Activity of the Enzyme Catalase with Hydrogen Peroxide

Activity of the Enzyme Catalase with Hydrogen Peroxide

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Activity of the Enzyme Catalase with Hydrogen Peroxide

Background Information:

An enzyme (Catalase) is a biological catalyst. It can modify the rate
of reaction without being changed itself. Enzymes are also globular
proteins that have a three-dimensional specific shape; among with a
pocket known as the active site, this has a precise shape to link with
the exact substrate. Every enzyme are capable of converting one kind
of substrate molecule into one kind of product molecule, which forms
the enzyme-substrate complex. There are the minority factors that
affect the rate of reaction that include:

1. Temperature:

If the temperature was to increase so would the rate of reaction up to
a decisive point, as the enzyme will start to denature; this usually
occurs at temperatures around 50 to 60 Celsius. The rate of reaction
increases because enzymes and substrate have more energy permitting
them to move around more rapidly, resulting with more collisions and
ensuring the enzyme-substrate complex. When the enzymes denature, the
weak bonds that hold the tertiary structure of the enzyme together
vibrate at an excessive rate, due to the kinetic energy, that they
break as well as altering the shape of the active site to a degree
that the substrate can no longer link to it.

2. PH:

The formation of the enzyme-substrate complex relies on an exact
complementary shape and charge. If there is a change in pH, it will
corrupt the charges so that no enzyme-substrate complex can be formed.
All enzymes have an optimum pH depending on where they are based,
intracellular or extracellular.

3. Substrate concentration:

The higher the concentration that faster the rate of reaction, which
is until they are working as fast as they possible can. This increase
in rate of reaction occurs because the more substrate there is the
more chance there is of collisions resulting in the formation of
enzyme-substrate complex. If the reaction is working fast as possible,
the limiting factor is no longer the substrate but the enzyme
concentration.

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4. Enzyme concentration:

This factor also enhances the rate of reaction because there are a
greater number of active sites accessible for the enzyme-substrate
complex to be formed.

5. Inhibition:

The inhibitor decreases the rate of reaction because it alters the
shape of the active site or either blocks the active site so that the
enzyme cannot form the enzyme-substrate complex.

Hydrogen peroxide is formed by a product of chemical reactions in the
living cells and is also toxic. If the cells do not deal with it
instantaneously it could kill the cells themselves. Catalase is a very
rapid reacting enzyme, which is formed in many living cells where it
breaks down hydrogen peroxide to water and produces oxygen as a
result. What's more is that one cell can deal with six million
molecules of hydrogen peroxide in one minute.

Planning

Subsequent to the previous information, I have analysed it and taken
it into consideration and I have come to the decision of conducting a
practical where I will compare the rate at which oxygen is developed
as a result of the breaking down of hydrogen peroxide, at different
substrate concentrations. I will use the enzyme catalase of potatoes
to see the affect that altering the substrate concentration has on the
rate of reaction or the rate at which oxygen is evolved.

I will now carry out a practical to investigate the effect of
substrate concentration on the rate of activity of the enzyme
catalase.

Below is a list of reasons why I have chosen to investigate substrate
concentration instead of the other factors that were mentioned:

· If I were to investigate the effect temperature has, I would find it
difficult to keep the temperature constant.

· I decided not to investigate pH because most enzymes only work on a
narrow range of pH and keeping these constant would be a struggle and
would result as inaccurate outcome.

Justification on the procedures selected:

For my practical I have chosen to investigate the use of potatoes as
my source of catalase. I will macerate the potato tissue in a
homogeniser to insure that I keep the surface area the same. I will
then take this material and weigh 2.0g of it using a weighing scale.

I will use 20-volume solution of hydrogen peroxide as my 100% at 20ml.
The reason being for this is that it allows me to take simple readings
at a 100%, 75%, 50%, 25% and 0% solution of hydrogen peroxide. I also
anticipate that these concentrations will give a clear indication of
the effect that substrate concentration has on the activity of an
enzyme. I've decided to perform for 0% because it will give a clear
indication if the practical is going according to plan. If the
practical is performing appropriately, the 0% solution should not give
off any gas. I also plan to use distilled water to make these
solutions complete. For example for 25% solution I will combine a
mixture of 15ml of hydrogen peroxide and 5ml of distilled water.

I will measure out 20ml of the substrate solution in a 50ml-measuring
cylinder and placed in 100ml conical flask including the potato
tissue. The reason I've chosen to use a 100ml conical flask is that it
will prevent any froth from emitting out.

I also want to prepare my diverse concentrations so that there is a
sufficient supply of each concentration for every experiment i.e. the
100% solution will need 80ml so that 20ml can be used for all four
experiments. I will also have supplementary hydrogen peroxide if for
any case I were accidentally to spill some.

