Laboratory Exercise 6: Enzyme Activity

Name: Richard Senser Section: BIO111607 Date Performed: 6/15/11

Name of Lab Partner(s): Shannon Lee Professor: Judith Boyle Date Report Written: 11/29/19

Introduction
A number of chemical reactions continue their processes due to the factors of time or an extra input of
energy, usually in the form of heat, being added to the reactants of any chemical reaction. If one was to rely on
the factor of time, the chemical reaction could require mere nanoseconds or millions of years to complete on its
chemical process. The implication of energy in the form of heat could vary as well, from the heat produced by
one’s body to that produced from the death of a near by star. These reactions can be speeded up, so to speak, or
made to react/proceed at a lower energy input, by the addition of a catalyst to the reactants. The formal
definition of a catalyst is described in paragraph one, line four, of page one of the “Laboratory Exercise 6:
Enzyme Activity” lab manual. To define a catalyst in words more easily understood, a catalyst lowers the
energy of activation, EA, of a reaction, i.e. the energy needed to start the reaction; thus allowing the reaction to
occur at a faster rate.
So what is an enzyme? An enzyme is a protein-based, biocatalyst or an organic catalyst. These
biocatalysts are specific to a certain chemical reaction within an organism, such as metabolism. The challenge
with metabolism is many of the reactions needed, need to be performed very rapidly and at a low enough
temperature so that the reaction will occur and prevent the killing of the organism. This is where enzymes, our
biocatalysts, come in. The biocatalysts will lower the EA , thus allowing the necessary reactions within the
metabolic process to occur at a lower energy input; making them occur faster and at an optimal temperature to
protect they reaction, but, most importantly, the organism from killing itself.
Being a protein-based catalyst allows one to understand the reasoning behind the specificity of these
biocatalysts. The underlying structure and formation of a protein gives us the answer. Once the protein has
formed into its three-dimensional shape or its native conformation, it will only catalyze a specific reaction. This
phanominon is due to the active site, a site located on the three-dimensional structure of the enzyme, which
matches the shape of the target reactant aka the substrate. When an enzyme is placed into the reactants of a
specific chemical reaction, the enzyme-specific reactant or substrate binds to the active site of the enzyme,
which activates it; allowing it to lower the EA and speeding up the overall reaction.
Like Superman's weakness to kryptonite, everything has a point or substance that renders then
ineffective. In biological terminology an enzyme that is rendered ineffective is know as denaturation, the
process of a permanent change in or destruction of the enzyme’s three-dimensional shape. Enzymes can be
denatured by either chemical or environmental factors. For example, if an enzyme is placed into a solution with
a pH or temperature that differs from its optimal pH or temperature significantly, it will result in denaturation of
the enzyme and leave it ineffective.
In our experiment we tested the affect of differing levels of pH, i.e. 2, 7, 14 and differing temperatures,
i.e. 2°C, 40°C, 80°C on the amount of O2 produced, in milliliters, over a period of ten minutes, in thirty second
intervals, from the enzymatically driven reaction of hydrogen peroxide to water and oxygen:
2H2O2 2H2O + O2
The reason as to why we are measuring the amount of oxygen produced is to see if the pH and/or
temperature of the solution in which the enzyme is placed will have an affect the enzyme by either slowing the
rate of the reaction or causing denaturation. If the amount of oxygen produced is little to none, we know the
enzyme has been denatured. If the amount of oxygen produced is high, then we know the enzyme is working
within this optimal environment. We will also be examining the rate at which the oxygen is produced; the same
principles are in effect. If the rate is high, the reaction is proceeding at a rapid pace, alluding to an optimal
environment. If the rate of oxygen production is slow to stagnant, the reaction is not proceeding and this result
alludes to a less than optimal or denaturing environment.
My two hypotheses for this experiment is as follows:

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 A. If the catalase is placed into a solution in which the temperature differs significantly, as in an
extreme of 5°C or more, from 37°C, a normal body temperature; then the rate of the
enzymatically-driven breakdown of hydrogen peroxide by catalase will be affected by causing
the enzyme to denature; thus rendering it ineffective and stopping/slowing the rate of O2
produced.
B. If catalase is placed into a solution that is at a pH level which differs significantly, as in an
extreme of 0.50 on the pH scale or more, from the body’s pH range of 7.35 – 7.45; then the rate
of the enzymatically-driven breakdown of hydrogen peroxide by catalase will be affected by
causing the enzyme to denature; thus rendering it ineffective and slowing/stopping the rate of O2
produced.

