The Hydrogen Peroxide Breakdown
7
The Hydrogen Peroxide Breakdown
Examining Factors that Affect the
Reaction Rate of Enzymes
Using a Graphing Calculator and Data Collection Device
There are thousands of chemical reactions that occur in an organism that make life possible. Most of
these chemical reactions proceed too slowly to occur by themselves. Enzymes are protein catalysts that
speed up chemical reactions in a cell. Catalysts are not changed by the reactions they control, and are
not used up during the reaction. Enzymes, therefore, can be used over and over again. Enzymes are
large complex proteins made by the cell and allow chemical reactions to take place at the temperature of
the cell. These catalysts are needed in only very small amounts because a single enzyme molecule can
complete the same reaction thousands of times in one minute.
Each enzyme is very specific and can only catalyze a certain reaction. The specific reaction catalyzed
by an enzyme depends on the molecular structure and shape of a small area of the enzyme’s surface
called the active site. The active site can attract and hold only its specific molecules. The target
molecule that the enzyme attracts and acts upon is called the substrate. The substrate and the active site
of the molecule must fit together very closely. Sometimes the enzyme changes its shape slightly to
bring about the necessary fit.
A chemical reaction requires that bonds in the reactants be broken. The initial energy that must be
absorbed in order to break the bonds of the reactant molecule is called the energy of activation.
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The Hydrogen Peroxide Breakdown
Enzymes work by lowering the energy of activation. For example, hydrogen peroxide decomposes to
form water, H2O, and oxygen gas, O2.
While this is a catabolic reaction, the rate at which it occurs is slow. Light and temperature affect the
reaction rate. As a result, bottles of hydrogen peroxide you may purchase at the drug store are sold in
light-blocking brown bottles and have instructions to store them in a cool dark place. Hydrogen
peroxide also comes with an expiration date because even with cool and dark storage, the breakdown of
H2O2 molecules will still occur to some degree. Hydrogen peroxide is toxic to cells. This property
makes it useful for treating open wounds since it kills invading bacterial cells. Interestingly enough,
hydrogen peroxide is a by-product of some biochemical pathways found in many cells. Yet, the
accumulation of hydrogen peroxide can kill a cell. Cells, therefore, cannot wait for hydrogen peroxide
to naturally decompose because that takes too much time. Most tissues produce the enzyme, catalase, to
increase the rate of hydrogen peroxide’s decomposition. Catalase lowers the energy of activation
needed for the decomposition and as a result, more molecules are able to be decomposed in a shorter
amount of time.
There are factors that can affect how fast an enzymatic reaction occurs. For example, an increase in the
amount of enzyme will increase the reaction rate. There are more enzyme molecules available to be
involved in the reaction. Another factor that can affect how fast the enzyme works is temperature. An
increase in temperature causes the molecules to move faster and engage more frequently in a chemical
reaction. At higher temperatures though, the enzymes will denature as the hydrogen bonding falls apart.
This lab investigates the effect of enzyme concentration and temperature on an enzymatic reaction.
The reaction used in this laboratory exercise is the decomposition of hydrogen peroxide and the enzyme
used is catalase. A pressure probe connected to an interface device and graphing calculator will measure
the amount of oxygen gas produced. The more oxygen gas produced the greater the increase in
pressure.
PURPOSE
The purpose of this laboratory exercise is to determine the reaction rate of the decomposition of
hydrogen peroxide when the concentration of catalase is varies or the temperature is varied.
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The Hydrogen Peroxide Breakdown
MATERIALS
pressure sensor probe
CBL or LabPro interface
3% hydrogen peroxide solution
forceps
10 mL pipette
2 ea 50 mL beakers
# 4 test tube stopper with eye dropper
7
graphing calculator
filter paper disks
catalase solution
test tube rack
10 mL graduated cylinder
80 mL test tube
link cord
Safety Alert
CAUTION: Take care not to spill any liquids on the interface or graphing calculator.
PROCEDURE
Note: You will either do Part I or Part II of this experiment and then share your results with another
group.
