Nam Nguyen

Research Questions: What is the effect of Substrate Concentration of Hydrogen

Peroxide ( H 2O 2 ) on the rate of activity of Enzyme Liver Catalase?

Table of contents

Research Question 2
Exploration 2
Analysis 5

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Research question

What is the effect of Substrate Concentration of Hydrogen Peroxide ( H 2 O 2 ) on the rate of

activity of Enzyme Liver Catalase?

Five different concentrations of Hydrogen Peroxide ( H 2 O 2 ) (6.0%, 5.5%, 5.0%, 4.5% and
4.0%) will be used to see how the rates of reaction catalysed by enzyme liver catalase change
within a set period of time. The change in the rates of reaction catalysed by enzyme liver
catalase will be measured by the rate of water displacement in a 50ml cylinder which is an
indication of the rate of reaction taking place when the substrate is mixed with the liver
contaning enzyme catalase. It is expected that increasing the concentration of hydrogen
peroxide will increase the average rate of water displacement, which suggests that the rate of
catalysation of enzyme liver catalase has increased.

Exploration

I. Background information

Enzymes are proteins which catalyse chemical reactions. Each enzyme fits a specific substrate
whose shape fits the active site of the enzyme. Catalase is a common enzyme found in nearly
all living organisms exposed to oxygen. It catalyses the decomposition of hydrogen peroxide
into water and oxygen. Hydrogen peroxide is a strong oxidizing agent which wil disrupt the
cell chemistry if it is accmulated too much by respiration. Catalase is thus a very important
enzyme in protecting the cell from oxidative damage, which can occur when cells or other
molecules in the body come into contact with oxidative compounds. 1 In mamals, catalase is
found predominantly in the liver.

The chemical reaction equation below illustrates how hydrogen peroxide is decomposed into
water and oxygen by the activity of enzyme catalase in the liver.

2H 2 O 2 catalase 2H 2 O 2 + O 2 ↑

According to Worthington publication, originally published in 1972 as the Manual of Clinical

Enzyme Mesurements, factors affecting enzyme activity are temperature, pH, enzyme
concentration, substrate concentration and the presence of any inhibitors or activators. 2 Thus,
the purpose of this experiment is to examine the relationship between the substrate
concentration and the activity of enzyme liver catalase and its effect on the rate of reaction.

The hypothesis is the higher concentration of hydrogen proxide (the substrate) increases
the rate of activity of enzyme liver catalase.

Why: If the concentration of hydrogen proxide is increased, then the amount of oxygen
produced and its rate of being released will also increase, which will lead to an increase in
water displacement. This is because the higher the concentration of hydrogen peroxide, the
more substrate molecules available to collide with enzyme liver catalase and then be catalysed
by it, hence the higher the rate of reaction that decomposes hydrogen peroxide into water and
oxygen. As oxygen is the product of this reaction, the higher rate the reaction, the more
oxygen that will be produced and thus the higher the average rate of water displacement to be
shown in the investigation. However, it is also noted that once the saturation point is reached,
1
Scienticfic American, Exploring Enzymes, viewed 6 November, 2019,
<https://www.scientificamerican.com/article/exploring-enzymes/>
2
Worthinton Biochemical Corporation, Factors Affecting Enzyme Activity, viewed 6 November,
2019,<http://www.worthington-biochem.com/introBiochem/factors.html>

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there will be no further effect on the rate of reaction. At this point, there will be an excess of
substrate molecules and not enough enzymes to bind to and so the enzyme concentration
becomes the limiting factor. At this point, there will be no water or negligible water
displacement.

II. Variable

Independent Variable

The independent variable to be changed, that will in turn affect the rate of reaction, is the
different concentrations of hydrogen peroxide that the investigation will be carried on. These
five different concentrations of substrate are 6.0%, 5.5%, 5.0%, 4.5%, and 4.0%. The
investigation will feature these different concentrations and examination will be conducted to
understand the effects of the concentration of hydrogen peroxide in the rate of reaction and
how well concentration levels come into play.

Dependent variable
3
The volume of water displaced will be measured in mililiter ( cm ) within a set amount of
time. This measurement will be taken by recording the difference between the initial place of
water and the final one in the measuring cylinder after a determined period of time. The time
set for this water displacement will be measured in second(s) with the use of a stopwatch.
With these being said, the rate of water displacement which implies the rate of reaction of
hydrogen peroxide catalysed by enzyme liver catalase will then be calculated in
3
milimeters/seconds ( cm / s ). Quantitative data will be recorded to carry out using valid
mathematical calculations.

