Hypothesis Enzymes

Hypothesis Enzymes such as Catalase are large protein molecules that are found
in living cells. They are used to speed up specific reactions in the cells. They
are all specific as each enzyme just performs one particular reaction. In their
globular structure, one or more polypeptide chains twist and fold, bringing
together a small number of amino acids to form the active site, or the location
on the enzyme where the substrate binds and the reaction takes place. Enzyme and
substrate fail to join together if their shapes do not match exactly. This
ensures that the enzyme does not participate in the wrong reaction. The enzyme
itself is unaffected by the reaction. When the products have been released, the
enzyme is ready to bind with a new substrate. Enzymes work to change the rate of
a reaction without being absorbed by the reaction. The reactant that an enzyme
acts on is called the enzyme's substrate. The product is what the enzymes
convert the substrate into. Substrate + Enzyme = Product Catalase is an enzyme
found in food such as potato and liver. It is used for removing Hydrogen

Peroxide from the cells. Hydrogen Peroxide is the poisonous by-product of
metabolism. Catalase speeds up the decomposition of Hydrogen Peroxide (which is
the substrate) into water and oxygen as shown in the equations below: Hydrogen
peroxide + Catalase Water + Oxygen H2O2 + Catalase H2O + O2 It is able to speed
up the decomposition of Hydrogen Peroxide because the shape of its active site
matches the shape of the Hydrogen Peroxide molecule. This type of reaction where
a molecule is broken down into smaller pieces is called an anabolic reaction. In
this experiment Apparatus ? Conical flask ? Rubber bung ?

Gas tube ? Gas syringe ? Measuring cylinders ? H2O2
? H2O ? Potato split into 4g tubes (enzyme catalase) ?

Clamp stand ? Cork borer ? Glass rod ? Thermometer ?

2 decimal place balance ? Pestle and Mortar ? Sand ? Beaker
? Scalpel ? White tile ? Safety goggles ? Forceps
? Stopwatch The apparatus was set up as shown: A pilot test was carried
out to figure out how much and in what form the apparatus would need to be in.

This is what was concluded; the potato should be ground down with sand (which is
unreactive with H2O2) into a paste. This gives a larger surface area that
permits the reaction to go faster because the substrate molecules can get to the
enzyme molecules faster and more efficiently. The reaction will be timed as soon
as the solution (H2O2 and H2O) is added. The oxygen that evolves will be
measured and recorded every 30 seconds until the reaction finishes. The
concentration of the solution will be variegated in the experiment. This is the
only element that is varied everything else must stay the same to keep the
investigation unbiased. 25cm3 of solution was chosen to be used because in the
pilot experiment this was all that was necessary to produce a sufficient amount
of oxygen in the gas syringe so that it could be easily measured. If there were
more solution it would take longer to pour in to the conical flask therefore gas
would escape before the rubber bung could be fixed into place to seal the
conical flask. If less solution were to be used the reaction would take longer
because there wouldn’t be as many substrate molecules to react with the
enzymes. The solution will be measured in a measuring cylinder. The range was
selected because it is easily multiplied into 100, which is what is needed to
calculate percentages. The measuring cylinder was chosen to be used over all
other methods of measuring because it is easily available. Table to show H2O2 :

H2O Percentage of H2O2 Volume H2O2 (cm3) Volume of H2O(cm3) 100% 25 0 80% 20 5

60% 15 10 40% 10 15 20% 5 20 0% 0 25 The enzyme catalase works best at a warm
temperature so to speed the experiment up the conical flask will be in a water
bath at the temperature range 40-45 oC. The water bath needs to be kept at a
constant temperature because the enzymes will then be working at the same
constant rate. The mass of the potato needs to be kept the same at 4g so there
is the same amount of enzyme in each experiment. The overall amount of solution
needs to be kept constant to ensure that the % concentration is correct. The
oxygen evolved will be measured every 30 seconds from the moment the solution is
added to the potato. This will be done so that the results from each experiment
can be compared at the end. The experiment will need to be repeated at least 3
times to allow for an average to be taken, and to be able to exclude any
anomalous results and still obtain an average. Overall the experiment will be
done 18 times. Although it will be permitted to carry the experiment out a few
more times if one proceeds to go wrong. The results will be recorded in a table
that is clear and easy to understand. Here is a sample of the table that will be
used: Time (s) Experiment 1 Experiment 2 Experiment 3 Average Rate of reaction

30 60 90 120 ETC The results will be plotted into an evolved graph and a rate of
reaction graph. Rate of reaction is measured by Average Time Once the graphs
have been drawn trends will be looked for and identified. Prediction Once the

Hydrogen peroxide is added to the enzyme catalase (potato) it should begin to
fizz violently. This is a sign that the two substances are reacting together.