PART I (EFFECT OF CONCENTRATION)
1. Answer the pre-lab questions on the student answer page.
2. Formulate two hypotheses: The first hypothesis should be about the effect of enzyme concentration
on the reaction rate, and the second hypothesis should be about the effect of temperature on the
enzyme reaction rate. Record this on the student answer page.
3. Slide the calculator and CBL or LabPro Interface in the bottom part of the cradle and it will click
into place. Snap the calculator into the top portion of the cradle.
4. Plug the short black link cable into the link port on the bottom of the TI Graphing Calculator and the
interface.
5. Plug the gas pressure probe into channel 1 of the interface. There should piece of plastic tubing
from the pressure probe. Some versions of certain pressure probes have valves for opening and
closing the sensor. If the pressure probe has such a valve, ensure that it is open in accordance to the
directions accompanying the probe.
for a TI 83+ or
for the TI 83 then press the number
6. Turn on the calculator and press
key that precedes the DATAMATE program. At this time the interface should have automatically
identified your pressure sensor. It will display the correct pressure in the upper right hand corner. If
the correct pressure is not displayed do the following:
• Select SETUP from the MENU by pressing
• Select “CH1” from the MENU and press
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.
.
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The Hydrogen Peroxide Breakdown
• Press
for more, press
• Select the correct type of probe.
again for pressure.
• Select “MMHG” by pressing
• Press
to indicate OK.
.
7. Select SETUP from the MENU by pressing
• Select “MODE” by pressing the
• Select "TIME GRAPH" by pressing
and then
and then
.
again to change the time settings
• Enter “3” as the time between sample, in seconds, press
.
• Enter “99” as the number of sample (the interface will collect data for approximately five
minutes), press
.
8. Another window will appear with the summary of the probes and the length of the experiment. Press
to indicate OK. Press
or OK again to return to the main menu.
9. A new window will appear and the calculator is now ready to start the experiment. DO NOT press
until you are ready to run the experiment.
10. Place the other end of the aquarium tubing found on the pressure probe onto the eye dropper sticking
out of the #4 test tube stopper.
11. Place the test tube in a test tube rack. Measure 10 mL of hydrogen peroxide with a graduated
cylinder and add it to the test tube.
12. Pour a small amount of the catalase solution into the 50 mL beaker. Keep the catalase beaker on ice.
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The Hydrogen Peroxide Breakdown
7
13. Obtain some paper filter disks and separate any paper disks that may be stuck together. Using the
forceps, dip one disk into the catalase solution. Drain the disk against the wall of the beaker several
times to remove the excess solution. With the forceps, transfer the disk to the test tube and place it
on the interior wall of the test tube. It will stick to the wall of the test tube. Using the forceps
carefully push the disks 1/2 to 1/3 of the way down the wall of the test tube. Repeat this procedure
until you have four disks stuck to the inside wall of the test tube.
14. Place the stopper that is connected to the pressure probe onto the test tube.
15. Press
. There should be 4 short beeps and the quick setup light will flash. The experiment
will run for approximately 5 minutes. You should notice that the data is being graphed as it is being
collected.
16. Immediately, tip the test tube slightly on its side so that the hydrogen peroxide will bring the disks
down into the solution and the reaction will begin. Right the test tube up again.
• CAUTION: Pressure is building in the test tube. During the course of the experiment, it is
important that you keep your thumb on the stopper to prevent it from popping off as the
pressure builds.
17. When the experiment is complete, 4 short beeps will sound and the quick setup light will flash. Now
a labeled, fitted graph will be displayed.
• Use the
and
keys to move the cursor. View the data points will be displayed at
the bottom of the graph. Use these keys to determine the minimum or initial pressure and the
maximum or final pressure. Record this information in your data table on the student answer
page.
18. Press
and the screen will tell you which Lists contain your data. Press
the program is done.
again and
19. Repeat the experiment three more times, but each time using one less disk. Trial 2 should contain 3
disks; Trial 3 should contain 2 disks, and Trial 4 should contain 1 disk. Be sure to wash the test tube
thoroughly between trials, making sure all the catalase is removed. Also when starting
, and it will retain the last parameters set, so that you do not have
DATAMATE do not press
to reset the program. Be sure to record the maximum/final and minimum/initial pressure
measurements in your data table.
