Enzyme Activity of Peroxidase
Enzyme Activity of Peroxidase
Overview
In lab this week we will investigate the activity of an enzyme called peroxidase. Each experiment
will be conducted by a group of four students. All members of the group will help to set up each
experiment. The group must also designate one student to be a timer, another student to be the
spectrophotometer reader, and another to be the data recorder. Each student in the group must have
a copy of the raw data (see Peroxidase Lab Handout) so that she/he can answer the questions for
next week's lab, and one member of the group must enter the data into the online database before
the end of lab today (see link at the bottom of this page). Next week we will analyze the data
collected during this week’s lab.
Introduction
One of the key characteristics of life is metabolism - chemical transformations that allow organisms
to grow, maintain their structures, reproduce, and interact with the environment. All life’s chemical
reactions are catalyzed by a specific group of proteins called enzymes. Enzymes increase the rates
of chemical reactions and involve a transient combination between the enzyme and the substrate.
This transient combination is called the "Enzyme Substrate Complex."
The velocity of an enzyme-catalyzed reaction depends on the number of combinations per unit time
between the enzyme and the substrate and these are influenced by a number of factors. Substrates
bind at a specific location on the enzyme called the reactive site and its characteristics will
influence the velocity of the reaction. The pH is particularly important in enzyme-catalyzed
reactions because of its effects on the stability of the enzyme and the fact that enzymes, like other
proteins, are multivalent dipolar ions that dissociate in accordance with the pH of the medium. Like
all chemical reactions there should also be dependence on temperature.
During this lab exercise you will investigate the naturally occurring enzyme peroxidase. Peroxidase
is a heme-containing enzyme found in peroxisomes (cellular organelles) and can be obtained from a
variety of plant tissues. Each cell uses oxygen in its metabolism. A resulting by-product, H2O2, is
highly toxic and must be removed immediately by peroxidase, or other enzymes, before the cell is
damaged. The reaction that will be monitored is:
peroxidase
RH2 + H2O2 --------------> R + 2H2O
where RH2 stands for a variety of H donors and R for the form of the molecule after having
donated hydrogen.
It is impossible to see the reaction in vitro because all of the reactants are colorless. We use the
reducing agent, guaiacol, as the H donor, because it changes color when it loses hydrogens, forming
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Enzyme Activity of Peroxidase
a brown product, tetraguaiacol. Because the process now involves a color change, it is possible to
use a spectrophotometer to monitor the rate of change. The absorbance is a direct measure of the
amount of substrate hydrolyzed.
This lab includes the formulation of hypotheses to be tested by the experiments. In forming an
hypothesis, the assumptions are stated and a tentative explanation proposed that links possible
cause and effect. A key aspect of an hypothesis, and indeed of the modern scientific method, is that
the hypothesis must be falsifiable, i.e., if a critical experiment is performed and yielded certain
information, the hypothesis would be declared false and discarded because it was not useful in
predicting any natural phenomenon. Science advances as a result of the rejection of false ideas
expressed as hypotheses and tested through experiments. Hypotheses that over the years are not
falsified and which are useful in predicting natural phenomena are called laws or principles - for
example, the Laws of Thermodynamics.
Hypotheses are made in mutually exclusive couplets called the null hypothesis (Ho) and the
alternative hypothesis (Ha). The null hypothesis is stated as a negative and the alternative as a
positive. For example, lets say that you work for NASA and you are interested in the effects of
gravity on the enzyme salivary amylase. Your null hypothesis is that gravity has no impact on the
rate of reaction by salivary amylase, and your alternative hypothesis is that gravity does impact the
rate of reaction by salivary amylase. Once you have generated your null and alternative hypotheses,
you design an experiment to test the null hypothesis, collect and analyze data, and report the results
of your experiment(s) to your peers. As you can see, rival hypotheses constitute alternative,
mutually exclusive statements; both cannot be true. The purpose in proposing a null hypothesis is to
make a statement that could be rejected if data were available.
Post a null hypothesis and an alternative hypothesis for each of the four following
experiments to your lab website before attending lab. You don’t need to post hypotheses for
Standardizing the Amount of Enzyme.
Procedure
Preparing an Extract Containing Peroxidase
1. Weigh 8 g of peeled turnip tissue on a balance.
2. Homogenize the tissue by adding it to 300 ml of cold (4°C) 0.1 M phosphate buffer at pH 7.
Blend it for 20 seconds at high speed in a cold blender. Filter the extract through several
layers of cheesecloth. Keep the extract on ice. The extract will keep for about ten hours in a
refrigerator.
Standardizing the Amount of Enzyme
The extract you prepared contains hundreds of different enzymes, including peroxidase. However,
only peroxidase, will react with H2O2. The enzymatic activity of each extract will vary depending
on the size and age of the turnip, the extent of the tissue homogenization, and the age of the extract.
