Claudia Serrano

AP Biology
October 4, 2012

Determining how a change in temperature affects the reaction rate of enzyme
peroxidase.

Research:

Enzymes are proteins that work as catalysts. Catalysts are chemical substances that
speed up chemical reactions, by lowering the activation energy needed for the reaction
to take place. However, during this process, enzymes are not consumed. Each enzyme
is specific to a chemical reaction as well as to a substrate. A substrate is the reactant in
which the enzyme works on. Each enzyme has a specific region that is called the active
site. The active site is where the substrate binds to the enzyme, in order to start the
chemical reaction. When this binding takes place, the shape of the active site, changes a
little bit in order to fit the substrate more perfectly and this change in shape is known as
induced fit. Then, the whole process is called the enzyme-substrate complex, where
products are created and released by the reaction occurring between the substrate and
the enzyme. Aside from that, enzymes also receive help from other components called
cofactors and coenzymes. Cofactors are non-protein helpers while coenzyme, are
organic cofactors. These coenzymes are molecules that usually come from vitamins,
which form part of the active site in enzymes, in order for the enzyme to work.

Peroxidase is a type of enzyme that acts in many processes. Just as this enzyme
donates electrons to substrates, it also breaks hydrogen peroxide into water and oxygen.
Hydrogen Peroxide is a chemical compound composed of two oxygen atoms and two
hydrogen atoms. However, as said before, this compound can easily be broken down
into water and oxygen. This process occurs faster if the enzyme peroxidase is found in
it.
2 H2O2 2 H2 O + O2
Peroxidase can be extracted from turnips through the process of homogenization, which
means to break down a substance into its components and disperse them in a fluid.
During the decomposing process of hydrogen peroxide into water and oxygen, mixing it
with a compound called Guaiacol can test the presence of peroxidase. The oxygen
produced by hydrogen peroxide with help of peroxidase, would eventually react with
Guaiacol, causing it to oxidize and produce a brown color.

Many factors can affect enzyme activity and they include factors such as temperature
and pH. Normally, chemical reactions increase as temperature increases, but until a
specific temperature. Enzymes can easily become denatured, which means that a factor
such as high temperatures, alters the enzyme shape not allowing the enzyme to keep on
working. Animal cells usually reach this state when found in temperatures higher than 40
C. However, not only high temperatures can affect enzymes, but also low temperatures
that freeze the enzyme. Usually, when there is an increase by 10 C to a temperature
that the enzyme can handle, the reaction rate would almost double. Even small changes
in temperatures such as 1 C or 2 C, can increase the reaction rate by 20 to 30%. In the
picture below, we can see the path of enzymatic reaction when there is a change in
temperature.


Purpose:
The purpose of this experiment was to find out how a change in temperature can affect
the reaction rate of the enzyme peroxidase in the production of oxygen.

Hypothesis: If the temperature increases or decreases by a large difference from the
baseline temperature (20 C), then the reaction rate of the enzyme would decrease
because the enzyme would become denatured, loosing its shape and therefore, its
function.

Experimental design:

Manipulated variable Responding
variable
Constants Control
Temperature
of substance
in C,
composed of
0.6 mL of
distilled water
and 1.5mL of
enzyme
peroxidase
Temperatures
tested:
o 10
o 30
o 40
o 50
o 60
o 70
Reaction
rate of
enzyme
in the
productio
n of
oxygen
(measure
d by color
scale
from 1-
10)
pH of solute
(pH of water,
7)
Volume of
mixture
(15mL)
Enzyme used
(peroxidase)
Solute used
(distilled
water)
Substrate
used
(hydrogen
peroxide)
Guaiacol as
indicator
Baseline:
Room
temperatu
re 20 C
0.3 of
0.1%
hydrogen
peroxide
0.2 mL of
Guaiacol
1.5mL of
peroxidas
e
13mL of
distilled
water
Concentration
of: hydrogen
peroxide
(0.1%)
Concentration
of: Guaiacol
(0.1%)
Substrate:
7mL of
distilled water
0.3mL of
hydrogen
peroxide
0.2mL of
Guaiacol
Enzyme:
1.5mL of
peroxidase
6.0mL of
distilled water