20. Clean up your area and return your equipment to its original condition.
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The Hydrogen Peroxide Breakdown
PART II (EFFECT OF TEMPERATURE)
1. Answer the pre-lab questions on your student answer page.
2. Formulate two hypotheses: The first hypothesis should be about the effect of enzyme concentration
on the reaction rate, and the second hypothesis should be about the effect of temperature on the
enzyme reaction rate. Record this on the student answer page.
3. Slide the calculator and CBL 2 or LabPro Interface in the bottom part of the cradle and it will click
into place. Snap the calculator into the top portion of the cradle.
4. Plug the short black link cable into the link port on the bottom of the TI Graphing Calculator and the
interface.
5. Plug the gas pressure probe into channel 1 of the interface. There should piece of plastic tubing
from the pressure probe. Some versions of certain pressure probes have valves for opening and
closing the sensor. If the pressure probe has such a valve, ensure that it is open in accordance to the
directions accompanying the probe.
6. Turn on the calculator and press
for a TI 83+ or
for the TI 83 then press the number
key that precedes the DATAMATE program. At this time the interface should have automatically
identified your pressure sensor. It will display their correct pressure in the upper right hand corner.
If the correct pressure is not displayed do the following:
• Select SETUP from the MENU by pressing.
• Select “CH1” from the MENU and press
• Press
for more, press
• Select the correct type of probe.
• Select “MMHG” by pressing
• Press
.
.
again for pressure.
.
to indicate OK.
7. Select SETUP from the MENU by pressing
• Select “MODE” by pressing the
• Select “TIME GRAPH” by pressing
.
and then
and then
.
again to change the time settings.
• Enter “3” as the time between sample, in seconds, press
.
• Enter “99” as the number of sample (the interface will collect data for approximately 5 minutes),
press
.
8. Another window will appear with the summary of the probes and the length of the experiment. Press
to indicate OK. Press
334
or OK again to return to the main menu.
Laying the Foundation in Biology
The Hydrogen Peroxide Breakdown
7
9. A new window will appear and the calculator is ready to start the experiment. DO NOT press
until you are ready to run the experiment.
10. Place the other end of the aquarium tubing found on the pressure probe onto the eye dropper sticking
out of the #4 test tube stopper.
11. Place the test tube in a test tube rack. Measure 10 mL of hydrogen peroxide with a graduated
cylinder and add it to the test tube.
12. Pour a small amount of the catalase solution into the 50 mL beaker. Keep this catalase beaker on
ice.
13. Obtain some paper filter disks and separate any paper disks that may be stuck together. Using the
forceps, dip one disk into the catalase solution. Drain the disk against the wall of the beaker several
times to remove the excess solution. With the forceps, transfer the disk to the test tube and place it
on the interior wall of the test tube. It will stick to the wall of the test tube. Using the forceps
carefully push the disks 1/2 to 1/3 of the way down the wall of the test tube. Repeat this procedure
until you have four disks stuck to the inside wall of the test tube.
14. Place the stopper that is connected to the pressure probe on to the test tube.
. There should be 4 short beeps and the quick setup light will flash. The experiment
15. Press
will run for 5 minutes. You should notice that the data is being graphed as it is being collected.
16. Immediately, tip the test tube slightly on its side so that the hydrogen peroxide will bring the disks
down into the solution and the reaction will begin. Right the test tube up again.
• CAUTION: Pressure is building in the test tube. During the course of the experiment, it is
important that you keep your thumb on the stopper to prevent it from popping off as the
pressure builds.
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7
The Hydrogen Peroxide Breakdown
17. When the experiment is complete, 4 short beeps will sound and the quick setup light will flash. Now
a labeled, fitted graph will be displayed.
or
keys to move the cursor and the data points will be displayed at the
• Using the
bottom of the graph. Use these keys to determine the minimum or initial and the maximum or
final pressure. Record this information in your data table on the student answer page.
