RESEARCH QUESTION

“What is the effect of increasing concentration of hydrogen peroxide on the rate

(0,0M – 1,0M) of reaction by the addition of the enzyme catalase
from the blood of Bos taurus as measured by the volume of oxygen produced?”
1. BACKGROUND When Catalase meets H2O2, it decomposes H2O2
a. Enzyme and substance. into H2O + O2. This reaction is called oxidoreductases
(The enzyme Oxidoreductase catalyzes the oxidation
Enzymes are biological catalysts (also known as reaction where the electrons tend to travel from one
biocatalysts) that speed up biochemical reactions in form of a molecule to the other) (Enzymes, 2020). So,
living organisms, and which can be extracted from the substrate is H2O2, and the reaction products are
cells and then used to catalyze a wide range of Water and Oxygen.
commercially important processes (Robinson,
Enzymes: principles and biotechnological
applications, 2015). To know what factor affects the
reaction speed of enzymes, we do an experiment with
Catalase – a key enzyme that uses hydrogen peroxide,
a non-radical ROS*, as its substrate. This enzyme is
responsible for neutralization through the
decomposition of hydrogen peroxide, thereby
maintaining an optimum level of the molecule in the
cell which is also essential for cellular signaling
processes. (Ankita Nandi, 2019).

*ROS: A type of unstable molecule that contains

oxygen and that easily reacts with other molecules in a
cell. A build-up of reactive oxygen species in cells may
cause damage to DNA, RNA, and proteins, and may
cause cell death. Reactive oxygen species are free
radicals. Also called oxygen radical. Catalase protects
cells by decomposing H2O2.
As an enzyme, Catalase also has the common
properties of enzymes. It is a compound made of
protein: four identical, tetrahedrally arranged subunits
of 60 kDa, each containing in its active center a heme
group and NADPH. Its functional sides such as the
active side – are where the substances are reacted.
Figure 2. Enzyme decomposes reaction.
b. Background knowledge of variables.
To define the independent variables, dependent
variables, and variables must be controlled. We must
know that enzymes are proteins. So, any factors that
can denature protein also can make the enzyme work
unnaturally. These factors are temperature, pH,
chemical substances, the concentration of soluted salt,
UV, etc which can change the charge of amino acids or
break the bond on protein. In this experiment, we tried
to prevent these factors to affect the result as much as
possible. Specific materials and methods are explained
later in this research paper.

c. Speed of reaction.

As it has been told in 1. a Enzyme and substance,

the enzyme will react if these are substances that are
fit to the react site of the enzyme. It means if these are
a number of enzymes, the speed of reaction is
maximum as long as these are the maximum react site
that bonded substances. A change in the concentration
of substances with a constant number of enzymes leads
to a change in the speed of reaction, as long as that
number is not more than the number of react sites, the
increasing of substance always leads to an increase in
the speed of the reaction.

