The Effect of pH on the Activity of Catalase

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The Effect of pH on the Activity of Catalase


Secondary resources: from my research I carried out, I have learnt
that:

Catalyse is an enzyme that is found in foods such as potato and liver.
Catalyse speeds up the decomposition of hydrogen peroxide into water
because the shape of the hydrogen peroxide molecule. This type of
where a molecule is broken down into smaller pieces is called an
anabolic reaction. This enzyme catalysis is the breakdown of hydrogen
peroxide and it forms oxygen gas and water.

Enzymes are very large and complex organic molecules that are
synthesized by the cell to perform very specific functions. These
biological catalysts are important because they speed up the rate of
the reaction they catalyze that would otherwise be too slow to support
life. Catalyse is an enzyme present in the cells of plants, animals
and aerobic bacteria. It promotes the conversion of hydrogen peroxide,
a powerful and potentially harmful oxidizing agent, to water and
molecular oxygen.

Catalase is located in a cell organelle called the peroxisome.
Peroxisomes in animal cells are involved in the oxidation of fatty
acids, and the synthesis of cholesterol and bile acids. Hydrogen
peroxide is a by-product of fatty acid oxidation. White blood cells
produce hydrogen peroxide to kill bacteria. In both cases catalase
prevents the hydrogen peroxide from harming the cell
itself.Peroxisomes in plant cells are involved in photorespiration and
symbiotic nitrogen fixation (the breaking apart of the nitrogen
molecule N2 to reactive nitrogen atoms). Hydrogen peroxide is produced
as an intermediate during these chemical processes and must be removed
to prevent damage to cellular machinery. Aerobic (oxygen requiring)
bacteria produce hydrogen peroxide as a by-product of metabolism. This
fact is used when identifying bacteria. If hydrogen peroxide is added
to a bacterial colony and bubbles are produced, this is evidence of
oxygen production and confirms that the colony is aerobic.

The activity of catalase can be measured by finding the rate at which
the oxygen gas is released from the decomposition of hydrogen

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peroxide.

The buffer solutions that I am using are pH - 4.4, 5.2, 6.5, 7.5 and
8.4.

Hydrogen peroxide = 2H2O2

Hydrogen peroxide + catalase -> oxygen + water

The books and resources I used were:

Energy and chemistry - p229

Gareth Williams, biology for you pages 29-31

www.google.com- enzymes, catalase, hydrogen peroxide

OCR staged assessment phase 1 module by Mary Jones

Pre-test:

* In the pre-test I done it by putting my pH and hydrogen peroxide
mixed solution in the syringe

* Then put the potato in the conical flask

* Put the bung on

* Put water in the measuring cylinder and put it upside down without
the water coming out

* I then put the delivery tube in the measuring cylinder

* I got the stop watch

* Started the time when I pushed the mixed solution in the conical
flask with the potato

* Stopped the time when the gas was collected 10cm3

* Recorded my result in a table

* Repeat that experiment another 2 more time

* Do the other pH solutions

Weight of the Potato (g)

Volume of pH

Trial 1 - time taken for the gas to reach 10cm3 (s)

Trial 2

Trial 3

Average

5

4.4

32.73

35.04

33.96

79.09

5

6.5

48.11

29.84

36.45

90.1

5

8.4

25.8

18.87

18.85

21.17

The second row in my table has got the anomalous result. It can't have
been 90.1 because that is too much to be the average set of results.
This happened because I let some gas out as I was holding the
cylinder, I didn't realise the tube came out and then I thought why is
it taking so long. In my pre-test I had some mistakes, they were
firstly I didn't use hydrogen peroxide with my pH, so I had to do the
whole experiment again. The second mistake was, the gas I was
measuring I wasn't too sure if I was doing 20 or 15 so then I decided
to do 10.

[IMAGE]

My graph that I have made from the results of my pre-test isn't
exactly right because looking at my results it doesn't seem right.
This happened because may be some of the gas escaped from the side of
the cylinder, which is why it took time for my gas to be collected.

Introduction:

Enzymes are biological catalysts; this basically means that they speed
up the chemical reactions in living things. Without enzymes, our guts
would take at least weeks and weeks to digest out food, our muscles,
nerves and bones would not work properly and so on, so we would not be
living.

A catalyst is any substance, which makes a chemical reaction go
faster, without is self being changed. A catalyst can be used over and
over again in a chemical reaction but I wont get used up. Enzymes are
very much the same except that they are easily denatured by heat. Our
enzymes work best at body temperature. Our enzymes also have to have
the correct pH.

