Spring BIOL-1106.001
The Rate of Reaction of Enzyme Peroxidase
Abstract
An enzyme is a chemical substance that acts as a catalyst to speed up metabolic reactions
by lowering the activation energy for a specific reaction. Under certain conditions, enzymes are
very effective. For example, as temperature increases so does enzymatic activity, but too high
temperatures can denature an enzyme. This experiment tests the enzymatic activities of
peroxidase, an enzyme found in plants when exposed to various conditions such as temperature,
pH level, concentration, and boiling. The experiment was divided into four experiments –
temperature, pH, concentration, and boiling of enzymes. The goal was to find the optimal
conditions from each of the four factors to produce a high enzymatic activity. Some results that
were found was that certain pH levels contributed to an increase in enzymatic activity. Also, at
32⸰C enzymatic activity increased. Overall, doing this experiment allowed for us to know how
ezymes are affected by different conditions and factors.
Introduction
Metabolism is the chemical processes by which our bodies go through to convert food
and water into energy that we can use immediately or store for later (Burman, 2015). An
example of how vital it is in all organisms is reproduction. Organisms need the energy to
reproduce making metabolism a vital necessity for an organisms’ survival. Take warm-blooded
and cold-blooded organisms for example. The way metabolism works for the two are different.
For warm-blooded organisms, since metabolism refers to all the body’s chemical reactions food
is a necessity for them, life or death (Rohrig, 2013). The metabolism of food produces energy
and heat which allows warm-blooded organism to live in extremely cold environments because
all they need is a thick layer of warm coating and enough food source to stay warm. Coldblooded organisms, on the other hand, obtain energy from the food they consume, but they use
the environment in which they live to regulate their body temperature (Rohrig, 2013). This why
cold-blooded animals aren’t dependent on food and can go without it for a while. Some reptiles
like pythons can go a year without eating because they don’t use food to stay warm (Rohrig,
2013). Enzymes are used to regulate the chemical reactions that take place an all forms of
metabolism. Enzymes speed up reactions in all living cells of the body (Agarwal, 2006).
Cyclophilin A is an enzyme that helps regulate diseases in animals and humans (Nigro et al.,
2013). But enzymes have an important characteristic about them that allows them to be so
effective.
Enzymes have a “lock-and-key” mechanism that allows them to work effectively by
combining onto a substrate if and only if the shape of substrate’s binding surface matches with
the enzymes binding surface (Agarwal, 2006). The spot where the enzyme and the substrate
combine is called the active site. However, different enzymes have different affinity for certain
substrates (Coleman, 2015). When an enzyme binds with its substrate it forms an enzymesubstrate complex, lowering the activation energy of the reaction, but they are very specific
meaning the shape, size, orientation, structures all must match for the reaction to happen. All of
this makes the enzyme highly specific because the active sites act like a puzzle where each
substrate must match the site perfectly (Coleman, 2015). There are some factors that can render
an enzyme useless: temperature and pH are major contributors. An increase in temperature can
affect the chemical bonds in enzymes and alter its shape, same goes for any change in pH levels.
When the enzyme changes shape it can no longer bind to a substrate, thus becoming useless and
no reaction occurs (Coleman, 2015).
In this experiment, the enzyme peroxidase was used. It is found in eukaryotic organelles
in peroxisomes (Coleman, 2015). This enzyme helps cells immediately remove hydrogen
peroxide before the cell gets damaged, that occurs as a by-product that is produced during
normal metabolic processes of oxygen (Coleman, 2015). This lab aims to understand the
enzymatic activities when exposed to various conditions such as a change in temperature,
concentration, pH, and boiling. Any change in its optimal conditions will cause the enzymatic
processes to slow down dramatically. There will be greater enzymatic activity with a higher
temperature because reactions will be sped up. During the concentrations experiment, when high
concentrations of peroxidase are added enzymatic activity will increase drastically due to more
enzymes present in the solution. When tested, it is predicted that a pH level that is slightly acidic
will also cause an increase in enzymatic activity. And there will be more active enzyme reactions
if put in boiling water longer than 50 seconds.
Materials and Methods
The task of this experiment was to conduct research on the enzymatic activity of the
enzyme, peroxidase, found in plants. The experiment was conducted indoors in a biology lab
classroom. Five experiments were conducted: effects of temperature, pH, change in enzyme
concentration, effects of boiling on peroxidase, and with the fifth one being standardizing the
amount of enzyme. The materials used were test tubes, spectrophotometer, cuvette, and lens
paper.
First, the enzyme was extracted from a turnip, then weighed 8 g of peeled turnip tissue
on a balance. After, a tissue was added to a 300 ml of 0.1 M phosphate buffer that was 4⸰C with a
pH of 7 to homogenize it. Then, filtered it using several layers of cheesecloth while making sure
to keep it on ice. When done, a disposable pipette was obtained and labeled “extract” (Coleman,
2015). Once the extract had been prepared, a controlled experiment was conducted where the
amount of extract was the only variable added.