Once the experiment has started I will obtain readings of the gas
produced every 20 seconds for 3 minutes using stopwatch. This time is
appropriate because it allows me to read the result on the syringe and
write them down.

I will obtain readings for the gas produced from the gas syringe, as
it is the most precise measurement of gas developing. I have chosen
not to use the method of conduct the gas developed from a large glass
pipe which is filled with water bubbled through a delivery tube
because I believe it is less accurate and is also more difficult to
utilize. The gas syringe allows me to acquire readings of the gas
produced up to 100cm3.

One requirement to the experiment is that I make sure it has been
conducted fairly, and I will do this by initially carrying out a trail
experiment and subsequently carrying out 3 repeats and lastly
calculating the averages. Furthermore, I will calculate the overall
gas developed at different concentrations from all four of the
practical since it allows me to create a respectable comparison.

To keep the pH constant, I will use a pH buffer tablet and this pH
will have an influence on the rate of reaction.

The experiment will be performing at room temperature, yet it is
difficult to maintain a constant temperature given that it is an
exothermic reaction. Keeping room temperature is a vital aspect while
the experiments in progress and this will be dealt by shutting all
windows and doors.

Once all four experiments have been completed, I again determine to
calculate averages and overall averages and as a consequence to
calculate the rate, at which oxygen is produced at different
concentrations, followed by a draw up of graphs.

Preparation of the concentrations:

When adding my potato tissue with different concentrations, I will
have to combine them in the following ways four times:

100% - 20ml Hydrogen Peroxide 0ml Distilled water

75% - 15ml Hydrogen Peroxide 5ml Distilled water

50% - 10ml Hydrogen Peroxide 10ml Distilled water

25% - 5ml Hydrogen Peroxide 15ml Distilled water

0% - 0ml Hydrogen Peroxide 20ml Distilled water

Equation:

[IMAGE][IMAGE]2H2O0 2H20+O2

[IMAGE]Hydrogen Peroxide (Substrate) Water + Oxygen (Products)

Variables:

Dependent variable- Oxygen Produced

Continuo's variable- Concentration of substrate

Fair testing:

Fair testing allows the experiment to be conducted moderately and
giving you accurate results, the following list is of all the fair
testing aspects that I will cover:

1. Making sure that I weigh 2.0g of potato tissue after it has been
homogenised accurately.

2. Macerate the potato tissue in a homogeniser to assure that the
surface area is kept comparable.

3. Making use of the buffer tablets so that the pH is kept constant.

4. Accurately measuring out 20ml of hydrogen peroxide and distilled
water, as well as pouring out all 100ml of the mixtures.

5. Drying the inside of the glass syringe is important, as it may be
moist and then closing the syringe entirely. Also the rubber pipe must
be all open and the rubber bung must be in reach inside 2 seconds, so
there is no excess of gas escaped.

6. Making sure that the stopwatch is on 0 seconds and using the
correct time and I will do this by asking someone to remind me when 20
seconds have elapsed.

7. Making sure that I or no one else shakes or shift the syringe as
well as the delivery tube at all times.

8. Clean out all flasks that the experiment will be repeated in and
dry them.

9. Take out any gas left in the gas syringe that had been produced.

Safety measures:

Safety precautions are a serious matter to the practical as a whole
and not carrying out any precautions cause danger to myself as well as
those around me. The following is a list of the procedures that I will
take out:

1. Wearing appropriate goggles, apron and safety gloves.

2. Work in centre of table with plenty of space.

3. Tie any loose ends such as shoelaces and keep bags and coats on the
hangers and not in the way.

4. Work standing up and with chairs under tables and not in the way.

5. Move any irrelevant objects away from the working area.

6. Not touch any equipment such as the gas syringe while in progress.

Apparatus:

1. 1x Retort stand, boss and clamp.

2. 1x Delivery tube.

3. 1x Rubber bung

4. 1x Stopwatch

5. 1x Weighing scale

6. 1x Homogeniser

7. 1x Tweezers

8. 1x Spatula

9. 1x 50ml measuring cylinder

10. 4x 100ml Conical flasks

11. 1x Pair of gloves

12. 1x Apron

13. 1x Goggles

14. 1x Pipette

15. 1x Plate

16. Hydrogen Peroxide

17. Distilled Water

18. Potatoes

Method:

1. Put all belongings on the hangers and put on safety goggles, apron
and gloves. Also clear table and put stools under the tables.

2. Gather all equipment and place it on one side of the table.

3. Set-up the retort stand along with the boss and clamp and firmly
fasten the gas syringe to it, and then attach the delivery tube and
rubber bung to the clamp.

