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Investigating the Effect of pH on the Activity of Catalase

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Investigating the Effect of pH on the Activity of Catalase

Planning:

Our aim is to observe what effect varying the pH would have on the
rate of reaction involving the enzyme catalase.

We have chosen to vary the pH of the solution, however there are a
number of different variables which need to be controlled, these
include temperature, enzyme concentration and the concentration of the
substrate. The temperature needs to be constant because if the
temperature increases of decreases it changes the amount of kinetic
energy the enzyme and substrate have and therefore will affect the
rate of reaction. When the temperature increases the enzyme will have
more kinetic energy so will be able to collide more often with a
substrate and over come the activation energy needed and so combine
and produce a complex. If temperature is increased further the heat
energy causes the molecules to vibrate because of the kinetic energy
from the heat, this will cause bonds to break particularly hydrogen
bonds as they are not very strongest bonds. This will therefore change
the globular protein shape, and so the active site, therefore no
substrate will fit into the site and the enzyme is no longer useful
and is called denatured. Another variable is the enzyme
concentration. The rate of a reaction will increase as the enzyme
concentration increases, this is because as the concentration
increases there is a great change of a substrate and enzyme colliding
and producing a complex. Therefore the rate of an enzyme-catalysed
reaction is directly proportional to the concentration of enzyme, as
long as there is excess substrate. The other variable needed to be
controlled is the concentration of the substrate. As the concentration
of the substrate solution increases the rate of reaction also
increases, this is because the increased concentration increases the
change of a successful collision between enzyme and substrate. However
the rate of reaction will start decreasing and level off as the
reaction is limited by another factor, the enzyme concentration.

Catalase is an enzyme found in almost all living tissue, because it
catalyses a added product of aerobic respiration, hydrogen peroxide to
produce water and oxygen. Hydrogen peroxide is a toxic substance and
catalase removes this product by catalysing it. I predict that as the
pH increases so will the activity of the enzyme increase until it
reaches its optimum pH, after this it will decrease. The graph will
have a bell jar shape to it, as enzymes pH ranges vary and some may
have very wide range of pH preferences. This is because the pH ionises
some of the amino acids in the side chains of the protein globular
shape. As the pH changes the ionisation of its constituents amino
acids changes and the ionic bonds, which help to stabilise, are
broken. As a result, the protein changes shape and therefore the
active site will also have changed shape and is denatured.

The activity of this catalase can be measured by the rate of oxygen
bubbles released from the reaction. We all decided to use minced
potato as a source of catalase however; you could find catalase almost
anywhere. To vary the pH we used a buffer solution using, citric acid
and sodium phosphate. We decided to use pH values of 4.4, 5.2, 6.5 and
7.5 because they show a variety of pH’s and so we could get a range of
results.

Apparatus:

* Citric acid-sodium phosphate buffer solutions. Theses were made up
by as the following. 21g citric acid H2O dm-3 and 28.4g anhydrous
disodium phosphate Na2HPO4 dm-3. The table below it shows the
composition of citric acid-sodium phosphate buffer solution at
various pHs.

pH

Citric acid/cm3

Sodium Phosphate/cm3

4.4

27.9

22.1

5.2

23.2

26.8

6.5

14.5

35.5

7.5

3.9

46.1

* Hydrogen peroxide solution, 20cm3

* Minced potato, 3cm3

* 5cm3 plastic syringe with end cut off to measure minced potato

* graduated pipette or syringe to measure buffer solutions

* conical flask, where reaction occurs

* bung, to seal reaction from escaping oxygen

* measuring cylinder to measure volume of oxygen bubbles released

* stop watch

Method:

The pH I was assigned to was 6.5. Firstly I made the buffer solution
which I measured out 14.5/cm3 of citric acid and 35.5/cm3 of sodium
phosphate using a measuring cylinder, which I then put into the
conical flask. I added the 3cm3 minced potato into the flask with the
already made buffer solution. I then swirled the solution gently to
ensure that they mixed. Then I set up my apparatus and filled the
measuring cylinder and beaker with water, I also ensured that tube
from my solution to the measuring cylinder was air tight. When
everything was ready and fully set up I measured out 5cm3 of hydrogen
peroxide into a syringe and injected it into the conical flask
containing the potato and buffer solution. I then immediately replaced
the bung to prevent as little as possible oxygen escaping, I recorded
the volume of oxygen bubbles every 30seconds for 5minutes in a results
table.