Controlled variables

Controlled variable Why it is controlled How it is controlled

1. The amount of liver The mass of liver used must All the trials will be
be the same for all the trials conducted with the same
of experiment for five mass of liver of 1.0g.
different concentrations of
hydrogen peroxide. This is
because the diffrence of
amount of liver, which means
the difference in the amount
of enzyme liver catalase
available, will affect the
validity of the data collected.
In this investigation, to
measure the effect of
different concentrations of
hydrogen peroxide on the
activity of enzyme liver
catalase, the amount of
enzyme must be the same
which means the same
amount of liver used in all
trials.
2. The amount of hydrogen The amount of hydrogen The volume of hydrogen
peroxide peroxide used will be peroxide for each type of
controlled because different 3
concentration will be 5 cm .

3

3. The amount of time for
the liver to fully submerge
into hydrogen peroxide
4. The number of swirls of
the test tube containing the
liver whose enzyme liver
calase catalyses the
hydrogen peroxide
volume of hydrogen peroxide
with different concentrations
will alter the trends of the
results collected. This
amount must be the same for
each type of concentration
despite different
concentrations.
The difference in the time
that the liver moves along
inside the test tube to
eventually submerge into the
hydrogen peroxide to
catalyse the reaction must be
minimized and should be the
same for every trial.
Within the catalysation
between the enzyme liver
catalase and the hydrogen
peroxide, the test tube
containing them must be
controlled and swirled with
the same number of times in
each trial to ensure that all
the surface of the liver will
submerge into the hydrogen
peroxide, which means all
the enzyme liver catalase will
have a chance to collide with
the substrate and catalyse it.
The liver must be moved
into the hydrogen peroxide
as fast as possible, ideally as
soon as the stopwatch starts
to measure the time. The
uncertainties in the time
taken for every trial will be
± 1 second.
Swirl the test tube three
times at intervals in each
trial during the catalysation
between enzyme liver
catalase in the liver and
hydrogen peroxide, when
hydrogen peroxide
decomposes into water and
oxygen led through the
rubber into the measuring
cylinder.

III. Method

1. To begin with, depending on the concentration of hydrogen peroxide wanted, there will be
an accordingly appropriate amount of water diluted with a certain existing amount of
hydrogen peroxide 6% for it to be produced. In this investigation, besides the existing
hydrogen peroxide 6% used to study the activity of enzyme liver catalase, hydrogen peroxide
with the concentrations of 5.5%, 5.0%, 4.5%, 4.0% need to be produced by the
aforementioned method to be used.

To calculate the appropriate amount of water needed to produce hydrogen peroxide with the
x
× 50 cm 3
wanted concentration, the formula used is 6 , with x being the concentration of
3
hydrogen peroxide wanted and 50 cm being the total volume of hydrogen peroxide 6% and
the water added.

Illustrated below is an example featuring how hydrogen peroxide 4.5% is produced.

4.5
× 50 cm 3 = 37.5 cm 3
6
By this calculation, the volume of hydrogen peroxide 6% needed to produce hydrogen
3
peroxide with the concentration of 4.5% is 37.5 cm and the according amount of water
3 3 3
needed to be diluted with it is 50 cm - 37.5 cm = 12.5 cm . These all will be mixed and

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stored using a 50ml measuring cylinder. Likewise, hydrogen peroxide with concentration of
5.5%, 5.0%, 4.5%, 4.0% will be produced using the same method.

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2. After this step, use a pipette to draw up 5 cm of hydrogen peroxide from the measuring
cylinder contaning hydrogen peroxide with the wanted concentration and release it into a test
tube held firmly into the stand and ring clamp.

3. Half-fill a trough, bowl or sink with water.

4. Fill another 50ml measuring cylinder with water. Invert it over the trough of water, with the
open end under the surface of the water in the bowl, and with the one end of the rubber tubing
in the measuring cylinder. The other end of the rubber tubing is connected with the cork of
the test tube to direct the oxygen produced from the decomposition of hydrogen peroxide in
the test tube into the measuring cylinder filled with water.

5. Record the initial place of the water filled inside the measuring cylinder.

6. Set the specific period of time for each trial with the assumption in mind that the higher the
substrate concentration, the greater the chance of substrate molecules colliding with the
enzyme and hence the shorter the time it will take for the decomposition of hydrogen
peroxide to complete. For example, in this investigation, the trial with hydrogen peroxide 6%
will have the shorter period of time than the one with hydrogen peroxide 4.5% for the
completion of the reaction.

7. Use the forceps to put the liver with the mass of 1.0g straight into the test tube containing
hydrogen peroxide and use the test tube cork connected with the rubber tube to cap the test
tube as soon as the liver is inside the substrate. This process must be done as fast as possible
and the liver surface must be ensured to fully submerge into the hydrogen peroxide as soon as
the set time starts to count down.