For the next part of the experiment, two test tubes were acquired and labeled - one of
them was BpH5 and the other was BpH7. The BpH stands for Buffer with the pH level of the
buffer. Each test tube was filled half full with the appropriate stock solution: test tube 1 had the
buffer solution pH 5 and test tube 2 had the buffer solution pH 7. Next, two disposable pipettes
were labeled with the corresponding test tubes: one with the BpH5 and the other with the BpH7.
Make sure to use them with the right buffer, otherwise, you could risk cross contaminate the
experiment and introduce errors. Later, three more test tubes were obtained and labeled from1-3.
Test tube 1 was used as the control with 2 ml of hydrogen peroxide added to it, while test tube 2
had the substrate and dye indicator, also with 2 ml of hydrogen peroxide, and test tube 3 had the
extract solution (Coleman, 2015). Furthermore, the three test tubes were prepared with the
solutions. With that in mind, test tube 1 was poured into a cuvette after wiping the cuvette with
lens paper while handling it by only the top 1/4th of the tube. This was mainly used as the
“blank” (Coleman, 2015) for the spectrophotometer, adjusting it to zero absorbance at 500nm
while using the blank. The contents of test tube 2 were poured into test tube 3, then the new
mixture was quickly poured into a clean cuvette and immediately placed it into the
spectrophotometer. The absorbance was observed at a 20-second interval and recorded the
results. The tube was removed from the spectrophotometer after 20 seconds and observed the
color change. Lastly, the experiment was repeated but this time with the times at 0, 15, 30, and
60 seconds.
This next experiment tested to see the effects that came from changing the extracted
amount. Five test tubes were obtained and labeled them from 1-5. Test tubes 2,4,5 had 1 ml of
buffer pH 5 solution added to them, test tube 3 had 2.5 ml of buffer pH 5 solution and if buffer
pH 7 added to it with 0.5 ml of extract added to it. Test tube 5 also had 1 ml of buffer pH 7
solution added to it with 2 ml of extract also added to it, and test tubes 2 and 4 had 2 ml of
hydrogen peroxide added to it with 1 ml of Guaiacol also added (Coleman, 2015). Another blank
was made from test tube 1, adjusting the absorbance to 500nm. Test tubes 2 and 3 were later
mixed into a cuvette and what was observed was recorded for 60 seconds at 15-second intervals.
Later, the contents of test tubes 4 and 5 were mixed and put into another cuvette, and recorded
what was observed for 60 seconds, but this time on 25-second intervals.
This next experiment tested the mixtures reactions from pH levels. Nine test tubes were
used, with test tube 1 being the control having 6 ml of buffer pH 5 solution, 1 ml of extract, and
1 ml of Guaiacol in it. Test tubes 2 and 3 had 4 ml of buffer pH of 3 solutions added to them with
test tube 2 also having 2 ml of hydrogen peroxide and 1 ml of Guaiacol added as well, and test
tube 3 only had an extra 1 ml of extract added to it (Coleman, 2015). Test tubes 4 and 5 had 4 ml
of buffer pH of 5 solution added to them with test tube 4 also having 2 ml of hydrogen peroxide
also added and 1 ml of Guaiacol added as well to it and test tube 5 only had an extra 1 ml of
extract added to it (Coleman, 2015). Test tubes 6 and 7 had 4 ml of buffer pH of 7 solution added
to them with test tube 6 also having 2 ml of hydrogen peroxide also added and 1 ml of Guaiacol
added as well to it and test tube 7 only had an extra 1 ml of extract added to it (Coleman, 2015).
Test tubes 8 and 9 had 4 ml of buffer pH of 9 solution added to them with test tube 8 also having
2 ml of hydrogen peroxide also added and 1 ml of Guaiacol added as well to it and test tube 9
only had an extra 1 ml of extract added to it (Coleman, 2015). Like always, the
spectrophotometer was adjusted using the control (blank) first. Test tubes 2 and 3 were mixed
together, test tubes 4 and 5 were mixed together, test tubes 6 and 7 were mixed together, and test
tubes 8 and 9 were mixed together and their absorbance changes were measured and recorded at
15-second intervals for 60 seconds.
Moving on, the reactions for boiling the extract was tested. A test tube was obtained and
3ml of extract was added to it and after placed in boiling water. After five minutes, the test tube
was removed and let to cool down to room temperature. To complete the experiment, three more
test tubes were obtained and labeled 1, 2, and 3 and the appropriate reagents were added to them.
