Enzyme Concentration & Rate of Catalase Reaction
Objective:
Describe the structures of proteins and amino acids. Explain the functions of proteins in living organisms. Identify
some reactions that amino acids undergo. Relate the structure and function of enzymes.
(Also addresses SC.912.L.18.1)
Purpose of Lab:
Investigate the effect of variations in enzyme concentration on rate of reaction.
Prerequisites: Review with students how enzymes affect activation energy. Describe how an enzyme’s shape is
important to its function. Ensure that students know the difference between the meaning of the word catalase and the
word catalyst. Some students may confuse these terms. It is important that students are familiar with all of the
vocabulary terms listed in the student section.
Problem Statement: How will different concentrations of hydrogen peroxide affect the reaction of an enzyme?
Background:
In chemical reactions atoms in compounds separate and are rearranged into new compounds. In order for this to
happen, bonds must be broken between the original compounds (reactants) and new bonds must be formed to form the
products of the reaction. While the breaking of chemical bonds often releases the potential energy stored in them, it
takes energy to make that happen (Like it takes a spark to detonate a bomb). This is called the activation energy of a
reaction. Some compounds serve the purpose of speeding up chemical reactions by lowering this activation energy.
They do this by facilitating the breakage of bonds. These compounds are called catalysts and the action they perform is
called catalysis. In biochemical pathways, special proteins often fulfill this function. These biological catatalysts are
called enzymes. They are usually globular in structure and have a region where their target chemicals can fit. In this
site, a micro-environment creates the perfect conditions and proximity for bond breakage and rearrangement, which
lowers the energy necessary to start the reaction. This “active-site” is design to fit only these target chemicals called
substrates. This happens either by “lock-and-key”, where the substrate fits perfectly, or by “induced-fit”, where the
enzyme folds over the substrate once it finds it. Therefore, each enzyme fulfills a specific job on specific substrates.
However, it is not consumed during the reaction and can be reused over and over. Because of this fitting procedure,
enzyme shape is very important to its function. Therefore, anything that affects its shape can disable the enzyme. For
example, changes in temperature and pH can “denaturate” or change the natural shape of the enzyme, making it
useless. This change is often irreversible. There are also other ways to regulate enzyme activity. Sometimes, although
the enzyme is designed to fit only one thing, a compound that has very similar shape to the substrate can bind to it and
disable it. This is called competitive-inhibition because this other compound competes with the substrate for the
enzyme. Another way is to bind to other areas of the enzyme (not the active site) causing the enzyme to change its
shape, which affects the shape of the active site and disables the enzyme. This is called non-competitive inhibition. The
body often uses this time of inhibition to turn enzymes on and off as needed. For example, you would not want flesheating enzymes that digest scabs to work at all times because they would eat through your skin. However, in the area
where a scab is ready to fall off, it is necessary to activate the enzyme. Therefore, the body secrets a chemical in the
area that combines with the enzyme to change its shape and make the active site accessible. This process is called
allosteric regulation of enzymatic activity. Sometimes enzymes also require the help of “assistance” chemicals call cofactors. For example, hemoglobin can only carry oxygen in red blood cells, if iron is in the molecule. Iron is the
cofactor that facilitates the hemoglobin activity. Enzymes also work better if there are more of them. However, this
only works to a point because there is no point in having thousands of enzymes and not as much substrate to act on.
Therefore, the concentration of substrate also matters. If the substrate is rare in the solution, it is harder enzymes to find
their target and act. Therefore, enzyme catalyzed reactions often speed up in the beginning, but after most of the
substrate is gone, the reaction rate slows down. Enzyme action is also slower in solutions where a lot of other materials
are dissolved and the substrate concentration is relatively low compared to this other things.
This lab focuses on one particular enzyme – catalase. Catalase plays an indispensable role in a living cell by preventing
the accumulation of toxic levels of hydrogen peroxide formed as a byproduct of metabolism. The enzyme, catalase,
speeds up the breakdown of hydrogen peroxide (H2O2) into water (H2O) and oxygen gas (O2). Oxygen produced forms
bubbles (that float on water). The reaction is described by the following equation:
2 H2O2 → 2 H2O + O2
Water is much more stable than peroxide. This reaction does tend to happen naturally. It is exergonic (releases energy).
However, without catalase, barely any peroxide degrades after a whole 24 hours. This is because the activation energy
necessary to jump-start the reaction is significant. Without catalase, the reaction would not happen fast enough in cells
and toxic levels of peroxide would accumulate as the cells perform their normal metabolic functions. With it, one
molecule of catalase can break 40 million molecules of hydrogen peroxide each second.
Vocabulary: activation energy, active site, enzyme, catalase, catalyst, denaturation, enzyme, induced-fit model, rate of
reaction, substrate
Safety:
 Hydrogen peroxide is an oxidizer and could bleach clothing.
