Practical task: Investigation of the effect of pH on enzyme activity

Research question: What is the effect of pH on catalase activity?

A. Objective
To investigate the effect of pH on catalase enzyme activity by measuring the gas pressure.

B. Introduction
All cells in our body produce hydrogen peroxide as part of the immune system to kill
bacteria, or as a by-product of metabolism.

Catalase is one of the enzymes found in liver that speeds up the decomposition of hydrogen
peroxide. Meanwhile, the pH value in blood and body cells is changing from time to time,
affected by different factors such as gas content and food we eat. Thus, the effect of pH on
the catalase activity will be investigated.

B.1 Biological principle

Enzymes are protein in nature and regarded as a natural catalyst found in human
body. They increase the rate of chemical reactions by lowering activation energy of
the reactions, and remain unchanged at the end of the reactions. The active site of an
enzyme is a polypeptide chain where substrates bind to, and form enzyme-substrate
complexes.

Enzymes are specific for a particular substrate since the shape of their active sites are
only suitable for specific types of substrate molecule to fit. The induced fit model
describes the active site changes shape slightly to form a complementary shape to the
substrate after binding in.

When a catalase is added to a hydrogen peroxide solution, it becomes water and

oxygen gas.
2H2O2 (l) + catalase → 2H2O (l) + O2 (g)

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 From the above equation it can be seen that since oxygen gas is released in the
decomposition of hydrogen peroxide, changes in gas pressure can be used to measure
the rate of reaction.

B.2 Hypothesis
It is hypothesised that the amount of gas produced is positively correlated with the pH
value. But once it has met the optimum pH, which is the time when the rate of
production of gas is the greatest, the rate of reaction will start falling.

B.3 Design of experiment

As mentioned above, oxygen gas is released by the decomposition of hydrogen
peroxide, and the gas released was trapped in a boiling tube closed by a stopper
connecting to pressure sensor to detect the gas pressure change inside the tube.

Thus, the rate of reaction could be deduced by measuring the increase in gas pressure
in the boiling tube after a 60 seconds reaction.

B.4 Variables
How to measure / control?
Independent variable The pH value of The pH solution with specific
different pH solution pH values is taken from the

2

Dependent variable
Controlled variables
Rate of gas produced
from the reaction
between hydrogen
peroxide and catalase
1. The amount and
concentration of
catalase
2. The amount and
concentration of
hydrogen peroxide
same container in every trial
and for each of the pH
solution, it is pipette with its
corresponding pipette to
prevent any mixture of
different pH solution.
The boiling tube is connected
with a data logger and
pressure sensor which track
the reaction happening inside
the tube for 60 seconds in
every trial.
It was taken from the same
container in every trial and
the same pipette is used to
pipette the amount required
(2 ml).
It was taken from the same
container in every trial and
immediately pipette 1 ml of it
to start the reaction, in order
to prevent exposure to air.

3. The amount and It was taken from the same

concentration of pH container in every trial and
solution the same pipette is used to
pipette the amount required
(2 ml).
4. Temperature of the The temperature was set in
surrounding room temperature of 25°C
for every trial.

B.5 Assumptions
In this experiment, it is assumed that through the measurement of gas pressure, we
can obtain the value of oxygen produced and thus the rate of reaction. It is also
assumed that the temperature of surrounding did not fluctuate such that pH is the only
independent variable which manipulate the amount of oxygen produced.
B.6 Safety measures
1. Wear safety goggles, a lab coat and long pants to prevent direct contact with
the corrosive and irritant1 hydrogen peroxide solution.
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Safety precautions while handling hydrogen peroxide 6%
https://jmloveridge.com/wp-content/uploads/2018/06/Hydrogen-Peroxide-6-Version-05.pdf

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 2. Handle fragile glassware with care.