The relationship between substrate concentration

Figure 1. Catalase structure, with 4 heme active sites where
and rate of reaction is shown in the following graph.
hydrogen peroxide reacted.
The speed of reaction keeps raising the fastest before
Km, then, it slows down and keeps the constant speed
 of reaction as all of the react sites are used. (University
of Birmingham, n.d.)
To measure the speed of reaction, we use H2O2
from 0,0M to 1M and reacted with 25ml of Bos Taurus
blood. Then measure the oxygen released after 60
seconds of swirling it.
3.
Speed of reaction:
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑔𝑎𝑠𝑒𝑠 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 (𝑚𝑙)
𝑇𝑖𝑚𝑒 𝑡ℎ𝑒 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑖𝑠 (𝑠)
In a reaction of enzymes, the maximum reaction
speed changed by many reasons: the property of
enzymes, number of substrates – enzyme complex at a
time and kinetic of environment. In an experiment,
sometime when only one of these value (number of
substrates or enzymes) increases, the speed of reaction
increases but stops later when all of enzymes has
bonded the substrates or there is no more substrate to
be reacted. It explains why controlled variables in 3.c
must be keep constant.
d. Method of calculation the result
To know that the final result is accuary and fair,
in scientific method, we use Standard deviation:
2. HYPOTHESIS.
a. MAIN HYPOTHESIS.
If the concentration of H2O2 to react with
Catalase increases, then the speed of reaction also
increases because more substrates are likely to meet
enzymes and react to produce water and oxygen gases.
b. OTHER HYPOTHESIS.
These are some hypotheses in cases the result is not the
same as the main hypothesis expects.
1. If there are no gases released during the
experiment, then there might be no Catalase in
Bos taurus blood.
2. If there is a big gap in data between trials, then the
result might be wrong because some variables
might not be controlled well or the volume of
H2O2 or Bos taurus blood might not be kept same.
VARIABLES.
a. Independent variables.
Concentration of Hydrogen perioxide (M)
The concentration of Hydrogen Perioxide are
0.0M, 0.2M, 0.4M, 0.6M, 0.8M, 1.0M. As it mentioned
in 1.c (…A change in the concentration of substances
with a constant number of enzymes leads to a change
in the speed of reaction…), because it is difficult to
know and control excatly the concentration of catalase
on Bos taurus blood, so Hydrogen perioxide will be a
better choice to measure effect on speed of reaction.
In this experiment, we use 10ml of Hydrogen
perioxide in different concentration for every trial.
b. Dependennt variables.
The reaction rate by measuring oxygen
released (ml/s)
When we drop the H2O2 into a constant amount
of Bos taurus blood (25ml), oxygen will be released
until there is no more H2O2 hasn’t been reacted yet as
the reaction by Catalase. All of the experiments finish
after 1 minute. To count the reaction rate, we divide
the volume of oxygen released for 60.
As the main hypothesis said, we supposed that
the reaction rate (volume of oxygen released by
second) increases as the concentration of H2O2
increases.
 c. Controlled variables. speed of reaction (ml oxygen released / second). We
need to prepare 1 controlled experiment (0.0M of
To get a fair test, these variables must to be H2O2), 5 trial experiments (0.2M, 0.4M, 0.6M, 0.8M,
controlled (keep them constant). 1.0M of H2O2) in test tubes. Then, pure one by one to
the arm flask (contains 25ml Bos taurus blood),
Variable Likely impact How will quickly close it and measure total volume of oxygen
upon the the released goes on graduated cylinder on 1 minute.
investigation variables
be b. Materials.
controlled?
Volume of Different volume Measure 1. Bos Taurus blood (500ml)
hydrogen of these values carefully by
perioxide. causes different suitable
(10ml ± 0.02ml) total volume of pipette. 2. Hydrogen Peroxide (0.2M – 1.0M)
Volume of Bos gases released
taurus blood and speed of 3. Distilled Water (100ml)
(25ml ± 0.05ml) reaction*.
Concentration of Use the same 4. Water (1000-2000ml)
blood (same blood from
sample) same sample
(prepared 5. Test Tube Rack
bottle).
Time of each trial Different time Use same 6. Buchner Flasks (1)
(60 seconds) causes different stopwatch on
total gases can be the same
released. Long time. 7. 25ml Test Tubes (6)
time is easier to
cause gas 8. 1000ml Beaker (1) (Wastage)
leakage.
*Explained in 1.c.
9. 25ml volumetric pipette (+/- 0.05ml) (1)

10. 10ml volumetric pipette (+/- 0.02ml) (1)

d. Uncontrolled errors.
11. Pipette pumps (2)
These factors are difficult to be controlled but not
affect too much to the result.
12. Rubber stopper (1)
Errors Likely impact How will
upon the the 13. Rubber tubing (1)
investigation variables
be 14. Beehive Shelf (1)
minimized?
The power of More power it is, Try to swirl 15. Graduated cylinder (1)
swirling arm the more likely the arm flask
flask. reactions happen. in the same
Sometime, way all of the 16. Trough (1)
overpower cause experiments.
bubbles on blood, 17. Masking Tape
which prevent
gases released to
the graduated 18. Marker
cyclinder.
Temperature At 37-degree, Try to do all 19. Timer
Catalase work at experiment at
the best. (SE, 2005) the same time 20. Safety goggles
and avoid
from heat,
cold. 21. Lab coats.