Test tube 1 had 5 ml of buffer pH 5 solution/1 ml of buffer pH 7 solution/1 ml of extract/1 ml
Guaiacol, test tube 2 had 1 ml of buffer pH 7 solution/2 ml of hydrogen peroxide/1 ml Guaiacol,
and test tube 3 had 1 ml of buffer pH5 solution/ 1 ml of buffer pH 7 solution/ 1 ml of extract
(Coleman, 2015). The spectrophotometer was adjusted with our blank. The contents of tubes 2
and 3 were later mixed together and poured into a clean cuvette and recorded the results of the
absorbance at a 25-second interval for 60 seconds.
Lastly, this experiment tested the reactions of enzymes when exposed to temperature.
Nine test tubes were used for this experiment. The appropriate solutions were added to them after
they were labeled: test tube 1 (control) had 6 ml of buffer Ph 5 solution/1 ml of extract/1 ml of
Guaiacol, test tube 2 at 4⸰C had 1 ml of buffer pH 5 solution/2 ml of hydrogen peroxide/1 ml of
Guaiacol, test tube 3 at 4⸰C had 2 ml of buffer pH 5 solution/1 ml of buffer pH 7 solution/1 ml of
extract, test tube 4 at 23⸰C had 1 ml of buffer pH 5 solution/2 ml of hydrogen peroxide/1 ml of
Guaiacol, test tube 5 at 23⸰C had 2 ml of buffer pH 5 solution/1 ml of buffer pH 7 solution/1 ml
of extract, test tube 6 at 32⸰C had 1 ml of buffer pH 5 solution/2 ml of hydrogen peroxide/1 ml of
Guaiacol, test tube 7 at 32⸰C had 2 ml of buffer pH 5 solution/ 1 ml of buffer pH 7 solution/1 ml
of extract, test tube 8 at 48⸰C had 1 ml of buffer pH 5 solution/2 ml of hydrogen peroxide/1 ml of
Guaiacol, test tube 9 at 48⸰C had 2 ml of buffer pH 5 solution/ 1 ml of buffer pH 7 solution/1 ml
of extract (Coleman, 2015). After, the test tubes were pre-incubated at the right temperature
before they were mixed. They were left to acclimate for at least 15 minutes. Test tube 2 was
mixed with test tube 3, test tube 4 was mixed with test tube 5, test tube 6 was mixed with test
tube 7, and test tube 8 was mixed with test tube 9 after each reached equilibrium one pair at a
time. The changes in absorbance were measured for 60 seconds at 15-second intervals. Lastly,
the changes recorded for all the experiments tested were put in their respective tables associated
with their respective experiments.
Results
A control variable was made before starting the experiment. The enzyme activity had a
linear trend that increased at a constant rate. The absorbance was observed for 120 seconds. The
control was tested one more for consistency and accuracy, but the absorbance was recorded at 0,
15, 30, and 60 seconds. The results were the same, thus making it safe to assume these were the
values to use.
Absorbance (500 nm)
Control
0,6
0,5
0,4
0,3
y = 0.0584x + 1.2135
R² = 0.9946
0,2
0,1
0
0
20
40
60
80
100
120
140
Time (seconds)
Figure 1. The enzymatic activity of peroxidase without any being exposed to any factor (120
seconds)
Control 2
Absorbance (500 nm)
0,3
0,25
0,2
y = 0.0584x + 1.2135
R² = 0.9946
0,15
0,1
0,05
0
0
10
20
30
40
50
60
70
Time (seconds)
Figure 2. The enzymatic activity of peroxidase without any being exposed to any factor
(60 seconds)
There was a higher production of tetraguaiacol from the 2.0 mL extract by having more
peroxide for peroxidase to react with. While observing the reactions for 60 seconds, it was
noticeable that the 2.0 mL extract increased at a much faster rate than the 0.5 mL extract.
Concentration
Absorbance (500 nm)
3
2,5
2
1,5
Control
0.5 mL Extract
1
2.0 mL Extract
0,5
0
0
10
20
30
40
50
60
70
Time (seconds)
Figure 3. behavior of different enzyme concentrations to produce tetraguaiacol
There were more concentrations of tetraguaiacol produced by peroxidase at pH 5 than at
any other pH level due to the high absorbances that were observed. pH 9 was the only one to
come close to have such high concentrations like pH 5. However, pH 3 and 7 were both not as
high, with pH 3 averaging an absorbance at about 0.7192 and pH 7 averaging an absorbance at
about 1.0602 according to Figure 4.
pH
3,5
Absorbance (500 nm)
3
2,5
Control
2
pH 3
1,5
pH 5
1
pH 7
0,5
pH 9
0
0
20
40
60
80
100
120
140
Time (seconds)
Figure 4. the enzymatic activities of peroxidase at different pH levels.