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Enzyme Activity of Peroxidase
To determine the correct amount of extract to use in Experiments 1-4, a trial run will be performed
in which the amount of extract (enzyme) added is the only variable.
Use the following directions to set up the chemical reactions and to conduct the experiment to
determine if the enzyme concentration of the extract is appropriate to conduct Experiments 1-4.
1. Label two test tubes as follows: buffer, pH 5; buffer, pH 7. Fill each about half full with the
appropriate stock solution. Label two pipettes to correspond with the solutions. Keep the
turnip extract in a beaker on ice. The 25 mM guaiacol and 10 mM hydrogen peroxide
solutions are in brown bottles.
2. Number three test tubes from 1 to 3. The contents of the tubes will be:
1. Control with no extract to be used in calibrating the spectrophotometer
2. Substrate and indicator dye
3. Extract solution
Tubes 2 and 3 will be quickly mixed together when it is time to measure a reaction. Mix them
only when you are ready to measure that reaction in the spectrophotometer. The exact
quantities to be added to each tube are listed in Table 1.
Table 1. Mixing table for trial run to determine extract concentration (all values in ml).
Tube
Buffer (pH 5) Buffer (pH 7) H2O2 Extract Guaiacol Total Volume
1 Control
4.0
2
3
1.0
2.0
1.0
1.0
2.0
1.0
8
2.0
1.0
4
4
1.0
3. Add stock solutions to each tube using the appropriate pipettes. Use of the wrong pipette will
cross contaminate your reagents and introduce errors into your subsequent experiments in
this exercise.
4. Adjust the spectrophotometer to zero absorbance at 500 nm. Pour the contents of test tube 1
into a cuvette. This tube is used to "blank" the spectrophotometer, so that any color caused by
contaminants in the reagents will not influence subsequent measurements.
5. Wipe a cuvette with lens paper and handle it by only the top 1/4th.
6. Pour the contents of text tube 2 into test tube 3. Quickly pour the mixture into the clean
cuvette and immediately place the cuvette in the spectrophotometer. Immediately start the
timer and read the absorbance. This is your 0 second reading.
7. Read the absorbance at 20-second intervals from the start of mixing and record the values in
Table 2. If you are a little late in reading the meter, change Table 2 to show the actual times.
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Enzyme Activity of Peroxidase
After 120 seconds remove the tube from the spectrophotometer and visually note the color
change. Discard the solution.
Table 2. Production of tetraguaiacol in the conversion of H2O2 into H2O by peroxidase.
Time (seconds) Absorbance (500 nm)
0
20
40
60
80
100
120
8. Using 1 ml of turnip extract should give a linear absorbance change from 0 to around 1 in
approximately 120 seconds. If this is so, then this will be the standardized amount of extract
to be used in Experiments 1-3. If this amount of extract does not produce the desired change
in absorbance, then repeat the experiment with different amounts of the extract and the pH 7
buffer until the appropriate change in absorbance is attained.
9. Repeat the experiment with the appropriate amount of extract and record the absorbance at 0,
15, 30, 45 and 60 seconds in Table 3.
Table 3. Production of tetraguaiacol in the conversion of H2O2 into H2O by peroxidase.
Time (seconds) Absorbance (500 nm)
0
15
30
45
60
Experiment 1: What is the effect of changing extract (enzyme) amount on the
reaction?
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Enzyme Activity of Peroxidase
State null (Ho) and alternative (Ha) hypotheses that relate change in enzyme activity to the amount
of extract used.
1. Number five test tubes 1 through 5. Set up extract amount effect tests by adding the reagents
to each of the tubes as described in Table 4.
Table 4. Mixing table for effect of changing extract (enzyme) amount (all values in ml).
Tube
Buffer (pH 5) Buffer (pH 7) H2O2 Extract Guaiacol Total Volume
1 Control
4.0
1.0
2
3
1.0
2.5
1.0
4
5
1.0
1.0
1.0
2.0
2.0
1.0
8
1.0
4
4
1.0
4
4
0.5
2.0
2.0
2. Mix the contents of tubes 2 and 3, pour into a cuvette, and repeat your measurements for 60
sec at 15-second intervals and record the results in Table 5.
3. Mix the contents of tubes 4 and 5, pour into a cuvette, and repeat your measurements for 60
sec at 15-second intervals and record the results in Table 5.
Table 5. Production of tetraguaiacol in the conversion of H2O2 into H2O by peroxidase for
different amounts of extract.
Time (seconds) 0.5 ml Extract (Tubes 2 & 3 A500) 2.0 ml Extract (Tubes 4 & 5 A500)
0
15
30
45
60
4. Mix the contents of tubes 6 and 7, pour into a cuvette, and record the absorbance in Table 5.
Experiment 2: What is the effect of pH?
State null (Ho) and alternative (Ha) hypotheses that relate change in enzyme activity to the pH of
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Enzyme Activity of Peroxidase
the solutions used.