4. METHOD
a. Ideas.

This experiment is about measure the affect of the

change in hydrogen perioxide concentration to the
c. Method.
1. Prepare all lab equipment and materials, making sure
that everything is clean. Follow correct Lab Set-Up
procedures being sure to wear eye-protection and lab
coats.
2. In preparation for the experiment, label 6 test tubes
using masking tape according to the concentrations
specified in the materials section.
3. Place the beehive shelf in the trough with the top
opening facing upward.
4. Fill your trough with approximately 2000ml of tap
water or till the water level is about 20mm from the top
of your beehive shelf.
5. Using the 10ml graduated pipette and suction device,
decant 10ml the hydrogen peroxide (at their various
concentrations) into its respective labelled test tubes,
being sure to rinse the pipette out before changing
concentrations. Place the test tubes in the test tube rack.
6. Connect your rubber tubing to the outlet on the
Buchner Flask and place the rubber stopper in the main
opening.
7. Using the water in your trough, fill your graduated
cylinder and invert it placing it above the opening atop
the beehive shelf, being sure to record the water level in
the gas jar.
8. Feed the rubber tubing through the opening at the
bottom of the beehive shelf, submerged under water and
directly below the opening below the gas jar.
9. Using the graduated 25ml pipette and suction device,
decant 25 ml of the bos taurus blood into the Buchner
flask, replacing the rubber stopper when done.
10. Perform a final check of your set -up, being sure
that the rubber tubing is properly connected and
underneath the opening where the gas jar is located.
11. Starting with your 0.0M Hydrogen Peroxide
(distilled water), decant the content of the test tube
into the Buchner Flask and replace the rubber stopper.
12. Start a timer for 1 minute and swirl the Buchner
flask at a steady rate.
13. Record the water displacement in your gas jar by
mL (it stands for the volume of gases released that
replaces water in cylinder), and if needed refill the gas
jar with water from the trough.
14. Clean the contents of the Buchner Flask
thoroughly, making sure to first pour the contents into
a wastage beaker.
15. Repeat steps 8 through 13 for each concentration.
16. For accurate results, repeat the experiment with
each concentration at a minimum of 2 times.
17. After all experimentation is complete, follow
correct lab clean-up procedures and follow any
directions given by the supervisor of the Lab.
5. CONSIDERING ABOUT SAFETY, ETHICAL,
AND ENVIRONMENTAL CONCERNS.
a. Safety
The experiment requests to use Hydrogen
perioxide which is strongly reducing and can be
harmful to eyes, wounded skin. We use the safety
goggles and lab coats to protect from it. Hydrogen
perioxide at high concentration (3%) causes
respiratory irritation in close contact. But in this
experiment, the concentration was low and safe.
Blood from Bos taurus can have bacterias and
parasitics, storage it on the fridge prevents these
dangerous living things reproduce. After doing the
experiments, all of them wash our hands carefully and
clean the lab.
b. Ethical
The experiment uses blood from a cow (Bos
taurus), this is an animal but can be eaten. We take it
from a dead cow, not do anything violence and there
is nothing not ethical.
c. Environmental concerns.
After doing experiment, all the trash (denatured
blood, hydrogen perioxide) are removed to a specific
glass bowl which is given to the teacher to throw it in
a suitable place. Nothing harmful was removed to
social sewer and also does bad effect to environment.
6. RAW DATA COLLECTION
a. Qualitative observation.
Denatured blood: as have mentioned 1.b,
hydrogen perioxide is a chemical substrate, although
it got reacted by Catalase but most of parts in blood
are different proteins and get denature. After 1 mintue
experiment, the blood on arm flask is denatured, with
darker colour, condensed, having bubbles as Firgue 3.
Because denatured blood are thick, lower
desnity, it can make a layer at the surface of liquid,
prevents oxygen released from the bottom and rate of
reaction. We dicided to swirl the flask during the
experiment to mix it, make sure the reaction fully
happened. But the bubbles made by denatured blood
keep some gases inside, we don’t have enough time to
wait until they are broken, so, the final result doesn’t
show the maximum volume of gases it can release.
 the hydroxide perioxide did not meet enzymes totally,
then, it started to react faster because of there were
more substrates – enzymes complex was made and
slow down when there is less substrates (Hydrogen
perioxide).