Materials and Methods

My lab partner was Shannon Lee and we were testing the affects of adding catalase to the
enzymatically-driven breakdown of hydrogen peroxide at three differing levels of pH, 2, 7 and 12. The pH level
of 2 is highly acidic and mainly found within vinegar, the pH level of 7 is neural or that of water and the pH
level of 12 is highly basic and mainly found in household ammonia. The amounts of O2 produced from the
enzymatically-driven reaction were recorded within the table 1 and 2 and are displayed within the “Results”
section. The set up of this experiment and a list of all the material can be found on pages two and three, as well
as on part C of page 5 of the lab manual. The set up involved the preparation of the hydrogen peroxide solution,
5 milliliters of hydrogen peroxide along with 4 milliliters of catalase and 2 milliliters of distilled water, a 50
milliliter graduated cylinder a reaction chamber and a tub filled with 4 inches of water. The water was set at
37°C, an optimal temperature for this metabolic reaction, and the set up for this experiment can be seen on page
4 of the lab manual.
Results
Below are both the tables and graphs from the above experiment:
Enzyme Reaction at Differing pH Levels
Time (Min) pH 2 (ml) pH 7 (ml) pH 12 (ml)
0.00 0.00 0.00 0.00
0.50 0.00 13.00 0.00
1.00 0.50 20.00 1.00
1.50 1.00 26.00 1.00
2.00 1.00 34.00 1.00
2.50 1.00 39.00 1.00
3.00 1.00 43.00 1.00
3.50 1.00 46.00 1.00
4.00 1.00 47.00 1.00
4.50 1.00 49.00 1.00
5.00 1.00 50.00 1.00
5.50 1.00 52.00 1.00
6.00 1.00 52.00 1.00
6.50 1.00 53.00 1.00
7.00 1.00 53.00 1.00
7.50 1.00 53.00 1.00
8.00 1.00 53.00 1.00
8.50 1.00 53.00 1.00
9.00 1.00 53.00 1.00
9.50 1.00 53.00 1.00
10.00 1.00 53.00 1.00

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 Enzyme Reaction at Differing pH Leves
60.00

50.00
mls of O2 produced

40.00

30.00 pH2 (ml)

pH7 (ml)
20.00
ph12 (ml)
10.00

0.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00
Time (mins)

pH of 2 0.05974026 Average rate of 0.06 mls of O2 produced every 30 seconds.

pH of 7 4.158441558 Average rate of 4.16 mls of 02 produced every 30 seconds.
pH of 12 0.049350649 Average rate of 0.05 mls of O2 produced every 30 seconds.

pH of 2 1.00 Max rate of milliliters of O2 produced over ten minutes.

pH of 7 53.00 Max rate of milliliters of O2 produced over ten minutes.
pH of 12 1.00 Max rate of milliliters of O2 produced over ten minutes.

pH of 2 1:30 Time at which milliliters of O2 produced leveled off.

pH of 7 6:30 Time at which milliliters of O2 produced leveled off.
pH of 12 1:00 Time at which milliliters of O2 produced leveled off.

pH of 2 1.00 Total amount of milliliters of O2 produced over ten minutes.

pH of 7 53.00 Total amount of milliliters of O2 produced over ten minutes.
pH of 12 1.00 Total amount of milliliters of O2 produced over ten minutes.

Table 2 and Graph 2:

Enzyme Reaction at Differing Temperatures

Time (Min) 2°C 40°C 80°C
0.00 0.00 2.00 0.00
0.50 1.00 4.00 0.50
1.00 1.00 4.50 0.50
1.50 1.50 5.00 0.50
2.00 1.50 10.00 0.50
2.50 1.50 12.00 0.50
3.00 2.00 13.00 0.50
3.50 2.00 14.00 0.50

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 4.00 2.50 15.00 0.50
4.50 3.00 15.00 0.50
5.00 3.00 16.00 0.50
5.50 3.00 17.00 0.50
6.00 4.00 17.00 0.50
6.50 4.00 17.50 0.50
7.00 4.00 18.00 0.50
7.50 4.50 18.50 0.50
8.00 4.50 19.00 0.50
8.50 4.50 19.00 0.50
9.00 4.50 19.50 0.50
9.50 4.50 20.00 0.50
10.00 4.50 20.00 0.50

Enzyme Reaction at Differing Tempatures

25.00
mls of O2 Produced

20.00

15.00
2°C
10.00 40°C

5.00 80°C

0.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00
Time (Mins)

2°C 0.455844156 Average rate of 0.46 mls of O2 produced every 30 seconds.