8. During the reaction happening inside the test tube, slightly swirl the test tube three times at
intervals in each trial to ensure the liver will sit low down in the substrate so that all enzyme
liver catalase will catalyse the reaction.

9. After the period of time set, record the final place of water inside the measuring cylinder.

10. Calculate the water displacement inside the measuring cylinder by substracting the intial
place from the final place of water recorded.

11. Repeat the experiment at least three times for each type of concentration to enhance the
reliability of the data. Hence, hydrogen peroxide with five different concentrations (6.0%,
5.5%, 5.0%, 4.5%, 4.0%) will be examined with at least three trials of each.

IV. Safety consideration

To reduce the arsing risks of skin allergy to a specific substance, wear disposable gloves and
wear safety goggles to protect the eyes.

The extraction of 1.0g liver for the experiment using knife or any sharp material will be
conducted in a careful manner.

After each trial, chemical waste will be disposed of into the cleaning sink, which causes no
harmful environmental effect.

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Analysis

I. Data collection

Raw data

The table showing five different concentrations of hydrogen peroxide used as independent
variable and the dependent variables being the volume of water displaced as well as the time
taken for such displacement of water. The uncertainties of water displacement and the time

H 2 O 2 Concentration (%) Dependent variable

Volume of water displaced Time taken ( ± 1 second)
3
( ± 0.5 cm )
6.0 27 60
17 60
23 60
35 60
5.5 27 30
36 45
55 60
5.0 50 120
55 120
53 120
4.5 29 60
27 60
23 60
36 180
35 180
38 180
40 180
43 180
4.0 45 120
42 120
43 120
26 120
18 120
17 120
26 120
19 120
3
taken are ± 0.5 cm and ± 1 second respectively.

The table above represents the volume of water displaced within a specific set amount of time
in each trial with five different concentrations of hydrogen peroxide. This implies the change
in reaction rate when there is a change in the concentration of substrate catalysed by the same
concentration of enzyme among trials.

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Processed data

With the data about the volume of water displacement and the the time taken for it in the raw
3
data table, the table below illustrates the processed rate of water diplacement ( cm /s)
calculated by the division between the volume of water displaced and the time taken for this
displacement in each trial. The table also displays the average rate of water displacement
which is processed by averaging all the rates of water displacement caluclated in all trials of
each type of concentration of hydrogen peroxide. This average aims to highlight the
relationship between the concentration of substrate and its effect on the activity of enzyme
liver catalase, simplifying the process of finding such a relationship while there are varied
data collected for each trial. The standard deviation is also calculated to further compare the
data ranges.

H 2 O 2 Concentration (%) Rate of water Average value of each Standard deviation

3
displacement ( cm /s) H2O2 ( cm3 /s)
6.0 0.450 0.425 0.12600
0.283
0.383
0.583
5.5 0.900 0.872 0.06320
0.800
0.917
5.0 0.417 0.439 0.02070
0.458
0.442
4.5 0.483 0.298 0.12100
0.450
0.383
0.200
0.194
0.211
0.222
0.239
4.0 0.375 0.246 0.09960
0.350
0.358
0.217
0.150
0.142
0.217
0.158

To clarify the methods of calculation, the effect of substrate concentration ( H 2 O 2 ) on the

activity of enzyme liver catalase in the liver can be analysed by calculating the rate of water
displacement using the following formula:

volume of water displaced (cm 3 )

=
3
Rate of water displacement ( cm /s) time taken for the displaceme nt (s)

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For example, in 6.0% of H 2 O 2 , with the water displacement of water being 27 cm and the
3

time taken for the displacement being 60s, the rate of water displacement will be
27 cm3
= 0.450 (cm 3 / s)
60 s (3 s.f).

After calculating all the rates of water displacement in all trials for each type of concentration
of hydrogen peroxide, the average rate of water displacement for that type of concentration
can be calculated using the following formula:

3
Average rate of water displacement ( cm /s)
sum of all rates of water displaceme nt of all trials (cm 3 / s)
=
the number of trials conducted
For example, in 6.0% of H 2 O 2 , with the sum of all rates of water displacement of all trials
3
being 0.450 + 0.283 + 0.383 + 0.583 = 1.699 (cm / s) and the number of trials conducted being
1.699
= 0.425 (cm 3 / s)
4, the average rate of water displacement of all trials will be 4
(3 s.f).

II. Data presentation

The effect of substrate concentration (H2O2 ) on enzyme catalase

1
Average rate of reaction (cm3/s)

0.9
0.8
0.7
0.6 f(x) = 0.19 x − 0.48
0.5
0.4
0.3
0.2
0.1
0
3.5 4 4.5 5 5.5 6 6.5

H2 O2 Concentration (%)