Biology Scientific Investigation

What effect does increasing the temperature from 0°C- 81°C, have on the amount of oxygen
produced in a reaction between hydrogen peroxide and the enzyme catalase?

Brigid Mullins

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Personal Engagement

My interest in analysing how catalase can be used in an enzyme reaction with hydrogen
peroxide to enhance the volume of oxygen produced, stemmed from my general curiosity
of understanding how enzymes can be used to speed up chemical reactions. Interestingly,
according to (Science Buddies, 2020) enzymes are used constantly in our body to help break
down food in the digestive system, to destroy toxins and to build muscle, and catalase is
found in our liver and breaks down harmful hydrogen peroxide into oxygen and water. My
hope is that this experiment will be a close replication of this internal bodily reaction.

Exploration

Research question

What effect does increasing the temperature from 0°C- 81°C, have on the amount of oxygen
produced in a reaction between hydrogen peroxide and the enzyme catalase?

Background Information

As (Science Buddies, 2020) states catalase is a very common enzyme that is present in
almost all organisms that are exposed to oxygen. The purpose of catalase in living cells is to
protect them from oxidative damage, which can occur when cells or other molecules in the
body come into contact with oxidative compounds. This damage is a natural result of
reactions happening inside your cells. The reactions can include by-products such as
hydrogen peroxide, which can be harmful to the body, just as how a by-product of a
welcoming bonfire can be unwanted smoke that makes you cough or stings your eyes. To
prevent such damage, the catalase enzyme helps your body to rid itself of these compounds
by breaking hydrogen peroxide (H2O2) up into harmless water and oxygen.

According to (Activation energy, 2020) Enzymes speed up reactions as they lower the
activation energy, which is the minimum energy barrier needed to be overcome before a
reaction can occur by providing an alternative reaction pathway.

The reaction under study can be written as followed:

2H2O2 CATALASE
→
2H2O(l) + O2(g)

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This experiment will specifically investigate the effect of temperature on the rate of
reaction, which can be calculated by measuring the speed of production formation. In this
experiment, the production of O2 gas will determine the rate of reaction through the change
in pressure as the reaction progresses. The rate at various temperatures is acquired by
calculating the slope of pressure vs. time graph. Gas pressure inside the test tube will also
increase depending on the amount of oxygen gas produced over a given unit of time. If the
pressure changes or the steepness of slope is large, the rate of reaction is fast; and if the
change or slope is small, the rate is slow.

It should be noted that according to (How Does Temperature Affect Catalase Enzyme
Activity?, 2020) the optimum temperature for the catalase enzyme, is at temperatures close
to body temperature (37.5°C).

Hypothesis

Based on the above information, it can be inferred that the reaction will release the highest
amount of oxygen gas at 37 degrees celsius as (How Does Temperature Affect Catalase
Enzyme Activity?, 2020) states that this is the optimum temperature for catalase reactions.
This is most likely because it is similar to the temperature of the human body and this
reaction occurs within the human body, as (Science Buddies, 2020) states, The reaction
occurs in our liver and breaks down harmful hydrogen peroxide into oxygen and water.
Furthermore, it can be hypothesised that there will be an increase in the amount of oxygen
produced going from 0- 37 degrees celsius and then there will be a steady decrease in the
amount of oxygen produced as the temperature continues to increase to 81 degrees Celsius.

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Variables

Variables Impact on the investigation How the variable will be changed/

Independent variable
Temperature of Changing the temperature of the
hydrogen peroxide hydrogen peroxide solution will enable
solution (°C) analysis on how increasing the
temperature will affect the amount of
oxygen produced and assist in
supporting or rejecting the hypothesis
of 37 degrees Celsius being the
optimum temperature for the volume
of oxygen released in the reaction.
Dependent variable
Volume of Oxygen Measuring the amount of oxygen
produced in produced will allow an analysis on how
reaction over 1 increasing the temperature affects the
minute amount of oxygen produced when
reacting with catalase.
Controlled variables
Substrate (H2O2) Using 11 cm3 of hydrogen peroxide will
Quantity allow for measurements to not exceed
the 100cm3 limit of oxygen in the glass
syringe and will allow for accurate
results.
Volume of Enzyme Using consistent amounts and
and concentration concentrations of the catalase enzyme
(catalase) will allow for accurate results that do
not exceed the 100cm3 limit of oxygen
in the glass syringe.
Time of each trial Timing each trial for one minute will
allow for accurate measurements of
the amount of oxygen released from
reaction.
Uncontrolled variables
Temperature of To ensure consistency with the
Catalase temperature of the water baths, the
temperature of the catalase was not
changed. This may have had an
extremely minor impact on
investigation results.
measured/ controlled
The temperature will be measured
with a thermometer until the desired
temperatures of 0°C, 37°C, 52°C, 67°C,
81°C are reached. A water bath will be
used for each station ??? and 3 trials
will be completed.
Will be measured using a glass syringe
and one minute will be timed using a
stopwatch.
Using the same pipette (± 0.1mL), all 3
trials at each station will use exactly
11cm3 of the same H2O2 solution.
Each of the 3 trials at each station will
use 1.5cm3 from the same catalase
solution using a pipette (± 0.1mL).
Each trial will be timed for one minute
after the hydrogen peroxide has
reacted with the catalase
The temperature of the catalase was
not measured before each trial
however it will remain consistent
across each trial as it was taken from
the same beaker.