b. Quantitative observation.

Raw data: Data that observed after 1 minute.

Oxygen released (ml /

1 min)
(+/-0.1 ml)
Figure 3. Denatured blood.
H2O2 concentration (M) Trial 1 Trial 2 Commented [KG1]: Add the units to the heading and
0.0 0.00 0.00 delete the units from inside the data table.
Amount of time that enzymes reacted: We 0.2 0.00 0.00
0.4 0.50 1.50 Commented [KG2R1]: Check sig figs.
observed that at the beginning, these was no gas
released. Then, it began to release a lot of gas and slow 0.6 2.00 1.00
0.8 3.50 2.00
down later. It can be explained that at the beginning,
1.0 8.00 7.00

Processed data:

Volume of oxygen released per second Average Standard deviation

(ml/ s) reaction rate Commented [KG3]: Did you calculate this uncertainty?
H2O2 concentration (M) Trial 1 Trial 2 (ml/s)
0.0 0.00 0.00 0.00 0 Commented [KG4]: See comment above
0.2 0.00 0.00 0.00 0
0.4 0.01 0.02 0.02 0.00707
0.6 0.03 0.02 0.03 0.00707
0.8 0.06 0.03 0.04 0.0212
1.0 0.13 0.12 0.13 0.00707 Commented [KG5]: /
Sample of calculation Total volume 1 (ml) / 60 Total volume 2 (ml) / 60 (Trial 1 + trial 2) / 2 Noted on the background (1.d)
Commented [KG6]: I like the sample calculation, but
please move it below this data table.
Graph:

RATE OF REACTION (ml/s) ON

{0M, 0.2M, 0.4M, 0.6M, 0.8M, 1.0M} OF HYDROGEN PERIOXIDE
0.16
0.14
Average reaction rate (ml/s)