This will show that the catalase is decomposing the hydrogen peroxide so fast
that it can be seen. The enzyme catalase and the substrate hydrogen peroxide
will join together to make the products oxygen and water. This is called the
lock and key method. The enzyme catalase is specific to this reaction. Because
the reaction is being heated in the water bath it will go much faster and be
easier to see the increase in oxygen evolved. It will also allow for a third
replicate to be taken because the time allocated for the investigation is brief
and without the reaction being speeded up there may only be time for one or two
experiments for each concentration. As the substrate concentration increases,
the rate should go up at a directly proportional rate until the solution becomes
saturated with the substrate hydrogen peroxide. Once the saturation point is
reached, adding more substrate will make no difference. If the concentration of
hydrogen peroxide is doubled the rate of reaction should double as well. The
rate should steadily increase when more substrate is added because more of the
enzyme’s active sites are being used which results in more reactions so the
required amount of oxygen is made more quickly. Once the amount of substrate
molecules added exceeds the number of active sites available then the rate of
reaction will no longer go up. This is because the maximum number of reactions
are being done at once so any extra substrate molecules have to wait until some
of the active sites become available. Method To test out how the concentration
of hydrogen peroxide affects the rate of reaction this is what has been done:
? Grind up exactly 4g of potato with a pinch of sand (the amount of sand
doesn’t need to be accurate because it doesn’t affect the results) in the
pestle and mortar. Add this to the conical flask. ? Use the measuring
cylinder to measure out 25cm3 of hydrogen peroxide the 100% concentration. It is
important to measure the amount accurately to ensure the experiment is
impartial. ? Prepare the water bath by using hot water from the tap and
altering it using some cold water until the range (40-45oC) is reached. ?

Pour the hydrogen peroxide into the conical flask and immediately insert the
bung, at the same time start the stopwatch. ? Bubbles should start to
rise up the conical flask and fizzing will be noticeable. The gas syringe will
start to move quite rapidly. ? Shake the conical flask vigorously before
every reading is taken. ? Recordings will be taken from the gas syringe
every 30 seconds. ? When the reaction has finished wait for approximately

2 minutes to see if any more oxygen evolves then stop the stopwatch. ?

Repeat the experiment twice more for that concentration and note the results.
? Repeat the experiments 3 times for each concentration 80%, 60%, 40%,

20% and 0%, repeating the experiments will help to produce more accurate results
as any inaccuracies in one experiment can be compensated for in the other
experiments. ? The 0% concentration of hydrogen peroxide is used as a
control to show that at 0% concentration no reaction occurs. Safety To be
protected safety goggles must be worn at all times during the experiment. A lab
coat should be worn to protect from damage to clothing. Cleaning apparatus must
be kept near by in case of any spillage. Hydrogen peroxide is toxic and care
must be taken when using it because it will blister the skin easily. Results

100% concentration Time (s) Experiment 1 Experiment 2 Experiment 3 Average Rate
of reaction 30 35 22 24 27.00 0.90 60 49 44 42 45.00 0.75 90 62 54 52 56.00 0.62

120 66 59 56 60.33 0.50 150 68 62 59 66.00 0.42 180 72 64 62 66.67 0.37 210 73

65 62 68.33 0.32 240 74 68 63 69.00 0.28 270 75 69 63 69.33 0.25 300 75 69 64

69.33 0.23 330 75 69 64 69.33 0.21 360 75 69 64 69.33 0.19 390 75 69 64 69.33

0.18 80% Concentration Time (s) Experiment 1 Experiment 2 Experiment 3 Average

Rate of reaction 30 20 23 18 20.33 0.68 60 37 29 28 31.33 0.52 90 53 35 39 42.33

0.47 120 59 57 48 53.00 0.44 150 64 59 57 60.00 0.40 180 66 65 66 65.67 0.36 210

69 72 70 70.33 0.33 240 70 73 72 71.67 0.30 270 70 73 72 71.67 0.27 300 70 73 72

71.67 0.24 330 70 74 72 72.00 0.22 360 70 74 72 72.00 0.20 390 70 74 72 72.00

0.18 60% Concentration Time (s) Experiment 1 Experiment 2 Experiment 3 Average

Rate of reaction 30 14 18 25 16.00 0.53 60 37 39 39 38.00 0.63 90 54 50 69 52.00

0.59 120 66 57 75 61.50 0.51 150 67 65 82 66.00 0.44 180 72 67 90 69.50 0.39 210

73 70 94 71.50 0.34 240 74 74 96 74.00 0.31 270 74 74 98 74.00 0.27 300 74 74 98

74.00 0.25 330 74 74 98 74.00 0.22 360 74 74 98 74.00 0.21 390 74 74 98 74.00

0.19 Experiment 3 for 60% is anomalous. 40% Concentration Time (s) Experiment 1

Experiment 2 Experiment 3 Average Rate of reaction 30 15 16 15 15.33 0.51 60 30

33 32 31.67 0.53 90 40 46 45 43.67 0.49 120 51 57 57 55.00 0.46 150 63 67 69

66.33 0.44 180 68 77 78 74.33 0.41 210 74 82 85 80.33 0.38 240 79 84 89 84.00

0.35 270 82 86 90 86.00 0.32 300 84 87 91 87.33 0.29 330 86 89 92 89.00 0.27 360

86 89 92 89.00 0.25 390 86 89 92 89.00 0.23 20% Concentration Time (s)