 Protect clothing by wearing a lab apron.
 Wear safety goggles
Extension: Collision Theory Gizmo
Pre Lab
1. What are enzymes and what are they made of?
2. What do enzymes actually do?
3. How does the body use these enzymes?
a. Lipase
b. Amylase
c. Protease
4. What does the term catalyst mean?
5. What is the induced-fit model of enzyme action? How does it apply to this activity?
6. Explain the role of activation energy in a reaction. How does an enzyme affect activation energy?
7. Describe how a substrate interacts with an enzyme.
8. What is meant by denaturation of a protein? How can it happen?
9. List several ways to affect enzymes ability to perform its action.
10. What is the formula for hydrogen peroxide decomposition?
11. What are the reactants and products in the following equation? 2 H2O2 → 2 H2O + O2
12. What is the function of the enzyme in this investigation?
13. What is the substrate in this investigation? What is the enzyme?
14. Why is catalase so important?
15. Hypothesize as to what would happen to a reaction if one were to increase the concentration of catalase.
16. Based on your hypothesis draw a graph to represent how the reaction rate of catalase would change as you increase
enzyme concentration
Materials (per group):
 Beaker (250ml)
 Blender
 Distilled water
 Filter paper disks (Filter Paper + Whole
Puncher)
 Filtration Apparatus (Funnel, Cheesecloth of
filter paper, Container like a Beaker)
 Forceps
 Graduate Cylinder (100ml)
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Graph paper
Hydrogen peroxide (3%)
Ice
Ice bucket
Marking pencil
Paper towels
Potato
Yeast solution (Optional)
Procedures:
The enzyme solution 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.
ENZYME SHOULD BE KEPT ON ICE AT ALL TIMES!!
Alternative: Use yeast solution (rich in catalase). Or use crushed bovine liver crushed and then filtered in the same
manner as above
1. Label test tubes to represent the enzyme concentrations solution you will be creating: 0, 25, 50, 75, 100%
2. (Teacher may do this step to save time) Use a 100ml graduated cylinder to measure the volume of the test tubes
or vials you will be using. Fill the vials with water and then measure volume by pouring water into a graduated
cylinder (use funnel if necessary)
3. Prepare peroxide solutions in separate test tubes or vials as outlined below:
Concentration Hydrogen Peroxide Solutions
Concentration
Hydrogen Peroxide
Distilled Water
0% Peroxide Solution
No Peroxide
Full test tube
25% Peroxide Solution
Tube Volume x 0.25
Tube Volume x 0.75
50% Peroxide Solution
Tube Volume x 0.50
Tube Volume x 0.50
75% Peroxide Solution
Tube Volume x 0.75
Tube Volume x 0.25
100% Peroxide Solution
Full test tube
No water
4. Using the forceps, dip a filter paper disk into the beaker containing the enzyme solution. Keep the disk in the
solution for 4 seconds, and then remove it.
5. Place the disk on a paper towel for 4 seconds to remove any excess liquid.
6. Using the forceps, transfer the filter paper disk to the bottom of a rubber stopper.
7. Insert the stopper into one of the test tubes and quickly invert the test tube. Using a stopwatch, have one person
8.
9.
10.
11.
in your group measure how long it takes for the bubbles to carry the disk to the top of the test tube. Record the
time in a data table similar to the one shown below.
Repeat steps 3-7 for all test tubes.
Repeat trials: Wash test tubes and recreate solutions to repeat experiment. Do this 2 times to get 3 sets of data
and calculate averages. (Alternative: Use class data to generate averages and save time)
Calculate the average rising time for each of the peroxide solutions. Record this information in your data table.
Construct a graph plotting the concentration of hydrogen peroxide on the x-axis (independent variable) and
rising time on the y-axis (dependent variable).
Observations/Data:
Beaker
0% Peroxide
25% Peroxide
50% Peroxide
75% Peroxide
100% Peroxide
Trial 1
Table: Rising Time
Trial 2 Trial 3 Trial 4
Trial 5
Trial 6
Average
Data Analysis:
Construct and analyze a graph from your table. Remember to label axis, use units, and title your graph.
Discussion Questions:
1. Why was it important to use cold water and keep enzyme on ice when preparing the enzyme solution?
2. Why did some disks float?
3. Which concentration of catalase had the fastest reaction time?
4. Which concentration of catalase had the slowest reaction time?
5. How did you know that catalase was present in the above compounds?
6. What 2 substances form when catalase breaks down hydrogen peroxide?
7. What type of organic compound is catalase?
8. Suppose you had placed a filter paper disk in a 30% peroxide solution. Using your graph, predict how long it would
take this disk to rise to the top.
9. In a paragraph, describe how the concentration of peroxide affects the breakdown rate of hydrogen peroxide. Use
the results of this experiment to justify your answer.
10. The results reject of failed to reject your hypothesis? Why?