40°C 1.727272727 Average rate of 1.73 mls of O2 produced every 30 seconds.
80°C 0.012987013 Average rate of 0.13 mls of O2 produced every 30 seconds.

2°C 4.50 Max rate of milliliters of O2 produced over ten minutes.

40°C 20.00 Max rate of milliliters of O2 produced over ten minutes.
80°C 0.50 Max rate of milliliters of O2 produced over ten minutes.

2°C 7:30 Time at which milliliters of O2 produced leveled off.

40°C 9:30 Time at which milliliters of O2 produced leveled off.
80°C 0:30 Time at which milliliters of O2 produced leveled off.

2°C 4.50 Total amount of milliliters of O2 produced over ten minutes.

40°C 20.00 Total amount of milliliters of O2 produced over ten minutes.
80°C 0.50 Total amount of milliliters of O2 produced over ten minutes.
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 Discussion
The purpose of this of our experiment was to test the affect of differing levels of pH, i.e. 2, 7, 14 and
differing temperatures, i.e. 2°C, 40°C, 80°C on the amount of O2 produced, in milliliters, over a period of ten
minutes, in two minute intervals, from the enzymatically driven reaction of hydrogen peroxide to water and
oxygen:
2H2O2 2H2O + O2
The reason as to why we are measuring the amount of oxygen produced is to see if the pH and/or temperature of
the solution in which the enzyme is placed will affect the enzyme by either slowing the rate of the reaction or
causing denaturation.
My two formal hypotheses for this experiment were as follows:
A. If the catalase is placed into a solution in which the temperature differs significantly, as in an
extreme of 5°C or more, from 37°C, a normal body temperature; then the rate of the
enzymatically-driven breakdown of hydrogen peroxide by catalase will be affected by causing
the enzyme to denature; thus rendering it ineffective and stopping/slowing the rate of O2
produced.
B. If catalase is placed into a solution that is at a pH level which differs significantly, as in an
extreme of 0.50 on the pH scale or more, from the body’s pH range of 7.35 – 7.45; then the rate
of the enzymatically-driven breakdown of hydrogen peroxide by catalase will be affected by
causing the enzyme to denature; thus rendering it ineffective and slowing/stopping the rate of O2
produced.