18. Press
and the screen will tell you which Lists contain your the data. Press
and the program is done.
again
19. Repeat the experiment, but this time use hydrogen peroxide that has been cooled to 10o C. Be sure
to wash the test tube thoroughly, making sure all the catalase is removed. Also when starting
DATAMATE do not press
to reset the program.
, and it will retain the last parameters set, so that you do not have
20. Repeat the experiment, but this time use hydrogen peroxide that has been warmed to 35o C. Also
when starting DATAMATE do not press
you do not have to reset the program.
, and it will retain the last parameters set, so that
21. Clean all your equipment and return it to its original condition.
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The Hydrogen Peroxide Breakdown
7
The Hydrogen Peroxide Breakdown
Examining Factors that Affect the
Reaction Rate of Enzymes
Using a Computer
There are thousands of chemical reactions that occur in an organism that make life possible. Most of
these chemical reactions proceed too slowly to occur by themselves. Enzymes are protein catalysts that
speed up chemical reactions in a cell. Catalysts are not changed by the reactions they control, and are
not used up during the reaction. Enzymes, therefore, can be used over and over again. Enzymes are
large complex proteins made by the cell and allow chemical reactions to take place at the temperature of
the cell. These catalysts are needed in only very small amounts because a single enzyme molecule can
complete the same reaction thousands of times in one minute.
Each enzyme is very specific and can only catalyze a certain reaction. The specific reaction catalyzed
by an enzyme depends on the molecular structure and shape of a small area of the enzyme’s surface
called the active site. The active site can attract and hold only its specific molecules. The target
molecule that the enzyme attracts and acts upon is called the substrate. The substrate and the active site
of the molecule must fit together very closely. Sometimes the enzyme changes its shape slightly to
bring about the necessary fit.
A chemical reaction requires that bonds in the reactants be broken. The initial energy that must be
absorbed in order to break the bonds of the reactant molecule is called the energy of activation.
Laying the Foundation in Biology
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7
The Hydrogen Peroxide Breakdown
Enzymes work by lowering the energy of activation. For example, hydrogen peroxide decomposes to
form water, H2O, and oxygen gas, O2.
While this is a catabolic reaction, the rate at which it occurs is slow. Light and temperature affect the
reaction rate. As a result, bottles of hydrogen peroxide you may purchase at the drug store are sold in
light-blocking brown bottles and have instructions to store them in a cool dark place. Hydrogen
peroxide also comes with an expiration date because even with cool and dark storage, the breakdown of
H2O2 molecules will still occur to some degree. Hydrogen peroxide is toxic to cells. This property
makes it useful for treating open wounds since it kills invading bacterial cells. Interestingly enough,
hydrogen peroxide is a by-product of some biochemical pathways found in many cells. Yet, the
accumulation of hydrogen peroxide can kill a cell. Cells, therefore, cannot wait for hydrogen peroxide
to naturally decompose because that takes too much time. Most tissues produce the enzyme, catalase, to
increase the rate of hydrogen peroxide’s decomposition. Catalase lowers the energy of activation
needed for the decomposition and as a result, more molecules are able to be decomposed in a shorter
amount of time.
There are factors that can affect how fast an enzymatic reaction occurs. For example, an increase in the
amount of enzyme will increase the reaction rate. There are more enzyme molecules available to be
involved in the reaction. Another factor that can affect how fast the enzyme works is temperature. An
increase in temperature causes the molecules to move faster and engage more frequently in a chemical
reaction. At higher temperatures though, the enzymes will denature as the hydrogen bonding falls apart.
This lab investigates the effect of enzyme concentration and temperature on an enzymatic reaction.
The reaction used in this laboratory exercise is the decomposition of hydrogen peroxide and the enzyme
used is catalase. A pressure probe connected to an interface device and graphing calculator will measure
the amount of oxygen gas produced. The more oxygen gas produced the greater the increase in
pressure.
PURPOSE
The purpose of this laboratory exercise is to determine the reaction rate of the decomposition of
hydrogen peroxide when the concentration of catalase is varies or the temperature is varied.