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Apparatus

3
5

12
10

11
9
8

Figure 1.0 shows apparatus setup

1. 1 x Roll of parafilm
2. 1 x Glass thermometer ±0.05°C
3. 1 x Glass syringe (± 0.1cm3)
4. 1 x Rubber tubing
5. 1 x Test tube rack
6. 1 x Retort stand
7. 3 x Side-arm test tubes
8. 3 x Rubber test tube stoppers
9. 5 x Electric water baths (0°C, 37°C, 52°C, 67°C, 81°C)
10. 50cm3 x 6% catalase solution
11. 200cm3 x Hydrogen Peroxide
12. 2 x 10cm3 measuring cylinder (± 0.1mL)
13. 1 x Stopwatch
14. 1 x Safety glasses
15. 1 x Lab coat

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Methodology

1. The electric water baths were set up at the temperatures of 0°C; 37°C; 52°C; 67°C;
and 81°C.
2. The glass syringe was then set up on the retort stand with the rubber tubing ?.
3. 50cm3 of the 6% catalase solution, and 200cm3 of the hydrogen peroxide, was then
measured.
4. The test tubes and test tube rack was placed in the water bath as shown in figure
1.0.
5. Using the measuring cylinder and a pipette, 11cm3 of the hydrogen peroxide solution
was measured into each of the 3 side arm test tubes, and the solution was heated up
to the temperature of the water bath using a thermometer to check the
temperature.
6. The rubber tubing was connected to the side arm test tube.
7. Once the hydrogen peroxide was heated, 1.5cm3 of the catalase solution was
measured with a measuring cylinder and added to the test tube, and the rubber
stopper was quickly used to cover the top of test tube.
8. The 1 minute timer was started, and the volume of oxygen released within this time
was recorded for each of the 3 trials.
9. Steps 4 to 8 were repeated at each water bath.

Safety and Environmental Considerations

Multiple environmental and safety considerations were made throughout the experiment.
This included the use of safety glasses and lab coats throughout the experiment as eye
contact with the 6% hydrogen peroxide can cause serious eye irritation. However the
chemicals involved, including the catalase and hydrogen peroxide, are low risk. Caution was
taken when dealing with the high temperatures of some of the water baths. Furthermore all
chemicals were appropriately disposed of using a sink, or the chemical eco-friendly waste
bucket.

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Analysis
Qualitative Data

Solution at 0°C
Solution at 37°C
The solution has very slight bubbling at the top and
Solution displays excessive bubbling and clearly a
the test tube is cold to touch. Little reaction has
large volume of oxygen has been released.
occurred.

Solution at 67°C
Solution at 52°C Less bubbling is occurring at 67°C, however the
Solution is bubbling a lot however not as much as reaction has still clearly occurred. Test tube is hot
the solution at 37°C. Test tube is warm to touch. to touch.

Solution at 81°C

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 Little reaction has occurred, however slight
bubbling is evident. Test tube is quite hot to touch.
Quantitative Data

Temperature (±0.5°C)
Volume of 0 37 52 67 81
oxygen cm3
Trial 1 16 86 82 50 32
Trial 2 16 86 82 48 36
Trial 3 20 90 86 42 32
MEAN 17.3 87.3 83.3 46.7 33.3
STDEV 2.31 2.31 2.31 4.16 2.31
SEM 1.15 1.15 0.31 2.08 1.15

Figure 1.2 table showing results from investigation over 3 trials, the mean, standard
deviation and standard error of mean

Mean Volume of Oxygen Produced at Various Temperatures

100.0

90.0
Volume of oxygen produced (± 0.1cm3)

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0
0 10 20 30 40 50 60 70 80 90

