Divya Aishwarya Gandi

Enzyme lab report

Research Question:
How does increasing the concentration of the substrate, in this case, hydrogen peroxide
(0.75%, 1.50%, 3.00%, 4.50%, and 6.00%) affect the rate of enzyme activity of catalase
obtained from potato tissue, on decomposing the hydrogen peroxide?
Background Information:
Enzymes are biological catalysts that catalyses biochemical reactions in living cells. In an
enzyme-catalysed reaction, the substrate binds to the active site and forms enzyme-substrate
complex with the enzyme. The enzyme then breaks the bonds in the substrate. The product of
the reaction then leaves the enzyme, which remains unchanged after the reaction.
Catalase in an enzyme produced by our liver to break down hydrogen peroxide a common
end product of metabolism, but highly toxic if accumulated in the body into water and
oxygen. The equation of the reaction is as follows:
2 H2O2

O2 + 2 H2O
Catalase

When catalase is added to hydrogen peroxide, there is an initial rapid evolution of oxygen
which lasts for about two minutes, depending on the peroxide concentration. After this,
oxygen is given off at a steady rate which slowly decreases in the course of an hour. This
decrease in the rate is undoubtedly due to enzyme destruction. The rapid evolution and the
steady rate, however, are inherent features of the peroxide decomposition. 1In this
experiment, we obtain hydrogen peroxide solution and extract catalase from potato. Filter
paper discs are dipped into the catalase solution before they are submerged in hydrogen
peroxide solution. The oxygen produced from the enzyme reaction will form on the discs and
cause the disc to be buoyant enough to float upwards. Through this method we can
investigate the effects of substrate concentration on the rate of reaction. We manipulate the
substrate concentration by using different concentrations of hydrogen peroxide solution, and
measure the rate of reaction by measuring time taken for enough oxygen to be produced so
that the disc to float to the surface.
Hypothesis:
The time taken for the catalase to break down the hydrogen peroxide will decrease as the
concentration of the substrate increases, provided that all other external factors are kept
constant. This is because there will be a greater rate of reaction due to there being more
collisions and a lower activation energy. However, after a certain point there will not be an
increase in the rate of reaction as the enzyme concentration becomes the limiting factor.

1 http://www.nature.com/nature/journal/v160/n4054/abs/160041a0.html

Divya Aishwarya Gandi

Variables:
Independent
Dependent

Controlled

Variable
Concentration of the
hydrogen peroxide.
Time taken for the
filter paper to rise to
the top of the
solution.
Temperature

Value(s)
0.75%, 1.50%,
3.00%, 4.50%, 6.00%
In seconds (s)

Justification

Room temperature
(26C)

Type of tissue

Same potato used

Enzyme
concentration

1 filter paper soaked

in the potato solution.

Volume of hydrogen
peroxide

7.5 cm3

Size of the test tubes

Same width and

length

Diameter of the filter

paper.

Taken from the same

disk. Approx 0.5 mm

Enzyme reactions are affected

by external temperature, hence
the temperature is kept constant
so that the only factor affecting
the rate of reaction is the
substrate concentration.
To ensure that the same
concentration of enzyme is
obtained.
This is to ensure that only the
substrate concentration is
affecting the rate of reaction.
The crushing of a single potato
ensures that there is a
homogenous potato solution,
which means that the
concentration of catalase is
constant throughout.
This ensures that the same
number of hydrogen peroxide
molecules is available for the
reaction even though they vary
in concentration.
This is to ensure that the
distance that the filter paper
disc has to travel is the same.
This is so that the catalase
absorbed is of the same
quantity and so that the oxygen
required to lift it, is the same
during each trial.

In order to determine the rate

of enzymatic reaction.