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Laying the Foundation in Biology
The Hydrogen Peroxide Breakdown
7
MATERIALS
pressure sensor probe
catalase solution
test tube rack
10 mL graduated cylinder
80 mL test tube
paper filter disks
computer with Logger Pro installed
3% hydrogen peroxide solution
forceps
10 mL pipette
2 ea 50 mL beakers
# 4 test tube stopper with eye dropper
Lab Pro or serial interface box
link cord
Safety Alert
CAUTION: Electricity is being used.
Take care not to spill any liquids on any of the computer equipment or electrical outlets.
PROCEDURE
Note: You will either do Part I or Part II of this experiment and then share your results with another
group.
PART I (EFFECT OF CONCENTRATION)
1. Answer the pre-lab questions on student answer page.
2. Formulate two hypotheses: The first hypothesis should be about the effect of enzyme concentration
on the reaction rate, and the second hypothesis should be about the effect of temperature on the
enzyme reaction rate. Record this on the student answer page.
3. Plug the pressure sensor into Port 1 of the serial box or Ch 1 of the LabPro. This box should already
be plugged into the modem port and AC outlet. Plug the gas pressure probe into channel 1 of the
interface. There should piece of plastic tubing from the pressure probe. Some versions of certain
pressure probes have valves for opening and closing the sensor. If the pressure probe has such a
valve, ensure that it is open in accordance to the directions accompanying the probe.
4. Now click on the LoggerPro and open the file titled “Catalase”.
5. A graph should appear and you should notice that at the top you can read both time and pressure
readings. Make sure that the pressure probe is reading between 730 and 790 mm Hg. If it is not, tell
your teacher.
6. Place the other end of the tubing found on the pressure probe onto the eyedropper sticking out of the
#4 test tube stopper.
7. Place the test tube into a test tube rack. Measure 10 mL of hydrogen peroxide with a graduated
cylinder and add it to the test tube.
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7
The Hydrogen Peroxide Breakdown
8. Pour a small amount of the catalase solution into the 50 mL beaker. Keep this catalase beaker on
ice.
9. Obtain some paper filter disks and separate any paper disks that may be stuck together. Using the
forceps, dip one disk into the catalase solution. Drain the disk against the wall of the beaker several
times to remove the excess solution. With the forceps, transfer the disk to the test tube and place it
on the interior wall of the test tube. It will stick to the wall of the test tube. Using the forceps
carefully push the disks 1/2 to 1/3 of the way down the wall of the test tube. Repeat this procedure
until you have four disks stuck to the inside wall of the test tube.
10. Place the stopper that is connected to the pressure probe on to the test tube.
11. Press the enter key, or click on Collect. You should notice that the data is being graphed as it is
being collected.
12. Immediately, tip the test tube slightly on its side so that the hydrogen peroxide will bring the disks
down into the solution and the reaction will begin. Right the test tube up again.
• CAUTION: Pressure is building in the test tube. During the course of the experiment, it is
important that you keep your thumb on the stopper to prevent it from popping off as the
pressure builds.
13. When the experiment is complete, pull down the Analyze menu and highlight Statistics. This will
display the minimum or initial pressure and the maximum or final pressure. Record this information
in your data table on the student answer page.
14. Pull down the Data Menu and click on Store Latest Run. This will allow you to compare this run
to the subsequent runs of the experiment. The red line should become a thinner red line.
15. The effects of concentration of enzyme on the reaction rate can be tested by repeating the experiment
three more times. Each time reducing the number of disks using three disks, two disks, and one disk
respectively. Steps 7-12 are the steps that you should follow in repeating the experiment. Be sure to
wash out the test tube thoroughly making sure all the catalase is removed.
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The Hydrogen Peroxide Breakdown
7
16. After you have finished close all windows on the computer. Clean up your area and return your
equipment to its original condition.
PART II (EFFECT OF TEMPERATURE)
1. Answer the pre-lab questions on your student answer page.
2. Formulate two hypotheses: The first hypothesis should be about the effect of enzyme concentration
on the reaction rate, and the second hypothesis should be about the effect of temperature on the
enzyme reaction rate. Record this on the student answer page.
