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What effect does the enzyme concentration has on Hydrogen peroxide in regards to the activity
of potato catalase and Broccoli?
Catherine W. Barnes and Destiny Coward

Introduction
The purpose of conducting this investigation is to find out the effect that enzyme concentration
has on hydrogen peroxide in regards the activity of potato catalase and other plants with the same
temperature and pH in the breaking down of hydrogen peroxide into O2 and H2O. Catalase is an
enzyme that can be found in most living things including Humans. What are enzymes? In
biology and chemistry, enzymes are known as catalysts which speeds up chemical reactions in all
living things. Enzymes are chemicals molecules made up of proteins (Buchanan et al., 2000). An
Enzyme rate can be effected by many factors including pH, temperature, substrate concentration,
the presence of inhibitor and so on. The changes in pH alters the ionic charge of acidic and basic
groups of the enzyme, therefore, changing the shape of the enzyme, including its active sites,
where substrates momentarily bind (Alberts et al., 2008). Enzymes can regain its shape if it is
not expose to extreme pHs and returning to it maximum rate of activity when place back at the
perfect pH which is pH7. The main function of a catalase is to catalyze the decomposition of
Hydrogen peroxide. Where is hydrogen peroxide produce? Hydrogen peroxide is produce in
plant and animals as a toxic by-product that makes the catalase to speed up the breakdown of
hydrogen peroxide to water and oxygen. In the experiment, Hydrogen peroxide will be use as a
substrate and Catalase as an enzyme. Catalase break down hydrogen peroxide into oxygen which
will be measured as the volume, the activity of the catalase will be measure by finding the rate of

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oxygen release by from Hydrogen peroxide. Chop potato, onion, and broccoli are excellent
sources of catalase with the pH staying at 7.
Hypothesis: The concentration of catalase will affect the reaction of Hydrogen peroxide
oxygen and water.
Null hypothesis: As the concentration of catalase increase the rate of breakdown of
catalase from oxygen to water also increase. If the catalase concentration double then the rate of
hydrogen of catalase to water will also double.

Materials
For the experiment the following materials will be use: Goggles, gloves, petri-dish, stop
watch, mortar, hole punch, six micro-centrifuge tubes, beaker, one 250cm beaker (waste beaker),
forceps, permanent marker, tape, disc, hydrogen peroxide(H2O2), Filter paper, fine sand,
distilled water, plants (potatoes, carrots, broccoli, onion), pH7, electronic balancer, pipette,
thermometer
Method
As you begin the experiment, use goggles to protect your eyes from the hydrogen
peroxide and gloves to protect your skins from any spill of hydrogen peroxide. Gather the
materials that are to be use in the experiment. Place which ever plant is being use in a mortar,
pipetting two drops of pH7 and little quantity of fine sand and distilled water. Grind until the
plant turn into a paste. Scrape the paste into a micro-centrifuge tube using the spatula and place it
into the electronic balancer to spin. While the paste is spinning, cut several disc that will be use

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in the extract using the hole punch and filter paper. Transfer the catalase-containing the liquid
paste after spinning to a clean micro-centrifuge tube and label it using the permanent marker. Dip
a disc of cut filter paper into the extract with the help of the forceps and blot briefly for five
seconds on a sheet of filter paper. Measure 1-2cm of 3% hydrogen peroxide into 5ml beaker.
After briefly blotting drop the disc into the H2O2 to settle at the bottom and then rise up, observe
from the side and record the hydrogen peroxide and start the stop watch at the same time. Record
the volume of oxygen produce by the reaction. Use approximately 10 discs before placing H2O2
into the waste beaker. Plot a graph of volume of oxygen produce against time and the rate of the
reaction against the pH. Repeat the entire experiment with a different plants, temperature or pHs
(Evert et al., 2013)

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Results
pH buffer used was 7 and plant used was potato with a 3% of Hydrogen peroxide. The below
table shows the timing of 10 disc dropped in hydrogen peroxide individually after five seconds
of blotting from the potato extract showing the reaction between Hydrogen peroxide and catalase
producing a strong pressure in pH7.
Potatoes at room temperature with a pH-7
Discs

Time(secs)

10

Data Summary for the potato catalase observation

N
X
SS

X2

Discs
10
55

Time
10
38

Total
20
93

385

146

531

82.5

1.6

98.55

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Mean

5.5

3.8

4.65

The mean of the discs substrates from the time sum to 1.7 having a degree of freedom of 18, the t
value is 1.76. P---one-tailed equals 0.0476955 and p-----two-tailed equals 0.095391 which
assumes that the two samples have equal variances. The result is significant at p <.05.