Divya Aishwarya Gandi

Materials and Apparatus:

Digital stop watch, accurate to 0.01 s (x1) Uncertainty: 0.01

Test tubes (x6)

Test tube holder (x1)

Glass Rod

Electric blender

Beaker (500 cm3) Uncertainty: 1cm3

Knife

Pestle and Mortar

Sieve

Petri dish

Tweezers

Pipette (dropper) (x1) Uncertainty: 0.5cm3

6% hydrogen peroxide solution

Potato (x1)

Filter paper disks

Distilled water

Divya Aishwarya Gandi

Procedure2:
1. 0.75 % hydrogen peroxide solution is prepared by measuring 3.00 cm3 of 6 %
hydrogen peroxide using a measuring cylinder and then diluting it with 21.00 cm 3 of
distilled water. 1.50 %, 3.00 %, 4.50 % and 6.00 % hydrogen peroxide solutions are
prepared using the same method the with corresponding volumes of 6 % hydrogen
peroxide and water as shown in the table below

Concentration of hydrogen
peroxide solution
/%
( 0.02)

Volume of 6 % hydrogen
peroxide
/ cm3
( 0.01)

Volume of
water added
/ cm3
( 0.01)

0.75
3.00
21.00
1.50
6.00
18.00
3.00
12.00
12.00
4.50
18.00
6.00
6.00
24.00
0.00
Table 1: Dilution table for preparing different concentrations of hydrogen
peroxide solutions
The prepared hydrogen peroxide solutions are poured into test tubes using pipettes.
Each test tube should contain 7.5 cm3 of the solution. The test tubes with their
solutions are placed in the test tube holder, labelled and set aside as stocks.
2. A potato is peeled and cut into cubes. The potato cubes are then homogenized by
crushing them in water. The crushed potato is filtered using sieve and the filtered
liquid is collected in a beaker. Some of the filtered potato liquid is put into a Petri dish
to be used in the experiment.
3. A filter paper disc is soaked with potato liquid in the Petri dish and pushed to the
bottom of a test tube with 0.75 % hydrogen peroxide solution using a glass rod.
4. The stopwatch is started immediately when the filter paper disc touches the bottom of
the test tube.
5. The stop watch is stopped once the filter paper disc reaches the surface. The time
taken for the filter paper disc to float to the surface is recorded.
6. Steps 3 5 are repeated twice, using other new 0.75 % hydrogen peroxide solutions.
7. The average of the 3 readings for each hydrogen peroxide concentration is calculated
and recorded. The rate of reaction is calculated by the following formula:
Rate of reaction =
8. Steps 3 7 are repeated 5 times with 1.50 %, 3.00 %, 4.50 % and 6.00 % hydrogen
peroxide solutions.
2 http://www.slideshare.net/wkkok1957/ib-biology-on-decomposition-ofhydrogen-peroxide-by-enzyme-catalase

Divya Aishwarya Gandi

Safety Precautions:
1.
2.
3.
4.

Hydrogen peroxide is corrosive so wear latex gloves while handling the solutions.
Handle all glass apparatus with care.
Handle knife and scalpel with care. Exercise caution while using the electric blender.
Avoid parallax error while measuring the volume of solutions by placing eye
perpendicular to the lower meniscus

Raw data Table showing the amount of time taken for the filter paper disks soaked in
catalase to reach the surface of the test tube:
Concentratio
n of
hydrogen
peroxide
solution/ %
(0.02)
0.75
1.50
3.00
4.50
6.00

Time taken for the filter paper disk to reach the surface/s
(0.21)

Trial 1
19.69
11.60
10.75
10.00
7.41

Trial 2
20.75
10.72
11.90
10.00
7.63

Trial 3
23.12
11.53
11.16
10.72
6.00

Trial 4
21.32
11.21
11.13
9.81
6.23

Trial 5
20.15
10.89
11.75
10.32
6.71

Processed data table showing the average time taken for the filter paper to reach the
surface of the test tube for each concentration of hydrogen peroxide:
Concentratio
n of
hydrogen
peroxide
solution/ %
(0.02)

0.75
1.50
3.00
4.50
6.00
Uncertainties:

Time
taken for
the filter
paper disk
to reach
the
surface/s
(0.21)
21.01
11.19
11.34
10.17
6.80

Rate of
reaction/s1

0.048
0.089
0.088
0.098
0.147

Uncertainty in concentration of hydrogen peroxide: 0.02

Divya Aishwarya Gandi

Uncertainty in time taken for filter paper disk to reach surface: 0.01 + 0.2= 0.21
(Where 0.01 is due to the stopwatch and 0.2 is due to the human reaction time)
Percentage uncertainty in time taken for the filter paper disk to reach the surface/% calculated
using:
Average timetaken for t h e filter paper
0.21
reac h t h e surface 100
Absolute uncertainty for rate of reactionrat e of reaction percentage uncertainty time taken