All enzymes are made of protein, this is why they are sensitive to
heat, pH and heavy metal ions. Unlike ordinary catalysts, they are
specific to one chemical reaction, but an enzyme only works for one
specific reaction.

Enzymes must have the correct shape to do their job. They are made of
proteins, and are very easily affected by heat, pH and heavy metal
ions. Most people say an enzyme works like a key in a lock. If the key
has been twisted by heat, or dissolved in acid or stuck up with
chewing gum it will not work. Enzymes change their shape if the
temperature of pH changes.

Aim:

The aim of my experiment is to see the effect of pH on the activity of
catalase

Apparatus: I have decided to use the following equipment in order to
carry out my experiment:

* 1 Stopwatch

* 1 Beehive shelf

* 1 Trough

* 1 50ml Measuring cylinder

* 3 beakers 100mm

* 1 potato

* Hydrogen peroxide (H2O2)

* pH solutions

* Conical flask with bung

* Goggles

* Measuring scale

* 1 20ml syringes

* Delivery tube

* Petri dish

Method:

* 1st I got all of my apparatus

* Then grated my potato

* Get the hydrogen peroxide in a beaker

* Get the pH solution in a beaker

* Measure both of them 10ml in the syringe and put them in the other
beaker

* Get a trough with half filled with water

* Put the measuring cylinder in the trough and fill it with water
and turn it up side down in the water

* Put the delivery tube in the cylinder and hold the cylinder from
the top so that the cylinder doesn't tip over

* Measure the potato 5g in the Petri dish

* Once I have measured the potato I put that in the conical flask

* Get the mixed solution of hydrogen peroxide and pH solution they
should both add up to 20ml in the beaker

* Make sure the other end of the tube is blocked with the syringe

* Get the stopwatch

* Put the solution of the hydrogen peroxide and pH in the flask and
put the bung on

* As soon as you put the bung on start the watch

* I timed it until the gas reached 10cm3

* I recorded the time in a table

* I repeated this 3 more times so I got a set of results

* I then done it with the different types of pH

Fair test:

To make it a fair test I will need to have not let any gas escape, if
I do my test or time will take longer which won't give me accurate
results. I have to make sure that when I am measuring the pH solutions
and the hydrogen peroxide I need to ensure that I am using the same
amount for all of the experiments that I do for my actual experiment
and the pre-test. I need to also make certain that the amount of
potato that I am going to use, when I measure it, I need to use the
same amount every time, which were 5g.

I also need to make sure that when the oxygen has reached 10cm3, I
need to stop the watch straight away and record the time. I am going
to keep all of these things constant. I also require that I should
need to check that I am using the same equipment through out the whole
experiment.

The only thing I am changing is going to be the buffer solutions,
every time I do the 1 pH I made sure I repeated it a couple more time
preferably 3 times until I got a set of results that are not more than
2 seconds more than my 1st result.

If all of these factors are controlled it will make my test a fair one
and also lead me to gain a reliable results.

Safety:

To ensure that I carry out a safe experiment, I will be careful when
using the hydrogen peroxide because it tends to bleach clothes and
damage the skin, I will also be careful with hydrogen peroxide by
always closing the lid because the hydrogen peroxide can decompose if
it is not bottled in. I will wear goggles so that my eyes are
protected from the substrate. I will also wear protective footwear to
protect my feet from any spills.

Prediction:

I predict that as soon as I pour the pH solution and hydrogen
peroxide, I will have oxygen gas formed in my measuring cylinder. This
happens because the potato and the solution react fast to give me my
products. I also predict that the pH 4.4 would be less reactive than
the other pH's. I think this because as the pH value gets lower the
reaction time will go faster and the rate of reaction will increase so
when it gets lower from 7 to 6.5 and 4.4 the reaction time will
increase. This is because the collision theory says that chemical
reactions occur when particles of the reactants collide. They must
collide with a certain minimum energy, called the activation energy.

An enzyme is a protein molecule that speeds up chemical reactions in
all living things. Without enzymes, these reactions would occur too
slowly or not at all, and no life would be possible. All living cells
make enzymes, but enzymes are not alive. Enzyme molecules function by
altering other molecules. Enzymes combine with the altered molecules
to form a complex molecular structure in which chemical reactions take
place. The enzyme, which remains unchanged, then separates from the
product of the reaction. Therefore, an enzyme is a type of biological
catalyst. Those enzymes identified now number more than 700.

Enzymes are classified into several broad categories, such as
hydrolytic, oxidising, and reducing, depending on the type of reaction
they control. Hydrolytic enzymes accelerate reactions in which a
substance is broken down into simpler compounds through reaction with
water molecules. Oxidising enzymes, known as oxidises, accelerate
oxidation reactions; reducing enzymes speed up reduction reactions, in
which oxygen is removed.

