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Experiment #:

Date:

December 2, 2015

Topic:

Enzymes

Aim:

To investigate the distribution of catalase in a soaked pea,


and to determine the effect of different temperatures on its
activity.

Apparatus/
Chemicals:

A supply of soaked peas, hydrogen peroxide solution, test


tubes, test tube rack, water baths at 40C, 50C, 60C,
70C,80C and 100C, stopwatch, thermometer, scalpels,
forceps, test tube holder, white tile, glass rod.

Procedure:

1. The presence of catalase was tested by crushing a soaked


pea and adding a few drops of hydrogen peroxide solution.
2. Seed coats were removed from three soaked peas and
tested separately for catalase activity in both seed coats and
cotyledons.
3. Two test tubes containing distilled water were placed in a
water bath at 40 C.
4. Three whole peas were boiled in a test tube and then
placed in one of the tubes in the water bath.

5. Three whole unboiled peas were placed in other tube in


water bath.
Apparatus/
Chemicals:

6. Enough time was allowed for the peas to reach the


temperature of the water bath (at least 10 minutes).
7. Each pea was tested for catalase activity.
8. Experiment was repeated at 50C, 60C, 70C, 80C, and
100 C.
9. Observations were recorded and results commented on.

Observations:

TABLE SHOWING CONDITIONS OF SOAKED PEA WHEN


INTERACTED WITH HYDROGEN PEROXIDE SOLUTION
Condition of Soaked Pea

Observation when Hydrogen

Crushed
Coat of Seed
Cotyledon

Peroxide was added


A great foaming action observed
No observation
Foaming action observed

TABLE SHOWING CATALASE ACTIVITY OF BOILED AND


UNBOILED PEAS AT TEMPERATURE VARIED WATER
BATHS
Temperature/C
40
50
60
70

Boiled Peas
No observation
No observation
No observation
No observation

Unboiled Peas
Little foaming action observed
Moderate foaming action observed
Great foaming action observed
Moderate foaming action observed

80
100

Discussion:

No observation
No observation

Little foaming action observed


No observation

Enzymes are biological molecules (proteins) that act as


catalysts and help complex reactions occur everywhere in
life. Catalase is an enzyme which catalyses the
decomposition of hydrogen peroxide which provides water
and liberates oxygen gas as shown by effervescence:
2H2O2

2H2O + O2

Hydrogen peroxide is a toxic by-product of metabolism in


certain plant and animal cells and is efficiently removed by
catalase, which is one of the fastest acting enzymes known.
The experiment carried out involved the exposure of
temperature varied water baths to previously soaked boiled
and unboiled peas and their reaction with hydrogen
peroxide. Also the peas were faced in certain conditions and
their reactions observed with hydrogen peroxide.
The experiment focuses on the effect of temperature on
enzyme activity. As the temperature increases, the kinetic
energy of the substrate and enzyme molecules increases and
so they move faster. The faster these molecules move, the

more often they collide with one another and greater the rate
of reaction. As the temperature increases, the more the
atoms which make up the enzyme molecules vibrate. This
breaks the hydrogen bonds and other forces which hold the
catalase

Discussion:

molecules in their precise shape. The three-dimensional


shape of the enzyme molecules is altered to such an extent
that their active site no longer fits the substrate. The enzyme
is said to be denatured and loses its catalytic properties.
The enzyme found in red peas is catalase. The
pea in its dry state contains inhibitors which keep them
dormant until they are soaked and start to sprout. Therefore,
when soaked these inhibitors become neutralised and bring
about intensive metabolic activity as they begin to generate.
As a result, catalytic activity is at higher levels. This allows
the catalase in the peas to be more available so that it can
efficiently react with hydrogen peroxide.
When pea was crushed, a great foaming action
was observed. This observation proved the presence of
catalase as the foam was effervescence-oxygen and the liquid

which remained was water. The pea was crushed to increase


surface area in aid of increasing reaction rate as well as
making pea more available for reaction with hydrogen
peroxide. When separated, the seed coat showed no
observation when placed in hydrogen peroxide while a
foaming action was observed for the cotyledons when placed
in hydrogen peroxide. This therefore shows that the catalase

Discussion:

enzyme is specifically present in the cotyledons of the pea.


However, the difference in effervescence level between
crushed pea and cotyledons is due to the former having a
greater surface area.
The boiled and unboiled peas were placed in
temperature varied water baths. The boiled peas showed no
catalase activity when placed in all water baths. This due to
the fact that the catalase in the boiled peas have already
been denatured, therefore, it would not be able to respond to
the addition of hydrogen peroxide to them. The unboiled
peas, on the other hand, showed varied results. With
effervescence indicating enzyme activity, from temperatures

40-60 C showed an increase in enzyme activity and from


60-100 C showed a decrease in enzyme activity. Based on
this, the optimum temperature can be determined to be 60C
as it gave the greatest yield in effervescence thus showing
the most enzyme activity. Also, it can be said that as the
temperature increases, the enzyme activity increases to a
point then decreases due to denaturation. Therefore, it can
be said that the temperatures following 60C showed the
denaturation of catalase as the enzyme activity decreases at
this point hence the lower yield in effervescence noticed.

Discussion:

The theoretical optimum temperature of catalase


ranges from 40 50C while the experimental value came to
be 60C. This difference could have been brought about by
the strength of the hydrogen peroxide. The hydrogen
peroxide used in the experiment could have been weak thus
taking a longer time to achieve a desired result. Also, peas in
water baths may not have been in them long enough or too
long in addition to difficulty in temperature maintenance can
affect the result as well. The catalase in the peas can be

questioned as well as peas of varied origin were used thus


possibly having different catalase concentrations hence
exerting different catalytic levels.
Sources of
Error:

1. Temperatures were not properly maintained in respective


water baths creating inaccurate results.
2. Peas were not in the water baths long enough thus
allowing peas to not reach desired temperature affecting
observation readings.

Limitations:

1. Hydrogen peroxide used could have been weak allowing a


desired result to be achieved later than it should.
2. Peas were not from same plant creating the possibility of
having varied catalase concentrations among peas
influencing rate of enzyme activity with the hydrogen
influencing rate of activity wi
peroxide.

Conclusion:

Catalase is completely present in a soaked pea particularly


in the cotyledons. The temperature increases with the
catalase activity to a point- the optimum temperature, in this
case it is 60C. From this point, the catalase activity
decreases while the temperature still increases indicating the
denaturation of the enzyme.

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