Potato Catalase
12
10
8

Discs

Discs

Time

4
2
0
1

Time

The results above illustrate that the rate at which oxygen was developed increases as the
substrate concentration increases. Rate of reaction equals average overall gas produced.

pH buffer used was 7 and plant used was broccoli with a 3% of Hydrogen peroxide. The below
table shows the timing of 10 disc dropped in hydrogen peroxide individually after five seconds
of blotting from the Broccoli extract showing the reaction between Hydrogen peroxide and
catalase producing a strong pressure in pH7.

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Discs
1
2
3
4
5
6
7
8
9
10

Broccoli at room temperature with pH-7

Time(sec)
9
9
8
8
9
7
8
7
7
7

Data Summary of the Broccoli catalase

N
X
-

X2

Discs
10
55

Time
10
79

Total
20
134

385

631

1016

SS
82.5
6.9
118.2
Mean
55
7.9
6.7
The mean of the discs substrates from the time sum to 2.4 having a degree of freedom of 18, the
t value is 2.41. P---one-tailed equals 0.0134345 and p-----two-tailed equals 0.026869 which
assumes that the two samples have equal variances. The result is significant at p <.05.

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Broccoli Catalase
12
10
8

Discs

Discs

Time

4
2
0
1

Time

The results above illustrate that the rate at which oxygen was developed increases as the
substrate concentration increases. Rate of reaction equals average overall gas produced.
Discussion
The graph with the potato and broccoli show that with 3% concentration of Hydrogen peroxide,
oxygen is found. From this, it can be said that with a little percentage of Hydrogen peroxide
present onion catalase will be broken down into oxygen and water. There are possible errors that
the experiment may have encounter like the miscounting of the temperature, blotting of the discs
properly on the filter paper and timing. The pipetting of Hydrogen peroxide into the pipette
which often had air bubbles trap in the pipet that could not be remove leading to a lesser volume
than 2cm. Temperature may have a different effect on the experiment instead of the pH in a
different way. The experiment could be improve by using different concentration like HCL or
sodium hydroxide. A 7% of Hydrogen peroxide instead of 3% to give the reaction a faster rate.

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There should be no need for distilled water to be added on to the plant which dilutes the
concentration of the catalase. A transparent dish will be preferable for the hydrogen peroxide
than a smaller beaker giving it an open area that is larger. The pH7 is neutral making it perfect
for the reaction to take place. The potato does not need an alternative.

Conclusion
The result prove not to support my hypothesis on how the concentration of hydrogen peroxide
effects the rate of reaction between the different plants catalase. The higher the enzyme
concentration, the more oxygen was produced. This is because the higher the enzyme, there are
active sites where more successful collision will take place between the active site for the
enzyme and the substrate. The graph shows that as the catalase concentration was increasing
more, the rate for catalase activity was decreasing which indicates that is was nearing the
optimum rate of catalyze activity.
References
Evert, R. F., S.E. Eichhorn. 2013. Laboratory Topics In Botany. 8th ed. Biology of Plants.
University of Wisconsin, Madison.
Buchanan, B.B., W. Gruissem, and R. L. Jones (Eds.). 2000. Biochemistry and Molecular
Biology of Plants. American Society of Plant Physiologist, Rockville, MD.
Albert B., A. Johnson, J. Lewis, M. Raff, K. Roberts and P. Walter. 2008. Molecular Biology of
the Cell. 5th ed. Garland Science, New York. Oxford.