4. Macerate the potato tissue in the homogeniser for around 10
seconds, allowing it to macerate properly.

5. Weigh the macerated potato at exactly 2.0g and place them
separately into a plate using the spatula.

6. Combine and set up the 100%, 75%, 50%, 25% and 0% of 20-vol
hydrogen peroxide and separate them all into a 100ml conical flask and
use labels for each solution.

7. Begin with pouring out the macerated potato into separate flasks
and pour 20ml of the 100 % solution inside the flask and firmly push
the rubber bung into the flask, within 2 seconds.

8. At this point I will have also started the stopwatch taking
readings each 15 seconds for 2 minutes.

9. A table will already be made so once 15 seconds have elapsed, I
will enter that reading for each concentration. I will also calculate
the averages for the concentrations.

10. Repeat steps 7 to 9 with the 75% solution.

11. Repeat steps 7 to 9 with the 50% solution.

12. Repeat steps 7 to 9 with the 25% solution.

13. Repeat steps 7 to 9 with the 0% solution.

14. Repeat steps 7 to 13 for the next 3 experiments.

15. Clean out all objects I use with water and put them away after
use.

Analyse of the experiment:

With the results drawn together, the following results show that as
the substrate concentration increases so too does the quantity of
oxygen gas produced, therefore the rate of reaction increase with the
substrate concentration.

By using the 0% solution it showed that my results gathered from the
practical were correct as it showed no progress in the development of
gas produced. This is a clear sign that the practical was carried out
correctly.

The graphs produced on computer also show that each test with the
substrate concentration increasing, so too does the rate of reaction.

The last graph shows the variation of all averages of the gas
developed at each substrate concentrations and yet again showing that
when oxygen increases so does the substrate concentration. The
significant aspects of the graphs are that at 100% concentration the
gas developed is approximately the double the amount that is developed
at 50%. Another implication is that the gas developed at 25%
concentration is roughly quarter of the gas developed at 100%.

The science of enzymes shows in my experiment that when the rate of
reaction increases as well as the substrate concentration, there is a
greater amount of substrate where there is more apparent amount of
collisions occurring between the enzymes and the substrate, which
results in the enzyme-substrate complex that products are formed from.
The gas developed, oxygen, is quicker when substrate is increased. The
motive of gas developed at 100% concentration is approximately double
the amount of gas produced at 50% concentration is that there is
double the amount of substrate molecules vacant for more collisions to
occur. The same applies to the reason to the 75% solution, which is
three-quarters of the gas produced at 100% and results in less
collisions and less gas produced.

Rate of reaction:

In order to calculate the rate of reaction at which oxygen is
developed at different substrate concentrations, I will make use of
the formula below:

Rate of reaction = Average overall gas produced

Time

The averages of overall gas produced are taken from the tables.

100% Solution - Rate of reaction = 48.32 = 24.16

2

75% Solution - Rate of reaction = 35.47 = 17.73

2

50% Solution - Rate of reaction = 30.03 = 15.01

2

25% Solution - Rate of reaction = 20.94 = 10.47

2

0% Solution - Rate of reaction = 0 = 0

2

The results above illustrate that the rate at which oxygen was
developed increases as the substrate concentration increases. This is
what I expected before I started the experiment and it also shows I
carried it out correctly.

[IMAGE]

[IMAGE]


Trail experiment Experiment 1

[IMAGE]


[IMAGE]


Experiment 2 Experiment 3

[IMAGE]


Overall Averages

[IMAGE]Average Gas Produced (Trail Experiment)

Average Gas Developed (Experiment 1)

[IMAGE]


Average Gas Developed (Experiment 2)

[IMAGE]


Average Gas Developed (Experiment 3)

[IMAGE]




Average Gas Produced (Overall)

[IMAGE]




Evaluation:

The results obtained from the experiment shows that as substrate
concentration increases so does the rate of reaction, yet there are
many aspect that may result in more accurate results. This may include
the temperature, apparatus, different potatoes, etc.

If I were to do the experiment again in the future, I would take some
further factors into consideration, such as:

1. I would attempt to keep the temperature more constant. This is
because the room temperature may have varied and could have had an
effect on the results.

2. I may perhaps use a wider range of concentrations to acquire a
wider range of results to investigate and compare.

3. Maybe I'd use smaller flasks because this could have an effect on
the amount of collisions that occur and the rate of reaction would
possibly increase.

4. Perhaps I could use a technique of reducing the amount of gas
escaping from the syringe because I assume that some may have escaped.

5. I could use a gas syringe with a glass pipe, as an alternative
instead of the rubber pipe, as some gas may have been lost through it.

6. Maybe to use more potatoes in grams because it could increase the
amount of gas produced and are more apt.
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