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The Effect of Temperature on Catalase Enzyme

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The Effect of Temperature on Catalase Enzyme

Aim: Investigate the effect of temperature on the activity of catalase
(from potato).

Introduction: Enzymes are biological catalysts. They speed up
metabolic reactions in the body but remain chemically unchanged
themselves. Enzymes contain an active site. This is a region, normally
a depression or cleft, to which another molecule may bind. This
molecule is known as the substrate, and is usually specific to the
active site of the particular enzyme, which breaks it down. Substrates
will not usually fit into any other active sites other than that of
the enzyme it is specified to. This can be explained as a lock and key
model, where the lock and key are specific to each other, only, that
there are many of the same kinds of lock and key when it come to the
enzymes.

[IMAGE]

Just as lock and keys have three-dimensional shapes, proteins are also
three-dimensional. Usually, there is only one active site on an
enzyme; however there can be more. Some energy releasing reactions in
cells produce hydrogen peroxide. This is acidic, and can thus, kill
cells. Normally, hydrogen peroxide decomposes to form hydrogen and
oxygen:

[IMAGE] 2H2O2 2H2O + O2

However, this process is very lengthy. There is an enzyme known as
catalase in cells which dramatically increases the rate of
decomposition of hydrogen peroxide.

catalase

[IMAGE] 2H2O2 2H2O + O2

This type of reaction where a molecule is broken down into smaller
pieces is known as a catabolic reaction.

In order to investigate the effect of temperature on the activity of
catalase, I will record the amount of oxygen released when hydrogen
peroxide is broken down.

Variables: There are quite a few variables which can alter the rate of
reaction, and need to be kept constant. They are as follows:

a) PH: at too high PH, the enzyme is denatured due to the loss of H+
ions. The same applies for too low a PH level, where too many H+ ions
would attach to the negative regions of the enzyme, changes its shape
and causing it to denature.

b) Concentration of enzyme: The higher the concentration, the higher
the rate of reaction will be. With a larger number of catalase
molecules, the chance of successful collisions between enzyme and
substrate will be increased. In order to keep this constant, I will
make sure I use the same volume of tissue (potato) containing catalase
each time I conduct the experiment.

c) Surface area: The previous also applies to this.

d) Mass of tissue: Here it needs to be taken into account that
different potatoes will not give the same mass, even if equally sized
pieces are cut. Different potatoes will not have exactly the same
water and catalase content.

Arefin Khan

The mass will be kept constant in the same way as surface area and
concentration of enzyme.

Prediction: The higher the temperature, the higher the rate of
reaction up to a certain point. This is due to the fact that the
particles gain kinetic energy and subsequently move around more
vigorously. Thus, the chance of there being a successful collision
between the enzyme and substrate molecule increases as reacting
particles with collide more frequently with increased kinetic energy.

Enzymes have a very specific three-dimensional shape, held together by
ionic and hydrogen bonds. If the amino acids are too vigorous in their
motion, then, these bonds will brake. Once the bonds have been broken,
the enzyme is said to have become denatured. As a result of becoming
denatured, the enzymes' rate of activity becomes less because the
enzyme loses its specific three-dimensional shape. The enzyme will
start to become denatured after around 40ºC as enzyme activity is
usually at its optimum at this temperature. After this, the rate of
reaction will probably deteriorate. After 60ºC, there is likely to be
no reaction, as the enzymes would probably be completely denatured by
then. *(Above from: Source, Page 47)

Methodology:

1. Wear goggles for protection, and lab coat if available.

2. Arrange apparatus.

3. Get 250cm3 beaker and fill with water to about the 150cm3 mark with
water at a specific temperature. This temperature can be reached by
either cooling, via the use of ice, or by heating, via the use of a
Bunsen burner.

4. While the water is reaching the desired temperature, get potato and
bore out a cylinder with cork borer. To do this, place potato on tile
and stabilise by gripping potato by the sides with one hand. Then,
press down firmly with cork borer. To retrieve potato cylinder from
within cork borer, push it out with the flat end of a pencil.

5. Cut the cylinder of potato obtained, with a ruler so that it is
three centimetres long. Then, cut this three centimetre long cylinder
up into a further six equal pieces, each 5 millimetres wide. Make sure
to keep eyes level with ruler so as to minimise the chance of parallax
error. The diameter of the cylinder remains constant due to the use of
the cork borer.

6. Put the potato pieces into a boiling tube and put aside.

7. Measure out 30ml of hydrogen peroxide, using a 50cm3-measuring
cylinder. Pour this into a boiling tube.

8. Get the two boiling tubes containing one containing hydrogen
peroxide and the other potato. Put them both into the beaker of water
once the water has reached the desired temperature (this can be done
by heating or adding ice to water). After this, wait until the
temperature of the hydrogen peroxide and the water bath are equal.

