Biology Coursework - Enzymes.
Biology Coursework - Enzymes.
Aim: To investigate the effect of temperature on the enzyme catalase.
Prediction: I think that at 40°c, the catalase will produce the most oxygen, and at
20°c (the lowest temperature) and 80°c (the highest temperature) the catalase
will produce the least amount of oxygen.
Hypothesis: An enzyme is a catalyst; a chemical that speeds up a reaction while
remaining unchanged itself. This allows the enzyme to be used repeatedly. If not
for enzymes, these chemical reaction would still occur, but at such a slow rate
that the organism would die.
The substances that the enzyme works on is known as the substrate, and the
reaction itself takes places on the part of the surface of the enzyme known as the
active site. Each type of enzyme is specified to work on a certain type of
substrate. This can be known as the lock and key mechanism. It can be
described as so as one type of substrate can only 'fit' into one type of enzyme,
before it is either built or broken as needs require, and then released.
Enzymes can be split into two categories: builders and breakers.
Enzymes work by reducing the amount of activation energy required. Normally
the reactions that take place need activation energy to get the molecules to
react. Enzymes affect the amount of energy that is required. With enzymes, less
activation energy is required to get the reaction started. Since this energy usually
comes in the form of heat, the presence of enzymes means less heat is needed
to spark off the reaction. This means reactions can readily take place in lower
temperatures i.e. body temperature (37°c). This means there is more energy to
go around so more reactions can take place at once.
Body temperature is the optimum temperature for enzymes, as it is the highest
temperature in which the enzymes can function before they become denatured.
When enzymes become denatured, they change form and no longer remain
functional. They start to denature around 40°c, after which the rate of reaction
begins to slow down.
At temperatures below 37°c, this is not the case. Enzymes only start to denature
once they get too hot. However temperature still affects the rate of reaction. As
temperature decreases, the rate of the reaction slows down, as the enzyme
loses kinetic energy and therefore vibrate less. This means the chance of
collision with a substrate is decreased.
This information could be placed in a graph, which is roughly sketched below,
showing the general shape of the line. This shows how the rate of reaction
increases till it reaches the optimum temperature, before decreasing again once
the enzyme starts to denature.
This is why I predict that at 30°c, the most oxygen will be produced, as this is the
temperature closest to the optimum temperature, and one at which the enzyme
cannot denature.
At 20°c, the lowest temperature, I predict that the least amount of oxygen will be
produced, as at 20°c, there is much less kinetic energy than at higher
temperatures; therefore there are less collisions of catalase with the substrate,
hydrogen peroxide.
Although at 50°c, the enzymes ...
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the enzyme starts to denature.
This is why I predict that at 30°c, the most oxygen will be produced, as this is the
temperature closest to the optimum temperature, and one at which the enzyme
cannot denature.
At 20°c, the lowest temperature, I predict that the least amount of oxygen will be
produced, as at 20°c, there is much less kinetic energy than at higher
temperatures; therefore there are less collisions of catalase with the substrate,
hydrogen peroxide.
Although at 50°c, the enzymes have been most dramatically denatured, and the
enzymes will be unable to function properly so the reaction cannot take place as
effectively, the rate of reaction will still be faster than 20°c.
An experiment performed previously proves this. It was an experiment to
investigate the effect of temperature on the digestion of starch by amylase, in
which we filled test tubes with amylase and starch (separately at first), added
iodine to each test-tube, heated them in water-baths, and then mixed them and
placed them back in the water-baths in which they were heated. We used the fact
that iodine turned blue when it detected starch, therefore when the amylase has
digested the starch, it would leave glucose which would in turn, turn the iodine
yellow. We timed how long it took for this to happen.
These were our results :
Temperature
Time for iodine to go yellow
20°c
More than 27 minutes
40°c
45 seconds
60°c
3 minutes
80°c
0 minutes.
As you can see, the amylase in 20°c took the longest to digest the substrate,
starch. At 80°c, although the enzymes would have been denatured, the rate of
reaction was still much faster than the lowest temperature.
The current experiment however, deals with the enzyme catalase, one of the
fastest enzymes. We are going to react it with hydrogen peroxide, as this is a
reaction that occurs in our bodies. Hydrogen peroxide is a by-product of
respiration. However, it is very poisonous, so therefore it needs to be broken
down into less potent substances. This reaction occurs naturally, but it is such a
slow reaction that we would die before it was complete. This is the equation for
the natural reaction:
H202 2H2O + O2
Cells produce catalase to speed up this reaction, and give water and oxygen,
substances that are not harmful to our bodies at all.
The liver contains catalase naturally, which is why liver is being used for this
experiment.
Apparatus:
? Test-tubes (6 minimum)
? Test-tube holders
? Water-baths (for 3 different temperatures)
? Pipette
? Measuring cylinders (Two 10ml capacity, one 25ml capacity)
? Beaker
? Delivery tube with bung
? Stop-watch
? Mortar and pestle
Method:
Step 1: A liver dilute has to be made. This has to be enough for the whole
experiment, so that each experiment is done with the same concentration of liver
(catalase). The liver has to be ground using a pestle and mortar, with a bit of
sand to make it easier. Then you add water, keeping in mind you have to use the
same dilute for 15 experiments, making room for error. 170cm³ should be
sufficient.
Step 2: Fill 3 test-tubes with 10cm³ of liver solution each. Then fill 3 test-tubes
with 2cm³ of hydrogen peroxide. These should be measured with a measuring
cylinder. Leave the test-tubes in the appropriate temperature - if you are
performing 20°c, then leave it in room temperature. If you are performing 40°c,
then leave them all in a water-bath set at 40°c. Leave them in there till they have
reached the appropriate temperature.