To determine the effect of pH on peroxidase, perform the following experiment. Your instructor
will supply buffers at pHs of 3, 5, 7, and 9. Number nine test tubes 1 through 9. Set up pH effect
tests by adding the reagents described in Table 6.
Table 6. Mixing table for pH experiment (all values in ml).
pH
5
Tube
Buffer
H2O2 Extract Guaiacol Total Volume
1 Control 6.0 (pH 5)
3
2
3
5
4
5
7
6
7
9
8
9
1.0
2.0
4.0 (pH 3)
8
1.0
3
5
1.0
3
5
1.0
3
5
1.0
3
5
1.0
2.0
4.0 (pH 5)
1.0
2.0
4.0 (pH 7)
1.0
2.0
4.0 (pH 9)
1.0
1.0
After adjusting the spectrophotometer with the contents of test tube 1, mix pairs of tubes one at
time (2 and 3, 4 and 5, 6 and 7, 8 and 9) and measure absorbance changes at 15-second intervals for
60 seconds for each mixed pair. Record the results in Table 7 below.
Table 7. Production of tetraguaiacol in the conversion of H2O2 into H2O by peroxidase at different
pHs.
Time (seconds) pH 3 A500 pH 5 A500 pH 7 A500 pH 9 A500
0
15
30
45
60
Experiment 3: What is the effect of boiling on peroxidase activity?
Most proteins are denatured when they are heated to temperatures above 70°C. Denaturation is a
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Enzyme Activity of Peroxidase
nonreversible change in a protein's three-dimensional structure. If the shape of an enzyme is
significantly altered, what do you predict will happen to measured enzyme activity?
State null (Ho) and alternative (Ha) hypotheses that relate heat treatment of an enzyme (boiling) to
the expected effect on that enzyme's activity.
To test your Ho, follow these directions. Add 3 ml of extract to a test tube and place it in a boiling
water bath. After five minutes, remove the tube and let it cool to room temperature. Number three
test tubes and add reagents as called for in mixing Table 8.
Table 8. Mixing table for boiling extract (all values in ml).
Tube
Buffer (pH 5) Buffer (pH 7) H2O2 Extract Guaiacol Total Volume
1 Control
5.0
1.0
2
3
2.0
1.0
1.0
1.0
2.0
1.0
8
1.0
4
4
1.0
After adjusting the spectrophotometer with the contents of test tube 1, mix the contents of tubes 2
and 3, pour the mixture into a cuvette, and read the absorbance at 15-second intervals for 60
seconds. Record the results in Table 9.
Table 9. Production of tetraguaiacol in the conversion of H2O2 into H2O by peroxidase after boiling
the extract.
Time (seconds) Absorbance (500 nm)
0
15
30
45
60
Experiment 4: How does temperature affect enzyme activity?
To determine the effects of temperature on peroxidase activity, you will repeat the enzyme assay in
water baths at four temperatures:
1. In an ice bath at approximately 4°C
2. At room temperature (about 23°C)
3. At 32°C
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Enzyme Activity of Peroxidase
4. At 48°C
State null (Ho) and alternative (Ha) hypotheses that relate change in enzyme activity to the
temperature of the solutions used.
To test your Ho, you should use the following directions to set up the reactions and conduct the
experiment. Number nine test tubes in sequence 1 through 9. Refer to Table 10 for the volumes of
reagents to be added to each tube.
Table 10. Mixing table for temperature experiment (all values in ml).
Temperature
°C
Tube
Buffer Buffer
Total
H2O2 Extract Guaiacol
(pH 5) (pH 7)
Volume
1 Control
6.0
4
2
3
1.0
2.0
23
4
5
1.0
2.0
32
6
7
1.0
2.0
48
8
9
1.0
2.0
1.0
2.0
1.0
8
1.0
4
4
1.0
4
4
1.0
4
4
1.0
4
4
1.0
2.0
1.0
1.0
2.0
1.0
1.0
2.0
1.0
1.0
1.0
Preincubate all the solutions at the appropriate temperatures for at least 15 minutes before mixing.
After reaching temperature equilibrium and adjusting the spectrophotometer with the contents of
test tube 1, mix pairs of tubes (2 and 3, 4 and 5, 6 and 7, and 8 and 9) one pair at a time and
measure changes in absorbance for 60 seconds at 15-second intervals for each temperature. The
temperatures will not remain exact, but the effects can be overlooked.
Note: after the spectrophotometer is adjusted, the room-temperature experiment can be performed
immediately while the other tubes temperature-equilibrate.
Record changes in absorbance for each temperature in Table 11.
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Enzyme Activity of Peroxidase
Table 11. Production of tetraguaiacol in the conversion of H2O2 into H2O by peroxidase different
temperatures.
Time (seconds) 4°C 23°C 32°C 48°C
0
15
30
45
60
Before you leave lab today, please enter your data into the Peroxidase Database
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