9. Once the temperatures of the potato and hydrogen peroxide are
equal, clamp the boiling tube containing the hydrogen peroxide to a
clamp stand. Then add the potato into the test tube

Arefin Khan

containing the hydrogen peroxide and put bung on top of the test tube,
which now contains both potato and hydrogen peroxide, and make sure it
is in the beaker of water.

10. Now, measure the water being displaced from the 100cm3 measuring
cylinder every thirty seconds using a stop watch.

11. Take readings every thirty seconds for five minutes.

12. Repeat the above steps for different temperatures.

Risk assessment:

During this investigation, I will use catalase from potato cells to
speed up the breakdown of hydrogen peroxide. Hydrogen peroxide is an
oxidising agent, as oxygen will be given off during the reaction. This
implies that it could help the burning of fires. Due to this,
reasonable care must be taken to ensure that the oxygen is not
directly exposed to the flame of the Bunsen burner while water is
being heated. As well as this, hydrogen peroxide can be very harmful
if it enters the eyes. Thus, goggles must be worn. Also, if available
lab coats will also be worn.

The cork borer is also a potential hazard as it is quite sharp. When
cutting out the piece of potato, it must be ensured that the potato is
resting on the tile before it is cut. The methodology explains how it
will be cut.

The blade which will be used to cut the potato, is also another
hazard. It must be handled with appropriate care as it is sharp.

Glass apparatus must be handled with care so that they do not break.
If they do, that is also another hazard, and should be cleared up and
disposed of in an appropriate fashion.

Fair testing:

While conducting the experiment, it has to be taken into account that
a few variables will have to be controlled so as to ensure that the
test is fair.

1. The PH of the reactants should be kept constant. This will not be
hard to ensure as the PH of the reactants does not vary significantly
during the course of the reaction.

2. The temperature of the reactants will have to be kept constant
during the reaction. This is a variable which will be harder to
control. There will be used, a beaker of 150cm3 of water at the
appropriate temperature into which will be immersed, the boiling tube
containing the reactants once their temperatures have equalised and
stabilised. This will ensure that the temperature of the reactants is
stable for longer as the larger volume of water in the beaker will
maintain its temperature for longer. Also, the same volume of water
will be added to the beaker each time a new test is to be done, so as
not have any differences in how long the water maintains its heat for.

3. It must be ensured that when taking readings off the measuring
cylinders and thermometers etc, the readings are taken with the
graduated markings at eye level, so as to minimise the risk of
inaccurate readings.

4. All boiling tubes etc. must be washed thoroughly with water after
their use, so as to minimise the chance of contamination.

Arefin Khan

5. Making sure that all the potato pieces are cut to the same width,
and have the same diameter will control the surface area of the
potato.

While conducting the experiment, it must be ensured that the utmost
attention is paid to accuracy due to the fact that accurate
measurements will result in accurate results. Subsequently, the
evidence for or against my prediction regarding the enzyme activity in
relation to temperature will be more strong, and, more reliable. The
above mentioned methods of accurate measurement will be used as they
are most appropriate for the situation, with time factors being one of
the major reasons for the augmentation of such methods, as well as the
availability of equipment.

During the experiment, the following apparatus will be used:

Cork borer (size four)

Potato

Hydrogen peroxide

Water

Blade

Ruler (15cm)

Stop watch

Test tube rack

Boiling tubes

Delivery tube and rubber bung

Water container

Measuring cylinders (100 cm3 and 50cm3 )

Goggles

Cutting tile

Clamp and stand

Beaker (250cm3)

Graduated pipette (5ml)

ice

Thermometer (0 ºC-100 ºC)

Arefin Khan

During the experiment, the apparatus will be set up in the way shown
below:

[IMAGE]

During preliminary work, I found that the 100 cm3 measuring cylinder
was ideal for the measurement of water being displaced by oxygen
formed during the reaction. This is because it is graduated in
millilitres. Thus, it will be possible to measure the displacement of
water to the nearest 0.5 of a millilitre. Also, the measuring cylinder
is not so small that the volume of oxygen produced will be greater
than the cylinder can hold within the course of the reaction.

The 50cm3 measuring cylinder used to measure the volume of hydrogen
peroxide was ideal as the amount of hydrogen peroxide was wasn't too
little that it would be inappropriate and also inaccurate to measure
with a 50cm3 measuring cylinder, nor was it so large, that it would
just about be measured.

Boiling tubes are ideal for the reaction to take place in, as the
volume of oxygen produced is quite small. Thus, it will be quicker for
the oxygen produced to be able to displace the water in the measuring
cylinder. With a conical flask, it would take much longer.