Step 3: Set up the rest of the apparatus to collect the amount of oxygen released.
It should be set up as below:
Step 4: Take one test-tube of liver solution, and another of hydrogen peroxide.
Pour the hydrogen peroxide into the liver solution, quickly put the delivery tube on
the test-tube and start timing. You need at least 2 people to perform this. Time
for about 5 seconds, and then measure the amount of oxygen released into the
measuring cylinder. Record this result.
Step 5: Repeat for other experiments.
Fair Test:
* The same dilution of liver solution should be used throughout, so there are not
different concentrations of hydrogen peroxide.
* The same equipment should also be used throughout the experiment i.e. same
water-baths, same stop-watch. Measuring cylinders and pipettes should also
only be used for one type of solution; the hydrogen peroxide or liver solution.
* The experiment should be performed 3 times for each temperature, and then
an average calculated from those results to obtain more accuracy.
* The cork should be placed on the test-tube immediately upon contact of the
hydrogen peroxide and liver.
* The experiment should be carried out in the same environment and
atmosphere.
Results:
Temperature
(°C)
st
Attempt
(ml)
2nd Attempt
(ml)
3rd
Attempt
(ml)
Average
20
4
0
5
0
40
5
24
8
6
60
3
9
21
7
80
0
0
0
0
Conclusion:
These results turned out as we expected, according to our prediction., in which
we said at 40°c, the most oxygen would be released. This was true as 16cm³
was produced. We also said at 80°c (the highest temperature), the least oxygen
would be produced. We were also correct in saying this, as no oxygen at all was
produced within the 5 seconds for each of the 3 attempts. However we were
incorrect in saying that at 20°c, the least oxygen would be produced. At 20°c,
0cm³ of oxygen was produced, only 6cm³ less than at 40°c.
As was mentioned in my hypothesis, at 37°c is the optimum temperature at
which the enzymes function best, because this is the temperature at which they
work fastest, before they become denatured. Since 40°c was the closest
temperature to this optimum temperature, it is expected that this temperature
should produce the most oxygen. Although the enzymes at this point have started
to denature slightly, it is still not enough to override the loss of kinetic energy at
20°c, or the extent of the denaturing at 60°c.
At 20°c, the enzymes are not denatured, but they work slower because there is
less heat, therefore the enzymes have less kinetic energy. Since the enzymes
are vibrating more slowly, there is less chance of any substrate (hydrogen
peroxide) coming into contact with the active site of the enzyme. That is why less
oxygen was produced at 20°c than at 40°c.
Even less oxygen was produced at 60°c, because at this point the enzymes have
denatured to a point where only a few active sites remain in use. All the others
have been rendered useless. The same amount of hydrogen peroxide is having
to make do with less enzymes, therefore less oxygen is produced.
At 80°c, the end result was that no oxygen was produced. Very few, or no
enzymes were left to speed up the reaction as they had all been denatured,
therefore no oxygen was produced in the 5 seconds.
Evaluation:
Our results turned out to be quite accurate once we had taken the average, so
there appears to be no anomalous results on the graph. However, throughout the
experiment, there was several anomalous results.
At 20°c, our first attempt (4cm³) was anomalous, seeming a bit too little. At 40°c,
our third attempt (8cm³) also seemed smaller than what we expected, especially
compared to the previous two results. At 60°c, our first attempt (3cm³) also
seemed slight anomalous, but there is not such a difference between that an the
average as there were with the other anomalous results.
There could be several reasons for this. One could be the development of
technique as we performed the experiment. The experiment had to be carried
out over a week, so each lesson when we started the experiment, our reaction
was slow, but as we did the experiment more, we developed quicker reaction
and better technique. Better technique means less oxygen is released, so that
would explain why at 20°c and 60°c, the amount of oxygen increased with each
attempt.
Another reason could be the reuse of our test-tubes. Once we had completed
one attempt, we rinsed the test-tube out to use for the next experiment. Excess
water could perhaps have slightly altered the concentration of the liver solution,
causing less oxygen to be released.
Because the experiment was spread over a period of a week, the liver solution
had to be kept overnight. This meant that some solute would most likely have
sunk to the bottom, and since we didn't shake it up before transferring from the
beaker to the test-tube, perhaps different concentrations were used throughout
the experiment as well.
There was also a problem with one of the water-baths in the experiment,
because when we put the test-tubes into it, it turned out they were not at the right
temperature, so we had to wait till it heated up to the right temperature. This took
about 15 minutes, and in that time, some of the hydrogen peroxide may have
evaporated, or the liver solution been slightly cooked, which would have affected
the experiment slightly. The temperature at which this happened was 60°c.
We also didn't take the temperature of the liver solution and hydrogen peroxide
once they were removed from the water bath, as we simply assumed it was at
the right temperature. If they weren't, that would also have contributed to the
experiment.
Despite all these errors and flaws, other attempts managed to be successful
enough to balance out those anomalous results, so our end results turned out as
we expected. We were able to draw the conclusion that enzymes are affected by
heat as they gain more kinetic energy, up to a point (which was 40°c), after which
they started to denature, therefore decreasing the rate of reaction at which the
enzymes worked.
If I was to perform this experiment again, I would have made more liver solution,
to make room for error as well as to allow for some practises, to develop
technique. I would also have the appropriate amount of apparatus, which would
solve the problem of excess water in the test-tubes, and it would also save time
so more liver solution could be used while it was all the same concentration (no
solute left at the bottom). I would also be sure to take the temperature of the liver
solution and hydrogen peroxide, to make sure it was at the correct temperature.
To extend this experiment, I would go on to find out how other factors affect the
rate of enzymes, for example, the actual concentration of the substrate, or the
concentration of enzymes.
Rachel Dennis