Trial: #1

Materials:
9mL of Turnip peroxidase
1.8mL of 0.1% Hydrogen Peroxide
1.2 mL of Guaiacol
78mL of distilled water
14 test tubes
4 syringes (1,2,5,10mL)
Thermometer
Ice
Timer
Heating machines
At least 4 beakers of 50 mL
Parafilm

Procedure:
For each Substrate test tube (7 test tubes):
1. First add 7mL of distilled water.
2. Then add 0.3 mL of hydrogen peroxide by using a syringe
3. Add 0.2 mL of Guaiacol by using another syringe
4. Cover the test tube with a piece of Parafilm
5. Gently mix for approximately 10 seconds
For each Enzyme test tube (7 test tubes):
1. First add 6.0 mL of distilled water to act as the constant of pH 7.
2. Add 1.5mL of turnip peroxidase
3. Cover the test tube with a piece of Parafilm
4. Gently mix for approximately 10 seconds
5. Place water in a beaker and start heating it up by using the heater
6. Place the enzyme test tube inside the beaker and keep on heating it
7. Place the thermometer inside the test tube in order to record the temperature
wanted.
8. Repeat steps 5-7 for 5 test tubes
9. For cold temperature, place ice and water in a beaker
10. Place the enzyme test tubes inside the beaker and keep on cooling it
11. Place the thermometer inside the test tube in order to record the temperature
wanted
12. Repeat steps 9-11 for two test tubes
13. The last enzyme test tube keep at room temperature
14. When the enzyme tube reaches the temperature wanted, invert the substrate
solution in it
15. Record for every minute until reaching 5 minutes in total
16. Before recording, gently mix the solution and then compare it to color chart
17. Record data


Table:
Temperature C 1 minute 2 minute 3 minute 4 minute 5 minutes
10 (blue) 2 4 5 5 6
20 (Base line,
red) 3 4 5 6 6
30 (green) 2 4 5 6 7
40 (purple) 2 4 5 7 8
50 (turquoise) 2 4 5 5 6
60 (orange) 2 2 2 3 3
70 (light blue) 1 1 1 1 1

Graph:


0
1
2
3
4
5
6
7
8
9
0 1 2 3 4 5 6
T
u
r
n
i
p

p
e
r
o
x
i
d
a
s
e

c
o
l
o
r

c
h
a
r
t

Time (in minutes)
Reaction rate of peroxidase in different
temperatures
10C
20C
30C
40C
50C
60C
70C


Analysis:
In the chart and table above, we can see the reaction rate of enzyme peroxidase for
different temperatures recorded every minute, for a total of 5 minutes. The red line
represents the baseline at room temperature, in this case, 20 C. In all the tested
temperatures except for 70 C, we can see how the reaction rate and the production of
oxygen by peroxidase increased as time also increased. At temperature 70 C, the data
for the entire 5 minutes was 1 in the color chart, been the lowest reaction rate that the
enzyme reached, having no change and differing dramatically compared to the baseline.
By looking at both the chart and the table, we can also see that the highest reaction rate
that the enzyme reached, is when place at a temperature of 40 C, where it matches the
8 level in the color chart for both 4 minutes and 5 minutes. In temperatures 20 and 60
C, the red and the orange line, we can see how the reaction rate of peroxidase
maintained itself the same for the last two minutes. The second highest rate of change
was reached after 5 minutes in a temperature of 30 C. This graph also allows as to see
how the reaction rate started to decrease as temperature increases as it can be seen in
60 C, where the reaction rate stayed the same for the first three minutes, been only 2,
while for the last two minutes it went up to 3, maintaining itself low in reaction rate. The
only linear change between reaction rates can be seen in the green line that represents
30 C, where the change for every minute is always one level up in the color chart.

Conclusions:

The purpose of this experiment was to see how the reaction rate in the production of
oxygen, of the enzyme peroxidase, is affected by a change in temperature.