3. Plug the pressure sensor into Port 1 of the serial box or Ch 1 of the LabPro. This box should already
be plugged into the modem port and AC outlet. Plug the gas pressure probe into channel 1 of the
interface. There should piece of plastic tubing from the pressure probe. Some versions of certain
pressure probes have valves for opening and closing the sensor. If the pressure probe has such a
valve, ensure that it is open in accordance to the directions accompanying the probe.
4. Now click on the LoggerPro and open the file titled “Catalase”.
5. A graph should appear and you should notice that at the top you can read both time and pressure
readings. Make sure that the pressure probe is reading between 730 and 790 mm Hg. If it is not, tell
your teacher.
6. Place the other end of the tubing found on the pressure probe onto the eyedropper sticking out of the
#4 test tube stopper.
7. Place the test tube into a test tube rack. Measure 10 mL of hydrogen peroxide with a graduated
cylinder and add it to the test tube.
8. Pour a small amount of the catalase solution into the 50 mL beaker. Keep this catalase beaker on
ice.
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The Hydrogen Peroxide Breakdown
9. Obtain some paper filter disks and separate any paper disks that may be stuck together. Using the
forceps, dip one disk into the catalase solution. Drain the disk against the wall of the beaker several
times to remove the excess solution. With the forceps, transfer the disk to the test tube and place it
on the interior wall of the test tube. It will stick to the wall of the test tube. Using the forceps
carefully push the disks 1/2 to 1/3 of the way down the wall of the test tube. Repeat this procedure
until you have four disks stuck to the inside wall of the test tube.
10. Place the stopper that is connected to the pressure probe on to the test tube.
11. Press the enter key, or click on Collect. You should notice that the data is being graphed as it is
being collected.
12. Immediately, tip the test tube slightly on its side so that the hydrogen peroxide will bring the disks
down into the solution and the reaction will begin. Right the test tube up again.
• CAUTION: Pressure is building in the test tube. During the course of the experiment, it is
important that you keep your thumb on the stopper to prevent it from popping off as the
pressure builds.
13. When the experiment is complete, pull down the Analyze menu and highlight Statistics. This will
display the minimum or initial pressure and the maximum or final pressure. Record this information
in your data table on the student answer page.
14. Pull down the Data Menu and click on Store Latest Run. This will allow you to compare this run
to the subsequent runs of the experiment. The red line should become a thinner red line.
15. The effects of temperature on the enzyme reaction rate can be tested by repeating the experiment two
more times. The first time use hydrogen peroxide that has been cooled to 10o C and the second time
use hydrogen peroxide that has been warmed to 35o C. Steps 7-14 are the steps that you should
follow in repeating the experiment. Be sure to wash out the test tube thoroughly making sure all the
catalase is removed.
16. After you have finished close all windows on the computer. Clean up your area and return your
equipment to its original condition
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7
Name _____________________________________
Period _____________________________________
The Hydrogen Peroxide Breakdown
HYPOTHESES
DATA AND OBSERVATIONS
Experiment
Minimum Pressure
(mm Hg)
Maximum Pressure
(mm Hg)
Total Press Change
(mm Hg)
4 Disks
Part
I
3 Disks
2 Disks
1 Disk
Part
II
Room Temp.
Cold Temp.
Warm Temp.
PRE-LAB QUESTIONS
1. List four characteristics of enzymes.
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2. What is an active site and what is its relationship to an enzyme?
3. What is the energy of activation?
4. How does the enzyme affect the energy of activation?
5. What are some factors that can affect the reaction rate of an enzymatic reaction?
CONCLUSION QUESTIONS
1. What is the relationship between pressure and time as the hydrogen peroxide decomposes?
2. What gas is produced when hydrogen peroxide decomposes?
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7
3. What is the effect of concentration of enzyme on the reaction rate of the experiment?
4. What is the effect of temperature on the enzymatic decomposition of hydrogen peroxide?
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