Catalase is present in the peroxisomes (micro body organelles that
house various oxidation reactions in which toxic peroxides are
generated as side products) of nearly all-aerobic cells. It serves to
protect the cell from the toxic effects of hydrogen peroxide by
catalysing its decomposition into molecular oxygen and water without
the production of free radicals (An atom or a group of atoms with an
unpaired electron. Radicals are unusually reactive and are capable of
causing a wide range of biological damage).

The lower the concentration the less gas particles, the higher the
concentration the more gas particles collide.

From my research I should see my graph the same as the one that I have
from my results table. If I don't, then there is probably something
wrong with the pH or my results table wasn't as accurate.

Diagram of experiment:

Observation-results:

Potato (g)

Volume of pH

Trial 1 - time taken for the gas to reach 10cm3 (s)

Time taken for the gas to reach 10cm3 (s) Trial 2

Time taken for the gas to reach 10cm3 (s) Trial 3

Average

5

4.4

16.93

18.85

15.82

17.2

5

5.2

14.63

16.89

15.67

15.73

5

6.5

10.5

12.28

14.66

12.48

5

7.5

9.45

10.62

8.86

9.64

5

8.4

6.45

8.92

7.25

7.54

Conclusion:

From my results I can see that the experiment I carried out and my
prediction was correct because I predicted that pH 4.4 would be the
less reactive one as it took more time for the oxygen to be collected
10cm3. So pH 7.5 was the optimum pH at which the enzymes works best.
As the pH increases or decreases, the enzyme becomes less effective.

The higher the enzyme concentration was, the more oxygen was produced,
or in the case of my experiment, more bubbles were produced. This is
because the higher the enzyme concentration, the more active sites
there are where a collision between an enzyme and the substrate can
take place. As you can see in the graph, the amount of bubbles
increases as the amount of enzyme concentration decreases. This
indicates that it was reaching the optimum state of catalyse activity.

These set of results tell me that the best pH that effects the
catalyse to collect the oxygen using the hydrogen peroxide is 8.4
because it has the less amount of time in seconds to collect the
oxygen and it reacts really fast.

[IMAGE]Graph:

My graph looks good because the time is going from high to low. This
is because the pH 4.4 is the least reactive, as I done my experiment
with the catalyse.

According to the graph, there are no visible outstanding results.
However, if I had done the experiment more times and got a more
precise average, then there may have been a few outstanding results.
(By outstanding results I mean results that do not fit in with the
other results pre-test graph, for example they may be too high).
According to the graph the optimum pH, which is the most reactive, is
8.4 because it took the less time for it to react with the catalyse.

Evaluation:

The data that I collected was reasonable for the accuracy of my
experiment, it was fairly reliable meaning that the results we
collected were mostly expected but again with human error and
contamination there were a few different results in my pre-test, which
stood out quite a lot.

A major cause for concern on accuracy was the reading of the overall
measurement of the oxygen produced but also the measurements of
hydrogen peroxide and the pH used for the experiment. So if these
miss-readings were carried out through the whole investigation then my
results will be quite a long way out.

Looking back on experiment I believe that there are many ways to
improve it. One way is the accuracy of the measuring of the potato
this can be done by measuring accurately on the scale because it did
have 2 more decimal numbers which could have been just five.

Measuring the oxygen produced is a little harder, to measure this it
may include equipment that is not available to me and so cannot be
carried out. All that is best is that the same person reads it off
just in case someone else's vision is not as good. Also it was quiet
harder because I wasn't using a beehive shelf and the measuring
cylinder was a bit tilted because obviously I had to see and reading
and to collect the gas I needed the delivery tube to be there as well.

My graph is very basic and one way to improve this will be to increase
my range off tests. This will give me more results and so when I plot
my graph again there will be more points producing a greater accuracy
and a better curve with which I will be able to pull stronger
conclusions from.

I think the timing was as accurate and reliable as it could be, but
only a second or third experiment would back that up fully. The
equipment I used was reasonable. I think contamination was minor so
that did not cause a problem. One problem with our first experiments
was that I had to change the Hydrogen peroxide because I felt that it
was old, and was probably not as concentrated. This could of seriously
effected my main experiment with using one chemical but out of several
bottles, to eliminate this I must get one bottle big enough for the
whole experiment and use that one only; so that there is no change in
the quality of the H2O2.

My experiment has gone quite well, but I could do certain things in
order to get more accurate results. Firstly, if I had time, I would
certainly do the experiment more times, as this would give me a more
precise average.


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