As I conducted this experiment, I found many major findings. First, temperature affects
tremendously the way an enzyme works. However, I found out that 10 C more or less
from the optimum temperature that was 20 C in this experiment, does not have as much
change in the reaction rate as I expected. For example, when the enzyme was cooled
down to 10 C, the highest reaction rate that it reached, was 6, just as it had reached
when placed in room temperature. The same happen when we heat the solution 10 C
more, where the highest reaction rate even increased, reaching 7. I also found out, that
in the case of peroxidase, the reaction rate reaches its peak, when placed in 40 C
which is the double of the base line. Here, the reaction rate was classified as an 8 in the
color chart. At last, one of my major findings, was that when enzyme peroxidase is
heated up until 70 C, and eventually more, its reaction rate reaches 0, meaning that the
enzyme has stopped working. At last, the reaction rate for most temperature after both 2
minutes and three minutes, is the same, first been a level 4 in the color chart and then a
5.

My hypothesis was supported by my results. In my hypothesis I stated that If the
temperature increases or decreases by a large difference from the baseline temperature
(20 C), then the reaction rate of the enzyme would decrease because the enzyme
would become denatured, loosing its shape and therefore, its function. As we can see in
the data collected, when the temperature increases by just a few C, the reaction rate
didnt change much. For example, in 10 C, the reaction rate was almost the same as
the baseline. However, when the temperature increased for more than 30 C, for
example when it increased up to 60 C and 70 C, the reaction rate decreased
tremendously, affecting how much oxygen was produced.

My findings were similar to the data collected by the other group of the class that
manipulated temperature. In both experiments, as the temperature changed, the
reaction rate was also altered, sometimes increasing and sometimes decreasing. In both
cases, when the temperature increased by a lot compared to the baseline, the reaction
rate decreased dramatically. The lowest level in the color chart that the reaction rate
reached was level one. In the case of my lab, that reaction rate was reached when the
temperature was 70 C, while in the other experiment, it was reached when the
temperature was 60 C. Another similarity that I found between the two labs, was that
when the temperature was 10 C, for the first 3 minutes, the data was the same, but
these changed when the 5 minutes where completed. In the other groups experiment,
the reaction rate at 10 C after 5 minutes, reached level 8 in the color chart, been the
highest reaction rate, while in the experiment I conducted, the highest reaction rate that
the enzyme peroxidase reached, was level 8 when the temperature was 40 C.
However, in overall, both experiments were really similar, recording almost the same
data. For both labs, we can conclude that when the temperature changes a lot compared
to the baseline, 20 C, the reaction rate of the enzyme starts to decrease, producing less
oxygen and working less efficiently and slower.

There are many possible explanations that I can offer for my findings. First, as it is
known, shape determines function, therefore, a slight change in shape, can completely
affect the function of a molecule. There are many factors that can affect the shape of a
molecule and these include factors such as temperature and pH. Due to this, my
explanation can be that as a change in temperature occurred, the enzyme peroxidase
suffered denaturation, where the active site lost its shape, not allowing the substrate to
fit in it, and therefore, the enzymatic reaction slowed down and eventually stopped taking
place.

Errors in experiments, affect the data collected, making it less accurate than what it
should be and in this experiment, I do believe there were some errors. First, some
measurements when putting the amount of peroxidase, or Guaiacol or other substances,
could have not been completely exact, differing among the test tubes. This somehow
can change the results, since volume and concentration of substances was not kept the
same for all the test tubes. Another error that definitely took place in our experiment, was
that the temperatures in which enzyme peroxidase was tested, decreased throughout
the 5 minutes in which data was collected. This occurred because the recording of data
started when the temperature had reached the desirable temperature, but we were not
able to keep the temperature constant without cooling it down or heating it up,
throughout the whole time testing the reaction rate. Therefore, for the last minutes of
data recording, the temperature had decreased somewhat from the initial temperature.
At last, a timing error was also introduced since some data was not collected exactly
after a minute, but a little bit more or less.

In order to improve my results, I would first try to be more accurate with the
measurements and amounts of substances for each test tube and the time keeping. I
would also try to maintain the same temperature for the whole time that the data is been
recorded. Besides that, in order to have a more accurate experiment, I will record more
different temperatures, and for a longer period of time. For further study, I will
experiment with other variables that might affect the reaction rate such as the amount of
solute or the amount of enzymes.

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WiseGeek (2003-2012). What is a peroxidase? Retrieved October 2, 2012 from
http://www.wisegeek.com/what-is-a-peroxidase.htm
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ME.pdf
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http://www.thefreedictionary.com/homogenization
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October 2, 2012 from http://www.worthington-
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