0.12
0.1
0.08
0.06
0.04
0.02
0
-0.02 0 0.2 0.4 0.6 0.8 1 1.2
-0.04
Concentration of Hydrogen perioxide (mol / L) Commented [KG7]: Please delete the error bars in the x
direction.
Keep the units the same as the rest of the lab in label on the
x axis. Error bars do not seem correct.
7. ANALYSIS AND INTERPRETATION
a. Analysis
By basic observation, we can see the
minimum rate of reaction was 0 (ml/s) on
experiments with 0.0M and 0.2M.The
maximumm rate of reaction was 0.125 (ml/s) on
1M experiment. Note that this is the maximum
rate of reaction within the range of
hydrogenperoxidee concentration (0M – 1M).
The rate of reaction increases when the
concentration of H2O2 is higher than 0.2M, make
a linear trend line. The gap (or slope) between
the rate of reaction of 0.8M and 1.0M was big,
meaning that the chance that enzymes meet
substrates was perfect with this ratio.
The error bars (based on standard
diviation) was not too long, meaning that the
research was true and fair between trials, but on
0.8M, the error bar was big, putting a
questionable problem about trustable result.
Also, the error bars did not reach the trend line
(0.6M, 0.8M, 1.0M) meaning the trial range was
too big, need more smaller trials between each
experiment.
These are only 2 trials for each
experiment, which is quite a small number for a
certain conclusion. We can not guess the trend
line will keep on raising or turn to be a curve in
which concentration.
However, we can find out the
relationship between the rate of reaction and the
increasing of substrates. Which can answer the
research question that the effect of changes in
concentration causes the changes of reaction
rate by ratio.
b. Interpretation
The reason why points are far from trend line
might because of bubbles caused by thick blood.
As explained in 6.a that denatured blood
becomes thick, makes bubbles contains gases
more difficult to be broken and released gas. The
swirling boosted the speed and chance of
reaction, but caused more bubbles. So, we can
say that most of gases produced by 0.2 M H2O2
was in bubbles, the evidence that was not bubble
in 0.0M H2O2 controlled experiment.
Because of less time to do more trials, the gap of
data between 1 st and 2nd trial is big, leads to a big
of error bar and a line graph instead of a curve
graph.
8. CONCLUSIONS
Commented [KG8]: Check the right margin - words are cut
off.
Compare the graph we got in 6.b with the graph
in 1.c, we can see that 6.b graph is a line but 1.c Commented [KG10]: Start with the basic trends - what do
graph is a curve. It means this experiment did not you observe? What are the max and min values? What is the
reach the maximum reaction rate and keep shape of the trendline? The slope? Etc. Save the comparison
constant reaction speed. Also, there are 4 points to the research for the conclusion.
(at 0.0M; 0.2M; 0.6M; 0,8M H2O2) that far from
the trend line. It means there was something that
affected the experiment (which might be
uncontrolled variables) made it less accurate
(especially 0.2M).
Looking back to our hypothesis and research Commented [KG11]: Re-state the research question to
question, we can say that the final result can help your reader.
answer our expected questions that for a
constant number of enzymes; from 0, the more Commented [KG9]: Check this again after you fix them.
substrates is added, the faster and more
reaction will happen. So, the hypothesis that:
“If the concentration of H2O2 to react
with Catalase increases, then the speed of
reaction also increases because more substrates
are likely to meet enzymes and react to produce
water and oxygen gases” is partly true, with an
evidence that the trend line goes up as the
concentration of hydrogen perioxide increases (as Commented [KG12]: Support this with evidence from
0 – 0 < 0.02 < 0.03 < 0.04 < 0.12 (by ml/s) along your data. I need to see numbers.
to 0M – 0.2M < 0.4M < 0.6M < 0.8M < 1.0M).
The reason why it is only partly true will be
explained later.
Also, consider the research question:
“What is the effect of increasing
concentration of hydrogen
peroxide (0,0M – 1,0M) on the rate of
reaction by the addition of the enzyme
catalase from the blood of Bos taurus as
measured by the volume of oxygen
produced?” is the rate of reaction
increases along to the increasing of
concentration as the hypothesis
mentioned. But this statement is always
true with that range of concentration.
There is a research paper written by
Michaelis and Menten in 1913 proves
that there is a limitation of increasing in
rate of reaction.
Michaelis and Menten are famous names on
biochemistry community. Their research in
German is about the rate of an enzyme-catalyzed
reaction is proportional to the concentration of
enzyme-substrate complex predicted by the
Michaelis-Menten equation. In the following
Commented [KG13]: Delete Goody - just the last name is
firgue (Johnson, 2012), the ratio P/S0 which
needed
 stands for the rate of reaction has different
curves. Each of curves are different by the
concentration of Sucrose (which can be reacted
by Invertase into Glucose and Fructose). The
concentration is increasing from the triangle (∆)
curve to circle (•) curve.
Michales Menten research paper is the reason
why our hypothesis is only partly true, if keep
adding hydrogen peroxide until a specific
concentration, the rate of reaction will stay
constant instead of raising. It is because when the Commented [KG14]: Very nice! Now explain WHY based
number of substrates equals to the number of on the science you know or have researched. Tell me what is
available enzymes, the rate of reaction is happening at the molecular level. Why does the graph level
maximum. More substrates are added but the off?
number of enzymes stays the same, so there will
be left substrates that can not be reached by any
enzymes (now enzymes are all ‘busy’ with their
substrates) at the same time. But in the range
from 0.0M to 1.0M, the answer is true.