For this experiment, where the enzymatic activity after denaturation was tested, it was
found that the activity was lower than the activity of the controlled variable (Figure 5). It can be
concluded that long exposure to boiling water can drastically reduce the enzymatic activity of
peroxidase, causing it to occur at a much slower rate than it normally would.
Denatured
0,7
Absorbance (500 nm)
0,6
0,5
0,4
Control
0,3
Boiled Extract
0,2
y = 0.0584x + 1.2135
R² = 0.9946
0,1
0
0
50
100
150
Time (seconds)
Figure 5. The enzymatic activity of peroxidase after being exposed to boiling water and denatured.
The effect that temperature has on enzyme activity was tested using 4 temperatures – 4
⸰
C, 23 ⸰C, 32 ⸰C, and 48 ⸰C. It turns out that enzyme activity is greater at 32 ⸰C. The trend differed
for each group, with the 32 ⸰C having the most change in absorbance while the other
temperatures (i.e. 23 ⸰C and 48 ⸰C) being the next group to produce a high change in absorbance
(Figure 6.).
Temperature
2,5
Absorbance (500 nm)
2
y = 0.0584x + 1.2135
R² = 0.9946
Control
1,5
4 ˚C
23 ˚C
1
32 ˚C
48 ˚C
0,5
0
0
20
40
60
80
100
120
140
Time (seconds)
Figure 6. Shows the effect temperature has the enzyme activity of peroxidase. The graph shows
that 32 ⸰C has a large effect on the enzyme activity than the other temperatures.
Discussion
This lab aimed to determine the enzymatic activity of peroxidase when exposed to
various conditions. The conditions that tested were concentrations of peroxidase, different
temperatures, different pH levels, and boiling the tested solution. The control allowed for the
adjustments of an experiment if any issues arose and acted as a reference point to compare
experimental results with.
Looking at the data in the first experiment, it can be inferred that a higher concentration
of peroxidase present increases the enzymatic activity, thus supporting the hypothesis. This
supports the results because the more peroxidase present increases the amount of substrate
hydrolyzed in the reaction. This caused the reaction to occur at a faster rate based on Figure 3.
In the second experiment, the pH level determined whether there would be an effect to
occur based on the intensity of the pH. Based on the data, the optimal that seemed to produce
high activity was pH 5, supporting my hypothesis that a slightly acidic solution would produce
greater enzymatic activity. This is again supported by the results because certain enzymes have
specific specificity for pH. What is meant by this is that the acidic or basic a solution is will
determine how the enzyme will react with its substrate. The pH level of a solution cannot only
change the shape of the enzyme, but it could also change the charge and shape of the substrate.
This behavior could cause any reaction from occurring due to the enzyme no longer able to bind
the active site of the substrate (Urry et al., 2016).
For the third experiment, it was thought that as the time left in boiling increased, that the
enzyme activity would decrease and eventually become constant. The data in Figure 5 shows a
constant trend, however, compared to the control it can be inferred that the enzyme activity will
be lower in boiling water than what it would be when not exposed to a factor like that. The heat
acting on the test tube significantly denatured peroxidase causing to be so low with a nonincreasing rate. This behavior disproves my hypothesis, that being exposed to boiling water
longer than 50 seconds will show an increase in enzymatic activity. In the fourth experiment, it
was known that at some temperature enzymatic reactions will slow down or stop, but the exact
temperature was unknown. To test this, four temperatures were used to see the effect different
temperatures have on enzymes – 4 ⸰C, 23 ⸰C, 32 ⸰C, and 48 ⸰C. Looking at the data in Figure 6, it
is apparent that 32 ⸰C is the optimal temperature that is best suited for maximum enzymatic
processes with the maximum amount of products produced without any negative effect on the
enzyme. With this information, it can be inferred that if the temperature were to go past 32 ⸰C
will slow down or cease, while if you if the temperature is less than 32 ⸰C will produce a slow
reaction, thus producing fewer products.
During experimenting, four factors were tested to observe how much enzymatic activity
is affected – concentration, pH, boiling, and temperature. Using peroxidase as the enzyme, this
experiment was able to see how effective it was at hydrolyzing hydrogen peroxide when exposed
to the four factors tested. From all the data collected, it was concluded that higher concentrations,
a slightly acidic solution (pH 5), and a temperature of 32 ⸰C were the optimal conditions that
favored enzymatic reactions. However, it was concluded that enzymatic reactions slowdown in
boiling water rendering the reaction to become constant. Some errors that might have been
introduced could have been some solutions were spilled as test tubes were being mixed together
and might not have been calibrated correctly.
Literature Cited
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Nigro, P., Pompilio, G., Capogrossi, MC., (2006). Cyclophilin A: a key player for human
disease. Cell Death & Disease.
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