C. Materials and Apparatus

Materials and apparatus Quantity Materials and apparatus Quantity

400IU/mL Catalase solution 2 ml for each Marker 1

reaction
Buffer solution at different pH 2 ml for each Data logger with pressure 1
(pH 3,5,7,9,11) reaction sensor
6% Hydrogen peroxide 1 ml for each Timer 1
solution reaction
Boling tube 1 Pipette with pipette filler 1

Beaker 2 Boiling tube rack 1

D. Procedure of the experiment

1. Pipette 2 ml of pH solution with the pH 3 into beaker labelled “A”.
2. Pour hydrogen peroxide solution into beaker labelled “B”.
3. Pipette 2 ml of catalase solution and add it to a boiling tube.
4. Pour beaker A solution to the boiling tube and swirl it to mix the solution
well.
5. Pipette 1 ml of hydrogen peroxide and add it to the boiling tube, then
immediately close the boiling tube with a stopper connected to a data logger
with pressure sensor.
6. At the same time, start the data logger and record the gas pressure for 60
seconds.
7. Repeat step 1-6 for 4 trials.
8. Repeat step 1-7 with the pH solution at pH 5,7,9 and 11.

D.1 Drawing of the set-up

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 Figure 1: Drawing of the experimental set-up

E. Results of the experiment

D.1 Raw data table

pH values 3 5 7 9 11
Trial 1 Initial gas pressure (kPa) 101.86 102.30 101.80 102.73 100.39
Final gas pressure (kPa) 106.83 105.07 106.05 108.23 107.82
Trial 2 Initial gas pressure (kPa) 101.89 101.65 101.26 104.00 102.61
Final gas pressure (kPa) 106.69 105.86 105.64 111.56 109.98
Trial 3 Initial gas pressure (kPa) 101.29 101.53 102.44 101.29 102.24
Final gas pressure (kPa) 104.98 106.18 106.12 107.89 111.95
Trial 4 Initial gas pressure (kPa) 101.73 101.71 101.78 100.38 100.89
Final gas pressure (kPa) 105.16 105.64 106.32 102.83 108.35
Trial 5 Initial gas pressure (kPa) 102.10 101.63 102.34 101.74 102.89
Final gas pressure (kPa) 105.30 105.14 106.72 104.80 107.36
Table 1: Initial and final gas pressures under different pH (kPa)

D.2 Sample calculation

Change in gas pressure (trial 1 of pH3)
= (106.83−101.86) kPa
= 4.97 kPa
Average rate of change in gas pressure (trial 1 of pH3)
= 4.97 ÷ 60

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 = 0.083 kPa/s (cor.to 3 s.f)
Rate of reaction in terms of change in gas pressure over time (trial 1 of pH3)
= 0.083 kPa/s (cor.to 3 s.f)
pH values 3 5 7 9 11
Trial 1 Change in gas pressure (kPa) 4.97 2.77 4.25 5.5 7.43
Rate of reaction (kPa/s) 0.083 0.046 0.071 0.092 0.124
Trial 2 Change in gas pressure (kPa) 4.8 4.21 4.38 7.56 7.37
Rate of reaction (kPa/s) 0.080 0.070 0.073 0.126 0.123
Trial 3 Change in gas pressure (kPa) 3.69 4.65 3.68 6.6 9.01
Rate of reaction (kPa/s) 0.062 0.078 0.061 0.110 0.150
Trial 4 Change in gas pressure (kPa) 3.43 3.93 4.54 2.45 7.46
Rate of reaction (kPa/s) 0.057 0.066 0.076 0.041 0.124
Trial 5 Change in gas pressure (kPa) 3.2 3.51 4.38 3.06 4.47
Rate of reaction (kPa/s) 0.053 0.059 0.073 0.051 0.075
Average rate of reaction (kPa/s) 0.067 0.064 0.071 0.084 0.119

Average rate of gas collected

Average rate of change in gas pressure (kPa)

0.05
0.04
0.04
0.03
0.03
0.02
0.02
0.01
0.01
Table 2: Change
0 in gas pressure (kPa) and average rate of reaction (kPa/s) under different pH (cor.to 2 s.f.)
0 3 6 9
pH values

Figure 2: Graph showing the average rate of change in gas pressure under different pH
values

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 D.3 Error calculation
The error presented in the experiment is calculated using standard deviation. The
equation is:

∑ (x−x́)2
σ=
√ n
Sample calculation:
At pH 3,

(0.083−0.067)2 +(0.080−0.067)2 +(0.062−0.067)2 +(0.057−0.067)2+(0.053−0.067)2

σ=
√ 5

¿ 1.49 ×10−4 kPa/s

pH Standard deviation (10−4) (cor.to 2 d.p.)