I also found that the breakdown of hydrogen peroxide at room
temperature is very slow (Without a catalyst). No oxygen was given off
at all over the period of time I observed the hydrogen peroxide for
any reaction. However, I did not test whether this was true for higher
temperatures. If it was not, then there is the likelihood of major
inaccuracies in the conducting of the experiment.

Arefin Khan

Source:

Background information used during this investigation was obtained
from Cambridge Advanced Sciences. Biology 1 - endorsed by OCR: Chapter
3. (Page 42 onwards.)

Also, information on variables was taken from concepts learned in AS
chemistry: (Salters Horners Advanced Chemistry)

Arefin Khan

Results:

A Table to show the Volume of Oxygen Produced due to the Catabolic
breakdown of Hydrogen Peroxide in Relation to Temperature:

Key:

Time Volume of oxygen produced Temperature

(Seconds) (Cm3) (ºC)

10

20

30

40

50

60

70

0

0

0

0

0

0

0

0

30

0

1

2

3

2

1

0.5

60

0.5

2

2.5

3.5

2.5

1.5

0.5

90

1

3

4

4

3

2

1

120

1.5

4.5

5.5

5

4.5

2

1

150

2

5.5

6

7

5

2

1

180

2.5

6.5

7

9

5

2

1

210

3

7

8

10

5

2

1

240

3.5

7

10

11

5

2.5

1

270

4

8

11

12

5

3

1

300

4

8.5

12

13.5

6

3

1

A Table to show the Volume of Oxygen Produced due to the Catabolic
breakdown of Hydrogen Peroxide in Relation to Temperature (repeat
tests):

Key:

Time Volume of oxygen produced Temperature

(Seconds) (Cm3) (ºC)

10

20

30

40

50

60

70

0

0

0

0

0

0

0

0

30

0.5

1

1

2

1

0.5

0

60

0.5

2

3

3.5

2

1

0

90

1

3

3.5

5

2.5

1.5

0.5

120

2

3

5

5

3

2

0.5

150

2

4.5

6

7

3

2.5

0.5

180

3

5.5

7

7.5

3.5

2.5

0.5

210

3

6

8

9

4

2.5

0.5

240

3.5

7

9

11

4

3

0.5

270

4

7.5

11

12

4.5

3

0.5

300

4.5

10

12

13

4.5

3.5

1

Arefin Khan

From the graphs, the gradients were taken for the graph of oxygen
produced at each temperature. This gave the rate of reaction for the
breakdown of hydrogen peroxide, at the different temperatures the
experiment was carried out at, in volume of oxygen produced in cm per
second. The rate of reaction was then converted to the volume of
oxygen produced in minutes, by multiplying each gradient by sixty. The
two sets of gradients obtained for the graph of each temperature (one
for the initial experiment, and the other set of gradients for the
graph drawn from the repeat tests) were matched according to
temperature at which the hydrogen peroxide was catabolically broken
down at, and their average was taken for a more true picture of what
the rate of reaction really is at the different temperatures.

A table Showing the Rates of Reaction for the Initial Tests, the
Repeat Test, and also the Average Rate of Reaction

Temperature (ºC)

Rate of reaction for the results obtained in initial test (cm3/min)

Rate of reaction for the results obtained in repeat test (cm3/min)

The average rate of reaction (cm3/min)

10

5.30/270 = 1.06

5.20/294.5 = 1.04

(1.06+1.04)/2 = 1.05

20

9.90/300 = 1.98

9.95/300 = 1.99

(1.98+1.99)/2 = 1.99

30

13.1/300 = 2.62

12.65/300 = 2.53

(2.62+2.53)/2 = 2.58

40

13.4/300 = 2.68

13.5/300 = 2.7

(2.68+2.70)/2 = 2.69

50

5.50/300 = 1.1

4.85/300 = 0.97

(1.10+0.97)/2 = 1.04

60

2.85/300 = 0.57

2.35/300 = 0.47

(0.47+0.57)/2 = 0.52

70

0.70/300 = 0.14

0.6/300 = 0.12

(0.14+0.12)/2 = 0.13

Arefin Khan

Conclusion: The results obtained during the course of the experiment
seem to be quite conclusive. It possible to identify a pattern or
trend in the results obtained. From the rate of reaction graph, we can
see that the oxygen is produced more and more quickly via the
breakdown of hydrogen peroxide (when the reaction is catabolic) with
an increase in temperature. This is, however, only up to a certain
point. We see that the rate of reaction keeps on increasing until
40ºC, after which it starts to fall. There is still some oxygen being
produced after 70ºC, but only very little, almost zero.