9. EVALUATION

Evaluation of experimental weaknesses/errors and strength Commented [KG15]: Add strengths

Weakness/source of error and strengths
Independent variable
The amount of hydrogen peroxide on 5
experiments were so small to see the
curve graph of enzyme reaction.
Dependent variable
There was no error. This is a strength
because DV is an important value and we
measure it well.
Controlled variables
The accuraty of sample volume
Uncontrolled errors
The power of swirling arm flask
Qualitative data
The quality of blood after many days
Quantitative data
The range of concentration.
The range was so small, with big gap and
few.
Quantitative data
Number of trials
There are only 2 trials for each
experiment, which should be 5 instead.
Possible effect on data and
magnitude of weakness/error
Can not make the true graph of
enzyme activity, can not predict that
Catalase on blood could work
correctly or not.
Leading to the wrong result or
unbelievable values. Making the
main hypothesis wrong.
During the process of using pippete,
sometimes the blood turned to
bubbles and make it unaccurate.
Different speed and chance to react,
make bigger error bars on graph.
Blood is difficult to be preserved,
the more day it is, the lower quality
blood is, which can lead different
reactive effectiveness.
Make the final result (typical is the
graph) questionable. Also fewer
data to know how much is the
maximum rate of reaction.
Make the error bar untrustable, the
final value are not perfect leads to
wrong trend line.
Suggested improvement
Commented [KG16]: What other factors impact reaction
The distance between every concentration rate? What other controlled variables were difficult to
trial should be bigger, based on other control? Measurements like time are are large source of
researchs. But based on the graph today, error. What about the number of trials?
1.0M might be the Km of Bos taurus
blood’s Catalase, so 2.0M can be perfect.
There was no error
Commented [KG17]: Is this a strength? If not - then delete
this row.
Let the blood chill for a time, suck the
blood by pippete slowly and carefully to
avoid bubbles.
Swirling by machine or do the reaction on
a flat surface instead of a flask so we do Commented [KG18]: ?
not need to mix the blood with hydrogen
perioxide.
Do the experiment on 1 day only, and
closed by the day collect blood from Bos
taurus.
Do more experiments. Can be 10: 0 – 0.2
– 0.4 – 0.6 – 0.8 – 1.0 – 1.2 – 1.4 – 1.6 –
1.8 – 2.0 M
Do 5 trials for each experiment.

10. BIBLIOGRAPHY #:~:text=Catalase%20is%20a%20key%20enzyme,essential

Works cited
Ankita Nandi, L.-J. Y. (2019, November 11). National Center for
Biotechnology information. Retrieved from PubMed
Central:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885225/
%20for%20cellular%20signaling%20processes. Commented [KG19]: Is there more information for the
Enzymes. (2020). BYJU's BIOLOGY. Retrieved from first source? The last source must have an article title.
https://byjus.com/biology/enzymes/
Johnson. (2012, October 4). The Original Michaelis Constant:
Translation of the 1913 Michaelis-Menten Paper.
 Retrieved from NCBI:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381512/

Robinson, P. K. (2015, November 15). Enzymes: principles and

biotechnological applications. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692135/

Robinson, P. K. (2015, October 26). NIH National library of

medicine. Retrieved from PMC PubMed central:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692135/

SE, S. (2005). Retrieved from Science Magazine:

https://www.sinobiological.com/recombinant-
proteins/human-catalase-12084-
h07b#:~:text=The%20optimum%20PH%20of%20human,t
emperature%20is%20at%2037%20degree.

University of Birmingham. (n.d.). Retrieved from

https://www.birmingham.ac.uk/teachers/study-
resources/stem/biology/stem-legacy-enzymes.aspx