3 1.49
5 1.17
7 0.27
9 0.88
11 5.93

Table 3: Error for each data point

F. Conclusion and evaluation

F.1 Conclusion
According to the results shown in table 2 and figure 2, it can be concluded that part of
my hypothesis is correct. The trend in figure 2 shows that the rate of reaction
increases as the pH increases, but it does not drop after reaching the optimum pH,
which is known as pH 7.

The biological principle behind the positive correlation of gas pressure and pH values
before the optimum pH is due to the hydrogen ions and hydroxide ions in the acidic
and basic solution respectively. Enzymes are made of amino acids that are either
positively or negatively charged, and the charge of substrate must match with the one
in the specific enzyme in order to form an enzyme-substrate complex. When a

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 solution is extremely acidic, a relatively large amount of hydrogen ions will present,
bonding with the negative charge of enzyme or substrate, thus prevent proper charge
matching between the two, as the number of hydrogen ions dominated the binding
action. This is one of the reasons causing the enzyme to be less active and overall
decrease in rate of reaction. In extreme cases, the enzyme can be denatured, and
stopped the reaction.

F.2 Errors and improvements

The reaction time of human could be the error that primarily affect the results
obtained in this experiment. After adding the pipetted hydrogen peroxide to the
boiling tube, the reaction started immediately, at the same time, I had to insert the
stopper to the boiling tube and start the timer on the data logger. Unfortunately, no
matter how fast I carried out this process, there were still potentially some oxygen
leaking out before closing the stopper, thus affecting the data obtained. Moreover,
before I started the timer, the reaction may have already begun. As one of the random
errors, it cannot be eliminated but its effect can be minimised by having multiple
measurements. Performing at least 3 trials and taking the mean value can also
improve the preciseness of data.

Another possible source of error would be the re-usage of boiling tube. Due to the
constrained lesson period, I only had very limited time to carry out the experiment.
So, after every trial, I rinsed the boiling tube with running water, but I hadn’t had
enough time to wait for it to be completely dry. Some water droplets may stick on the
boiling tube and affect the concentration of other solutions. One improvement could
be made by requesting another boiling tube so that it was clean and dry for me to
carry out next trial while waiting for the previous boiling tube to dry.

F.3 Further investigation

To obtain a more accurate result, a same experiment can be carried out again, and
adding a few more trials with different pH values, such as pH 4,6,8 can aids our
investigation on a more accurate optimum pH.

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 If I had had enough time, I would also investigation
other factors that could affect the catalase activity, such
as temperature and substrate concentration.
Theoretically, the rate of an enzyme-catalysed reaction
increases as the temperature increases. As more kinetic
energy is provided to the particles, there will have more
successful collision between them and therefore an Graph 1: Expected graph obtained
from the investigation of effect of
increase in rate of reaction. But after the optimum temperature on catalase activity
temperature peak, the enzyme will be denatured and the graph will decrease sharply2.

And for substrate concentration, increasing the

factor will speed up the reaction, as long as there is
enzyme available to bind to. Once the collision has
reached the saturation, the graph will reach its plateau
too3. Above all, they are the two experiments that I Graph 2: Expected graph obtained
from the investigation of effect of
show keen interest in carrying them out in the laboratory. substrate concentration on catalase
activity

Reference

1. Safety Data Sheet Hydrogen Peroxide 6%. (2015, August) [PDF file]. Retrieved from

https://jmloveridge.com/wp-content/uploads/2018/06/Hydrogen-Peroxide-6-Version-
05.pdf

2
Diagram showing the relationship between temperature and rate of reaction of enzyme
https://ib.bioninja.com.au/higher-level/topic-8-metabolism-cell/untitled-6/enzyme-kinetics.html
3
Diagram showing the relationship between substrate concentration and rate of reaction of enzyme
https://ib.bioninja.com.au/higher-level/topic-8-metabolism-cell/untitled-6/enzyme-kinetics.html