With respect to the results obtained, I can now say that the
prediction I made earlier on was more or less correct, although not as
correct as I had hoped they would be, as I had predicted that the rate
of reaction would be zero after 60ºC, and this is more true of enzyme
activity at 70ºC. However, the rest of my prediction seems to be in
support of my hypothesis. Thus, it would seem that the enzyme activity
(in other words, the rate of reaction) increases with temperature up
until around 40ºC as the enzyme and substrate molecules gain more and
more kinetic energy. As a result, the reactants move around with
increased vigour. This results in there being an increased number of
effective collisions. Subsequently, the rate of reaction increases.
After 40ºC, the rate of reaction deteriorates. Although the kinetic
energy increases, and in essence, the rate of reaction should keep on
increasing, this is however not true. This due to the fact that after
40ºC, the optimum temperature for catalase enzyme activity, the weak
bonds that hold together the enzyme structure, start to break (this is
especially true of hydrogen bonds), due to the increased kinetic
energy. Resultantly, the rate of reaction deteriorates as the enzyme
becomes denatured due to the fact that its active site, and ultimately
its whole structure, is lost due to the breaking up of the bonds that
hold it together. This means that the substrate molecule can no longer
fit into the active site of the enzyme as the shape of the active site
changes.

Evaluation: Although the results obtained from the experiment were
more or less support in my prediction, I was not totally satisfied
with the experiment. There were a lot of errors, both in the
conducting of the experiment, and in the results obtained.

First of all the way I conducted the experiment was quite flawed. For
example, the measuring cylinder used to measure the volume of oxygen
produced, was quite inappropriate, and probably resulted in many of
the readings being taken being quite inaccurate as it is easy to make
a mistake in reading off the value while trying to hold the measuring
cylinder straight, and also trying to keep the bottom of the meniscus
of the water at eye level, all at the same time. Also, it was only
possible to measure to the nearest 0.5 of a centimetre cubed, as the
measuring cylinder was only graduated in 1cm3

The method used to keep the temperature of the reactants constant at
certain temperatures was also improper, as water in the beaker which
was used for the above purpose, was not in a large enough volume or
quantity to retain its heat for more than a minute or two. Thus, the
temperature of the reactants was fluctuating during the experiment.
Also, the water in the beaker did not cover all the reactants in the
boiling tube, and some of the reactants were not immersed by water.
Thus, there were regions of unequal temperatures in the reactants.

The controlling of the surface area of the potato was also inaccurate
as it was impossible to measure the lengths of potato to exactly 5mm
throughout.

Also, it was assumed that the PH of the reactants would remain
constant throughout. This may not have been the case

Considering the above, it is feasible to say that the results obtained
during the experiment are neither likely to be very reliable nor very
accurate.

Considering that the there was so much possibility for inaccuracy,
there were not however, any major anomalies in the results obtained,
although the results obtained in the repeats at

Arefin Khan

20ºC and 50ºC, were on the doubtable side, as there was quite a
difference when the volumes of oxygen obtained were compared with
those obtained during the first test:

Volume of oxygen produced (in cm3) at:

20ºC 50ºC

1st test: 8.5 6

2nd test: 10.0 4.5

These slight anomalies may have arisen may have arisen due to a number
of reasons:

a) Improper measurement off measuring cylinder

b) Improper surface area of potato, and effectively enzyme

c) Difference in temperature due to loss of heat (kinetic energy)

d) PH level may have altered

If I was to conduct the experiment again, I would make sure that it
was more accurate overall. This would ensure that the results obtained
were more reliable and accurate. I would do this in the following
ways:

1) I would use a graduated syringe, instead of a measuring cylinder as
this would ensure that it was easier to measure the volume of oxygen
produced. It would also be more accurate as there would be a clear
line to mark the amount of oxygen produced and there would not be a
need to observe where thw meniscus of water touched on the graduation
marks.

2) I would use a greater volume of water to ensure that the
temperature of the reactants remained constant, due to the fact that
water is such that, the greater the volume, the greater its ability to
retain heat.

3) I would use a buffer to control the PH of the reactants as it would
ensure that the Ph remained constant.

4) I would crush the potato to a paste, using a mortar ant pestle, and
drain the filtrate for use as the catalase sample. A measure of the
volume would be made.In this way, the exact amounts of enzyme could be
calculated. If not, then I would use yeast as my enzyme, as it can be
measured out to very precise quantities.

5) I would also use a graduated syringe to measure out the amounts of
hydrogen peroxide, and other fluids. The reasons for this are the same
as the ones mentioned above.

With the above taken into account, I would say that my conclusion is
in fact, not very safe due to the fact that there were too many errors
and uncertainties concerning the results obtained. Subsequently, the
results do not provide a very stable evidence for support of my
hypothesis. Although the percentage error of individual equipment may
have been at first glance, small, they add up to large percentage
errors which then render useless, the legitimacy of the results
obtained.

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"The Effect of Temperature on Catalase Enzyme." 123HelpMe.com. 20 Apr 2014
    <http://www.123HelpMe.com/view.asp?id=120339>.




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