Temperature (±0.5°C)

Figure 1.3 graph depicting the mean value of the 3 trials

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Evaluation
The investigation successfully examined the research question by discovering the correlation
in increasing the temperature and the amount of oxygen produced in a reaction between
catalase and hydrogen peroxide. Data is very precise as 3 trials were carried out and all
values are close to each other as there are no major outliers. This is displayed clearly in
figure 1.2 where the standard deviation and standard error mean values are all noticeably
low; the standard deviation values for 0°C, 37°C, 52°C and 81°C are all 2.31, whereas at 67°C
it is raised to 4.31, this increase in standard deviation may be due to a human or
experimental error.

The graph in figure 1.3 displays how the hypothesis was proved correct when an increase
from 0°C -37°C was followed by a steady decrease up to 81°C in the amount of oxygen
produced. The graph depicts the mean value over the trials, at 0°C 17.3 cm3 of oxygen was
produced which increased to 87.3 cm 3 of oxygen at 37°C. The volume of oxygen then
decreased to 83.3cm3 at 52°C, to 46.7cm3 at 52°C, to 33.3cm3 at 81°C. These results are most
likely because 37°C is roughly the temperature of the human body where this reaction
occurs.

In conclusion, the investigation successfully examined the research question by discovering

the correlation in increasing the temperature and the amount of oxygen produced in a
reaction between catalase and hydrogen peroxide. Data is very precise as 3 trials were
carried out and all values are close to each other as there are no major outliers, as shown in
figure 1.3. The investigation also has high accuracy to the estimate that 37 degrees celsius
will have the highest volume of oxygen. The standard deviation and standard error of mean
values are all low so we know the data is reliable. The hypothesis was supported by the
experiment as the value reaches the highest point at 37 degrees and decreases away from
that value.

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 Weaknesses
Data collection stretched over several days.
Although the solution was preserved in the
refrigerator, time still influences enzyme
activity. As enzymes in a solution lose
“freshness” and become inactive as time
passes, data collected during the later days
may not be as accurate as the first day
because of the gradual decrease in activity.
Hence, data may be slightly inconsistent.
The possibility of unreliable measurements
due to human error such as inaccurate
measurements using the measuring cylinder.
The uncertainty value of the equipment used
may have slight effects on results.
Improvement
All the data collected should be
experimented on in one day, and carried out
at one spot. In this way, the experiment can
be controlled and consistent with all enzymes
having the same level of activity.
Use a micropipette (low uncertainty of ±µl) to
transfer the catalase solution directly into the
hydrogen peroxide solution.
The uncertainty value of the equipment used
needs to be taken into account when
analyzing the results. This value can be
reduced by using the micropipette.

Future Investigations

Improvements to maximise reliability of data for future experiments include the experiment
taking place over one day. Further experiments could also include analysing the time it takes
for a certain volume of oxygen to be obtained at each temperature rather than the volume
of oxygen produced. How catalase works in plants such as the ginseng could also be
measured. How changing the concentration of the catalase solution effects the volume of
oxygen produced can also be analysed.

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Bibliography

Sciencing. 2020. How Does Temperature Affect Catalase Enzyme Activity?. [online] Available
at: <https://sciencing.com/temperature-affect-catalase-enzyme-activity-7776025.html>
[Accessed 9 July 2020].

Csef.usc.edu. 2020. Effects Of Temperature On Catalase Enzymes. [online] Available at:

<http://csef.usc.edu/History/2016/Projects/J0513.pdf> [Accessed 9 July 2020].

Ib.bioninja.com.au. 2020. Enzyme Experiments | Bioninja. [online] Available at:

<https://ib.bioninja.com.au/standard-level/topic-2-molecular-biology/25-enzymes/enzyme-
experiments.html> [Accessed 9 July 2020].

Science Buddies, S., 2020. Exploring Enzymes. [online] Scientific American. Available at:
<https://www.scientificamerican.com/article/exploring-enzymes/#:~:text=Catalase%20is
%20a%20very%20common,that%20are%20exposed%20to%20oxygen.&text=To%20prevent
%20such%20damage%2C%20the,into%20harmless%20water%20and%20oxygen.>
[Accessed 9 July 2020].

2020. Activation Energy. [online] Available at:

<https://www.khanacademy.org/science/high-school-biology/hs-energy-and-transport/hs-
enzymes/a/activation-energy> [Accessed 9 July 2020].

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