.
5. Measure the distance from the bottom of the test tube to the top of the foam in millimeters and
record below.
Part 3: Catalase Experiments

Your teams will be provided with the following materials
● Yeast solution and dilutions
● Dilutions of 3% H2O2
● 1 cup for yeast extract collection (only groups testing substrate concentration)
● 1 cup of 2% H2O2 (only groups testing catalase concentration)
● 100 ml graduated cylinder
● Pre-labeled cups for your reactions
● Forceps (tweezers)
● Filter paper & hole punch
● Stop watch (cell phone timers are handy here!)
One side of the table will complete the Substrate Concentration experiment.
Research Question: How does altering the H2O2 substrate concentration in an enzyme
(catalase) catalyzed reaction impact the reaction rate?
Procedure: Various Hydrogen Peroxide Concentrations

1. Ensure you have the following pre-labeled hydrogen peroxide reaction cups: 2%, 1.5%, 1.0%,
0.8%, 0.6% and 0.3%.
2. Your teacher made substrate dilutions according to these instructions:
Test Substrate Volume of H2O2 Volume of Water
Concentration
1 2.0 % 20 ml 10 ml
2 1.5 % 15 ml 15 ml
3 1.0 % 10 ml 20 ml
4 0.8 % 8 ml 22 ml
5 0.6 % 6 ml 24 ml
6 0.3 % 3 ml 27 ml
3. You need to pour 25 ml of each of the appropriate dilutions into the prelabeled hydrogen peroxide
reaction cups.
4. You should also have a cup of fresh 100% yeast catalase solution at your table.
5. Locate several small paper disks. Use forceps to pick them up. You may need to make more so
paper and hole punchers have been supplied.
6. Using forceps dip a disk in the 100% yeast catalase solution for 5 seconds Then, using forceps,
place the filter disk (containing enzymes) into the cup of the 2.0% H 2O2.
7. Time how long it takes from the disk touching the liquid to sink then rise back to the the top of the
liquid in the reaction cup.
8. Record the time in seconds in the appropriate space on the data chart that follows.
9. Obtain another disc and repeat steps 5 - 7 exactly as before.
10. Repeat the experiment a third time. Now you have triplicate measurements of the rate of oxygen
production. Average these three values and record on the chart.
11. Repeat this procedure for all assigned concentrations of H 2O2.
12. You will share your average data on a Google Form. This link is available here:
https://docs.google.com/a/hartdistrict.org/forms/d/17gVoVzjNtZpA0aonIeA-
Wgg6vLCbJrceG3DzDTuShzQ/viewform
13. We will generate a class average graph to include in your lab report.
Data Chart 1: Catalase Action in Various Hydrogen Peroxide Concentrations

Test Trial 1 (sec) Trial 2 (sec) Trial 3 (sec) Ave. (sec)
2% 2.34 2.00 0.80 1.71
1.5% 1.46 3.85 1.01 6.32
1.0% 1.12 2.38 1.09 3.86
0.8% 1.44 4.44 2.93 2.94
0.6% 0.34 1.91 1.20 1.15
0.3% 3.71 5.01 0.80 4.68
7. Summary of Learning/Conclusions from the graph.

The graph shows the relationship between hydrogen peroxide concentration and the reaction rate. The
higher the H2O2 concentration, the higher the reaction rate is until the concentration reaches about 16%.
The class data has a more steady reaction rate with the higher concentrations unlike the group data
which has its sharp up and downs.
One side of the table will complete the Enzyme Concentration experiment.
Research Question: How does altering the catalase concentration in an enzyme (catalase) catalyzed
reaction impact the reaction rate?
Procedure: Various Catalase Concentrations