The above hypotheses are accepted in my opinion. The reasoning behind this would is displayed within
the table and graph. When looking at the affect of temperature on an enzymatic reaction, we see that at a
temperature of 2°C and a temperature of 80°C, both differing with an extreme of 5°C from 37°C, the
enzyme was denatured and rendered ineffective. This can be seen by both the low level of O2 produced
and the slowed to stagnant rate of O2 produced these temperatures. When all three values are compared,
it is both visually apparent and supported by the data that the optimal temperature at which this
particular enzyme can produce the most amount of O2 and work at its fullest poetical, is at a temperature
of 40°C; thus supporting hypothesis A. As with hypothesis A, hypothesis B is supported as well. One
can visually verify the support of hypothesis B by glancing within the table and graph. When looking at
the affect of pH on an enzymatic reaction, we see that at a pH of 2 and a pH of 12, both differing with an
extreme of 0.5 on the pH scale from the body’s pH range of 7.35 – 7.45, the enzyme was denatured and
rendered ineffective. This can be seen by both the low level of O2 produced and the slowed rate of O2
produced at both pH levels of 2 and 12. When all three values are compared, it is both visually apparent
and supported by the data that the optimal level of pH at which this particular enzyme can produce the
most amount of O2 is at a pH of 7; thus supporting hypothesis B.
Discussion Questions:
1. What do the rate of oxygen production and/or the amounts of oxygen tell us about the chemical
reaction?
a. The oxygen production and/or the amount of oxygen tell is if the enzyme is working within an
optimal environment. If the amount of oxygen produced is little to none, we know the enzyme
has been denatured. If the amount of oxygen produced is high, then we know the enzyme is
working within this optimal environment.
2. Given the conditions tested, which are the best for catalase activity?
a. A pH of 7 and a temperature of 40°C, which is not surprising seeing as this reaction occurs
within many organisms and the above levels known as near standard.
3. How does the 80°C treatment affect catalase? What experimental data supports your answer?
a. At a temperature of 80°C, we can see that the average rate of O2 produced is 0.01 mils every 30
seconds and the time at which the reaction leveled off was after 30 seconds. This tells us that at
this temperature, the enzyme cannot function, at first the substrate binds to the active site but
then the temperature is too high and the overall three-dimensional structure falls apart, or
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 denatures. This all happened within the span of 30 seconds and then the amounts of O2 produced
levels off; alluding to the inactivity of the present enzyme. For without catalase, there is not
reaction.
4. How does the 40°C treatment affect catalase? What experimental data supports your answer?
a. At a temperature of 40°C, we can see that the average rate of O2 produced is 1.73 mils every 30
seconds and the time at which the reaction leveled off was after 9 minutes and 30 seconds. This
tells us that at this temperature, the enzyme is in an optimal environment, at first the substrate
binds to the active site and the enzyme continues to proceed with the reaction until there all the
substrate has been used up. This happened within the span of 9 minutes and 30 seconds and then
the amounts of O2 produced levels off alluding to the depletion of the substrate within the
solution. For without the presence of a substrate, there is nothing to bind to the active site, and so
the enzyme will shut off.
5. How do various pH treatments affect the catalase? What experimental data supports your answer?
a. By looking at data, one can visually verify the affect of pH on an enzymatic reaction; we see that
at a pH of 2, the average rate of O2 produced was 0.06 mils every 30 seconds and the time at
which the production leveled off was after 1 minute and 30 seconds. With a pH of 12, we see
that the average rate of O2 produced was 0.05 mils every 30 seconds and the time at which the
production leveled off was after 1 minute. Both values differ with an extreme of 0.5 or more on
the pH scale from the body’s pH range of 7.35 – 7.45, this enzyme was denatured and rendered
ineffective due to the extreme environmental factors i.e. extreme basic and acidic nature of the
solutions. To put it simply, these environments are simply too acidic and basic for the enzyme to
function properly and resulted in a slowed or stagnant rate of O2 production.
6. Why does the production of oxygen slow down over time?
a. The enzyme is using up the substrate within the solution, thus the reaction slows and the rate of
O2 produced slows as well. The substrate is like gas and the enzyme is like the car, if the car has
little to no gas, it cannot function properly nor can it accelerate or continue to drive down the
road.
7. If you added more catalase to the 37°C reaction that had stopped producing oxygen, what do you expect
would happen?
a. Nothing. If you add more catalase to a solution that has no substrate, then the catalase that is
added will remain inactive, due to the lack of substrate binding to the active site.
8. If you added more hydrogen peroxide to the 37°C reaction that had stopped producing oxygen, what do
you expect would happen?
a. The reaction would continue. This is due to the addition of the substrate. If the reaction has
stopped producing O2 this means that there is not substrate left within the solution to bind to the
active site and activate the enzyme. If more substrate is added, it will bind to the active site of the
enzyme and allowing the enzyme to proceed with the break down of hydrogen peroxide to water
and oxygen; thus the production of oxygen would continue.

Though my both my hypotheses were supported, my lab partner and I came across some errors. For the
readings within the pH of 12, we noticed that the amount of O2 produced was far from those of other classmates.
After further investigation, we noticed that during the trial run, the airtight seal and rubber hose were not fixed
into the 50 mil-graduated cylinder. It was due this fact that our values for this trial were skewed.

Based on this experiment and the data collected one is able to conclude that the optimal environment in
which this specific catalase is able to function is around 40°C and in a pH around 7. This type of environment
allows the enzyme to function at its fullest potential and produce the maximum amount of product allotted by
the implemented amount of substrate present without the hindrance of environmental factors.

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