Concentration of
hydrogen
peroxide
solution/ %
(0.02)
0.75
1.50
3.00
4.50
6.00

Average time
taken for the
filter paper to
reach the
surface/s
(0.21)
21.01
11.19
11.34
10.17
6.80

Percentage
uncertainty in
time taken for
the filter paper
disk to reach the
surface/%
0.99
1.87
1.85
2.06
3.09

Absolute
uncertainty in
rate of reaction

Rate of reaction
with
uncertainty/s-1

0.475
0.166
0.162
0.202
0.454

0.048 0.475
0.089 0.166
0.088 0.162
0.098 0.202
0.147 0.454

Qualitative data:
There was immediate formation of bubbles when the filter paper soaked in catalase was
added to the hydrogen peroxide. This is because of the formation of oxygen which pushes the
filter paper up to the surface.

Graph showing the concentration of hydrogen peroxide/% (0.02) against the rate of
reaction/s-1(0.21)

Divya Aishwarya Gandi

Analysis of graph:
The graph shows a steady increase in the rate of reaction until the concentration reaches
3.00% where there is a small drop of 0.001s-1, this anomaly can be said to be on account of
human error, either while preparing the solution or while timing the filter papers rise to the
surface. After this the rate of reaction continues to increase steadily. However, in general the
trend of the graph shows that as the concentration of the substrate (hydrogen peroxide)
increases so does the rate of reaction. There are large error bars for the rate of reaction which
shows that the experiment was not very accurate. This is due to human error and human
reaction time while taking the time.
Conclusion:
From the data collected and the trend of the graph, a positive correlation can be seen with an
increase in the rate of reaction as the substrate concentration increases. This proves my
hypothesis to be right although the concentration of the hydrogen peroxide did not increase to
the extent to make the catalase concentration the limiting factor. This can be explained by the
fact that there are more substrate molecules colliding with the enzyme molecules providing a
greater probability for a reaction to take place and therefore increases the formation of the
product, in this case the oxygen which pushes the filter paper to the surface. However there

Divya Aishwarya Gandi

is an anomalous value for the 3.00% hydrogen peroxide concentration in which there is a
decrease of 0.001s-1. Again this shows that the experiment was not very accurate.
Evaluation:
Strengths:

The controlled variables were kept constant throughout the experiment increasing
reliability of the results obtained.
Several trials (5) were conducted to give more accurate readings for the time taken
and therefore the rate of reaction.
The experiment was conducted in one session so there was no room for spoiling of the
potato or change in concentration of the solutions.

Limitations
Filter paper disk was not of the same
circumference.

Improvements
Use a cutter or template to make sure each
disk is the exact same size and circumference
to reduce error.
Use more concentrations of hydrogen
peroxide in order to have a larger spread of
data and to study the effect in detail.

There was not a great enough parameter in

the concentrations of hydrogen peroxide
which did not allow for the graph to reach a
steady point at which the enzyme
concentration would become the limiting
factor and the substrate concentration would
have no effect.
A control trial was not carried out which
A control trial would ensure that no oxygen is
could result in inaccuracies going undetected. being produced and the filter paper would not
rise allowing there to be a comparison
between the enzyme catalysed reaction and
enzyme non-catalysed reaction.
Disparity in the way the air bubbles are
produced which meant that the rise of the
filter paper was not always constant.
The filter paper sticking to the walls of the
Be extra careful while placing the filter paper
test tube meant that the catalase rubbed off
at the bottom of the test tube, maybe use a
on the walls and would not take part in the
test tube with a wider mouth to ensure that
reaction, reducing the amount of oxygen
the filter paper does not get stuck to the

Divya Aishwarya Gandi

produced.
There could have been human error in the
preparation of the different hydrogen
peroxide concentrations which would have
lead to anomalous readings for an entire
concentration.

walls.
Using ready-made solutions or preparing the
solution for each concentration more than
once and conducting several trials would
make it easier to detect human error and
would also reduce inaccuracies, making the
data more reliable.