1. Ensure you have the following pre-labeled catalase reaction cups: 20%, 40%, 60%, 80%, & 100%
2. You should also have a cup of 2% Hydrogen Peroxide (H2O2).
3. Fill each of these cups with 25mL of 2% H2O2 from the cup at your table.
4. You should also have an extra cup labeled Catalase (it may say 100% catalase - that’s ok!).
5. Your teacher has made the appropriate yeast catalase dilutions for you according to these
instructions:
Test Extract Concentration Volume of Enzyme Volume of Water
1 20% 2 ml 8 ml
2 40% 4 ml 6 ml
3 60% 6 ml 4 ml
4 80% 8 ml 2 ml
5 100% 10 ml 0 ml
6. Take your extra Catalase cup to the center table and locate the 20% Catalase solution. Stir the
solution with the pipette then place a few pipettes full into your Catalase cup. Take this back to
your lab station.
7. Locate several small paper disks. Use forceps to pick them up. You may need to make more so
paper and hole punchers have been supplied.
8. Using forceps dip a disk in the 20% catalase for 5 seconds. Then, using forceps, place the filter
disk (containing enzymes) into the cup of the 2.0% H2O2.
9. Time how long it takes from the disk touching the liquid to sink then rise back to the the top of the
liquid in the reaction cup.
10. Record the time in seconds in the appropriate space on the data chart that follows.
11. Obtain another disc and repeat steps 5 - 7 exactly as before.
12. Repeat the experiment a third time. Now you have triplicate measurements of the rate of oxygen
production. Average these three values and record on the chart.
13. Empty your Catalase cup into the waste container.
14. Now repeat this procedure for all assigned concentrations of Catalase.
15. You will share your average data on a Google Form. This link is available here:
https://docs.google.com/a/hartdistrict.org/forms/d/1YfQ66aGyVR9rdEKjM63A5singUx_N3k6F2S-
G7OwzHc/viewform
16. We will generate a class average graph to include in your lab report.
Data Chart 2: Catalase Action Using Various Catalase Concentrations

Test Trial 1 (sec) Trial 2 (sec) Trial 3 (sec) Ave. (sec)
20& 5.86 5.98 9.13 6.99
40% 6.20 7.63 8.06 7.30
60% 2.95 5.10 4.71 4.25
80% 3.75 5.45 2.80 4
100% 2.86 2.70 4.35 3.30
8. Summary of Learning/Conclusions from the graph.
Explain (tell what and why) what the class graph shows. Include as much vocabulary from your reading
and our class lecture as possible in your discussion.
The class graph shows an increase in enzyme activity with the increase in catalase concentration. This is
because the increase in catalase concentration causes an increase in reactivity with the enzymes. The
increase in catalase concentration allows for a higher rate of reactivity with the enzymes because there
are more enzymes breaking the hydrogen bonds and forming new bonds.
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Names: Claire Fellbaum, Kiera Bae, Dhanya Rodrigo, Sara Molidor Date: 10/12/2016 Per: 4

AP Inquiry Lab: Catalytic Activity of Enzymes

Background for this Lab

2H2O2 --> 2H2O + O2

Part 2: pH Influence on Enzyme Activity (Teacher Demo & Warm Up)

pH solutions of 3-5-7-9-11 peroxide 100% solution catalase

Metric ruler Graph paper

Part 3: Catalase Experiments

Procedure: Various Hydrogen Peroxide Concentrations

Data Chart 1: Catalase Action in Various Hydrogen Peroxide Concentrations

2% 2.34 2.00 0.80 1.71

1.0% 1.12 2.38 1.09 3.86

0.8% 1.44 4.44 2.93 2.94

0.6% 0.34 1.91 1.20 1.15

0.3% 3.71 5.01 0.80 4.68

7. Summary of Learning/Conclusions from the graph.

Procedure: Various Catalase Concentrations

Data Chart 2: Catalase Action Using Various Catalase Concentrations

20& 5.86 5.98 9.13 6.99

40% 6.20 7.63 8.06 7.30

60% 2.95 5.10 4.71 4.25

80% 3.75 5.45 2.80 4

100% 2.86 2.70 4.35 3.30

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