Experiment 1 Experiment 2 Experiment 3 Average Rate of reaction 30 14 16 12

14.00 0.46 60 27 22 25 24.67 0.41 90 32 31 29 30.67 0.34 120 35 33 31 33.00 0.28

150 40 39 36 38.00 0.26 180 46 42 39 42.33 0.24 210 52 48 47 49.00 0.23 240 55

52 51 52.67 0.22 270 58 56 56 56.67 0.21 300 61 60 60 60.33 0.20 330 65 62 61

62.67 0.19 360 65 63 61 63.00 0.18 390 65 63 61 63.00 0.16 0% Concentration No
reaction for 0% Rate of reaction at 5 minutes 100% concentration 0.23 80%
concentration 0.24 60% concentration 0.25 40% concentration 0.29 20%
concentration 0.20 0% concentration No reaction The replicates are all written
down to the nearest whole one on the gas syringe, because sometimes the gas
syringe was moving so fast it could not be recognised if it was at .5 of a cm3.

From these results it was able to plot a graphs of oxygen evolved and rate of
reaction. Interpretation It would have been expected that when the concentration
doubled so would the rate of reaction but this didn’t occur. After 270 seconds
the rate of reaction slows almost to a halt. The straight lines show this on the
graphs. At this point virtually all of the active sites are occupied so the
active sites are said to be saturated with hydrogen peroxide. Increasing the
concentration at this point will not cause the rate of reaction to go up any
more. . All the active sites are being used so any extra Hydrogen Peroxide
molecules will have to wait until an active site becomes available. The
theoretical maximum rate of reaction is when all the sites are being used but in
reality this theoretical maximum is never reached due to the fact that not all
the active sites are being used all the time. The substrate molecules need time
to join onto the enzyme and to leave it so the maximum rate achieved is always
slightly below the theoretical maximum. The time taken to fit into and leave the
active site is the limiting factor in the rate of reaction. There is something
obviously wrong with the results because the rate of reaction should be highest
at 100% concentration. These could be some of the explanations to illustrate why
the experiment went wrong Limitations 1. The experiment was repeated 3 times to
and an average was taken improve accuracy. The results were used to plot graphs
with a lone of best fit. It was unrealistic to think that all the variables
could be kept the same. 2. There was a slight delay between pouring the hydrogen
peroxide into the conical flask, inserting the bung and starting the stopwatch.

This will slightly affect the results but the three steps were carried out in
the same way for each experiment so it shouldn’t have made a very big
difference in the overall results. 3. One of the most inaccurate pieces of
apparatus used was the measuring cylinder this is extremely inexact because if
the meniscus isn't exactly on the line the measurement is wrong. This brings in
a fairly large percentage error. 4. The volume of gas in the conical flask is
very slightly affected by how far the bug is pushed down in each experiment, if
the bung is pushed down further then the volume in the conical flask will be
less so more oxygen will be pushed into the gas syringe early in the experiment
and this might have affected the results. 5. The gas syringe only had
measurements for 1 whole number this could have affected the results because no
decimal places could be used. 6. The amount of catalase in each potato
couldn’t be measured this would affect the results because some parts of the
potato could have more catalase than the rest. 7. The water bath was difficult
to keep at the constant temperature due to heat loss to the surroundings more
hot water would have to be added to heat it up again but this is imprecise. 8.

The hydrogen peroxide could have been at different temperatures due to the room
warming and cooling. 9. The potato ran out so another potato was used this could
have influenced the results because this potato could have contained a different
concentration of catalase. 10. The balance only measures to 2 d.p. this might
have affected the results because slightly different masses of potato could have
been used. 11. The gas syringe may have been slightly sticky making it harder
for the oxygen to push, to register the true result. Improvements 1. To gain a
more accurate result more replicates could be taken to get a more actual
average. 2. There is no way to rectify the time taken between pouring in the
hydrogen peroxide, putting in the bung and starting the stopwatch. Although it
does help if there are assistants to help so each person only has to do one job.

3. Instead of using a measuring cylinder a pipette or a burette would have been
far more precise pieces of equipment. 4. A conical flask with a pressure line
could have been used this would show how far to push in the bung. 5. A decimal
place gas syringe would give more accurate readings. 6. The catalase can’t be
measured in potato a much better catalase substitute would be yeast this can be
weighed and measured. Also once the potato is used another would have to be used
and this could produce catalase of a totally different concentration. 7. An
electrically monitored water bath would be useful this could keep the water in
the correct temperature range accurately. 8. The hydrogen peroxide should be
kept at a constant temperature by putting it into an electrically monitored
water bath and taking and recording its temperature before it is added to the
catalase. 9. A different source of catalase should be used one that’s
concentration can be measured e.g. yeast. 10. A balance with more decimal places
would have made the results more exact. 11. The gas syringes should be made so
that it has a non-stick surface inside so it doesn’t become stiff. The plotted
results on the graph go up to produce a curve of best fit levelling off at the
end. The main anomaly is the fact that the rate of reaction at 40% concentration
is far higher than that at 100% concentration. Another one of the anomalies in
the results was the 3rd experiment at 60% concentration this went completely
wrong. These were probably due to and experimental error involving some of the limiting factors mentioned above.