To ensure fair testing and no cross contamination I washed all my
apparatus first with distilled water and then the solution that was
going to be used in it.

Risk assessment:

The risks involved in this experiment were citric acid which is an
irritant as it causes irritation to your skin, and could damage your
eyes if it came into contact. Hydrogen peroxide is a corrosive
solution and very oxidising so therefore great care needs to be taken
when using these solutions. To prevent damage to skin or eyes wear
goggles and a lab coat, and washing hands afterwards.

Observation:

Time (min)

Volume of O2 cm3

pH 4.4

pH 5.2

pH 6.5

pH 7.5

0.5

16.0

45.0

6.0

30.0

1.0

16.0

45.0

7.0

28.0

1.5

16.0

45.0

8.0

26.0

2.0

16.0

45.0

8.0

24.0

2.5

16.0

44.0

9.0

22.0

3.0

16.0

45.0

10.0

21.0

3.5

16.0

45.0

11.0

20.0

4.0

17.0

43.0

11.0

20.0

4.5

18.0

42.5

12.0

19.0

5.0

18.0

42.5

12.0

18.0

Total oxygen evolved

2.0

2.5

7.0

13.0

Average Results:

pH

O2 volume in 5min (cm3)

Rate / min

4.4

2.0

0.4

5.2

2.5

0.5

6.5

7.0

1.4

7.5

11.0

2.2

I decided to plot rate / min because then I get the rate of reaction
per minute. However in this experiment plotting the oxygen evolved
will be similar in shape of graph, so therefore I will plot both these
lines on one graph which enables me to compare the two.

Analysis:

As the pH increases so does the rate of reaction, this is because as
the concentration of hydrogen ions increase the amount of amino acids
side chains which are ionised which also increase, therefore more
enzymes will be at their optimum pH and have their active site
specific for the substrate. As the hydrogen ions form hydrogen bonds
with the amino acids it changes the shape of the globular protein
because it causes the ionic bonds which hold the structure together to
break. This ensures that each enzyme has its own optimum pH because
each enzyme will “look” differently in different pH’s. At the pH of
7.5 the rate of reaction was at the highest, which suggests that
catalase prefers a higher pH. If we had continued we might have seen a
decrease in the rate of reaction of we could have found out if 7.5 was
the actual optimum pH, therefore this shows that because we only have
4 points on our graph that it is not very accurate and because we only
have one decimal place it is not very precise.

Evaluation:

Our collective results are somewhat accurate but not as accurate as it
could possibly be because of several limitations either by the method
used or because of human fault. Other problems might have been caused
by

* Cross contamination could occur very easily as we used equipment
which has been used for many different experiments and solutions
this could have contaminated our solutions.

* The reading taken every 30 seconds. It is very challenging to take
a reading at the 30 second especially if the rate of reactions was
constant.

* Measurements where hard to accurately measure, for example
accurately measuring 3cm3 minced potato, and also everyone was
using a different size surface area for the potato.

* It was a quantitative experiment which meant it was more accurate
than a qualitative experiment but was still left for human
judgement.

From the above you can see that our results were of a tentative nature
because even though we tried to be as accurate as possible by
controlling all the other variables, the things listed above still
made our experiment very inaccurate.

If I was to do the experiment again I would controlling or overcoming
more of the problems listed above, like used a more accurate means of
measuring the volume of oxygen evolved, I could use an airtight glass
syringe the oxygen would then move the syringe as more was produced. I
would also like to have a more varied range of pH’s especially where
the rate of reaction increases dramatically towards the optimum
temperature.

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"Investigating the Effect of pH on the Activity of Catalase." 123HelpMe.com. 23 Apr 2014
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