The Effect of Surface Area, Temperature, and pH on the Enzyme Catalase

Erin Simms
Biology 110: Molecular & Cell Biology
Section EG1
November 2
nd
, 2013









Plagiarism is the use of another persons hard work without giving proper credit. I have
properly cited my sources and given credit when appropriate. What follows behind this title page
represents my own work and not the work of another person.





Introduction:
Enzymes, which are typically made from protein, tremendously speed up the rate in
which chemical reactions occur in cells (Hershey et al., 2012.) They do this by lowering the
activation energy, or energy needed to initiate the reaction. The substance in which an enzyme
acts upon is the substrate (Solomon, Berg, and Martin, 2012.) Enzymes are very specific. They
each have distinct activation sites, or places where the reaction occurs, which allows them to
attach to the proper substrate. Catalase, an enzyme found in living tissue, is responsible for the
decomposition of hydrogen peroxide (H
2
O
2
), a substance that is extremely toxic to the body
(Solomon, Berg, & Martin, 2012.) Without this enzyme, hydrogen peroxide would be
decomposed at a much slower rate, causing a build-up of the deadly substance in cells. Due to
the high toxicity of hydrogen peroxide, organisms with an excess of the substance would
eventually face more complex issues, including potential death. Catalase works by breaking
hydrogen peroxide into two non-toxic substances: water and oxygen gas (Solomon, Berg, &
Martin, 2012.) The chemical equation is as follows: (2)H
2
O
2
+ catalase

= (2)H
2
O + O
2
(Hershey
et al., 2012.) In this lab, students were instructed to measure the amount of oxygen gas produced
as the result of enzyme reaction. The greater the amount of oxygen that was produced indicated
the greater the level of enzyme activity. By recording the oxygen levels produced, students were
able to find the preferred conditions for enzyme reactions.
The purpose of this experiment was to gain a better understanding of condition that affect
enzymes. Each enzyme is specific to the environment in which it is designated; for example, an
enzyme found in the stomach to help digest food would be able to withstand extremely acidic
environments. Therefore, each enzyme has favorable circumstances, including surface area
volume ratio, temperatures, and pH levels. By measuring the milliliters of oxygen gas (O
2
)
released at these various conditions, this lab proved how different factors affect the rate at which
enzymatic reactions occur.
With prior knowledge of the effects that increased surface area has on cells, it was easy to
hypothesize that the mashed banana, or enzyme with increased surface area, would also increase
the rate of enzyme reaction, or O
2
released. Many cells are not meant to withstand extreme
temperatures, and with this information it was natural to form a hypothesis: extremely high
(boiling) or extremely low temperatures (freezing) would entirely denature the enzymes; but less
extreme changes in temperature would have little to no effect on enzyme activity. Understanding
that an environment with a basic pH either adds hydroxide ions or takes away hydrogen ions, and
an environment with an acidic pH adds hydrogen ions, it was assumed that any change in pH
would alter the chemical structure of catalase, causing the enzyme to denature. In short, it was
hypothesized the enzyme catalase to be generally sensitive to environmental changes.








Results:
Graph I & Table I: The Effect of Surface Area on the Decomposition of Hydrogen Peroxide
with the Enzyme Catalase Using Banana:
Banana O
2
Gas
Collected
(mL)

0 min 1 min 2 min 3 min 4 min 5 min 6 min
Whole
(W)
0.5 mL 1 mL 1.5 mL 2 mL 2.4 mL 2.6 mL 2.9 mL
Mashed
(M)
0 mL 0.8 mL 1.1 mL 1.9 mL 2.6 mL 3.4 mL 4.4 mL



Graph II & Table II: The Effect of Temperature on the Decomposition of Hydrogen Peroxide
with the Enzyme Catalase Using Chicken Liver:

Time in Minutes
Liver
Temperature
O
2
Gas
Collected
(mL)

0 min 1 min 2 min 3 min 4 min 5 min 6 min
Iced 0 mL 2.8 mL 4.3 mL 5.6 mL 6.7 mL 7.7 mL 8.5 mL
Room 0 mL 5.5 mL 8.1 mL 12.3 mL 16.2 mL 19.7 mL 22.9 mL
37C 0 mL 4.1 mL 6.9 mL 9.4 mL 11.1 mL 12.9 mL 14.6 mL
Boiled 0 mL 0 mL 0 mL 0 mL 0 mL 0 mL 0 mL
Graph IV & Table IV: The Effect Iced Liver Returned to Room Temperature on the
Decomposition of Hydrogen Peroxide with the Enzyme Catalase Using Chicken Liver:

Time in Minutes
Iced
Liver
O
2
Gas
Collecte
d (mL)

0 min 1 min 2 min 3 min 4 min 5 min 6 min
Iced 0.9 mL 2.8 mL 4.3 mL 5.6 mL 6.7 mL 7.7 mL 8.5 mL
Room 0.5 mL 3.2 mL 3.8 mL 4.2 mL 5.3 mL 6.0 mL 6.5 mL


Graph V: The Effect of pH Levels on the Decomposition of Hydrogen Peroxide with the
Enzyme Catalase Using Chicken Liver:

Time in Minutes
Liver pH O
2
Gas
Collected
(mL)

0 min 1 min 2 min 3 min 4 min 5 min 6 min
3 0 mL 2.1 mL 2.5 mL 2.8 mL 3.1 mL 3.3 mL 3.4 mL
7 0 mL 5.5 mL 8.1 mL 12.3 mL 16.2 mL 19.7 mL 22.9 mL
10 0 mL 4.6 mL 5.2 mL 5.5 mL 5.9 mL 6.4 mL 6.9 mL

Experimental Procedures:
Materials:
For this experiment, a device for measuring gas levels was designed and set up for
Procedures I, II, VI, and V. The apparatus was placed in a bin of water, attached into place, and
consisted of a collection cylinder threaded with a tube that had a stopper attached to the opposite
end. Water filled the collection cylinder and the stopper was hung over the spot of attachment.
Students measured gas levels by observing the water level in the tube.

Procedure I: The Effect of Surface Area on the Decomposition of Hydrogen Peroxide with the
Enzyme Catalase Using Banana:
5 mL of pH 7.0, 3% H
2
O
2
was measured into two test tubes labeled W and M. A 1-
cm slice of banana was then cut and trimmed into two, 1 gram, equally shaped, wedges. One
wedge was kept whole and designated to the W test tube, while the other wedge was mashed in
and designated to the M test tube. 5 mL of pH 7.0, 3% H
2
O
2
was added first to the W test
tube, and the airtight stopper of the gas collection device was immediately placed on top of the
tube. The timer was set and the O
2
level of the W tube was measured and recorded each
minute, for six minutes. After six minutes, the W tube was removed from the device. 5mL of
pH 7.0, 3% H
2
O
2
was added to the M test tube, the stopper was placed and attached to the
device, and the gas levels were measured for another six minutes. Waste and tubes were poured
and disposed of in its proper receptacle.

Procedure II: The Effect of Temperature on the Decomposition of Hydrogen Peroxide with
the Enzyme Catalase Using Chicken Liver:
Four pieces of 0.5 gram pieces of whole liver were measured and designated to one of
four test tubes: Iced, Room, 37C, and Boiled. The piece of Iced liver was placed on a tray over
ice, the Room liver was left to sit on the lab table, the 37C was placed in a water bath with a
small amount of distilled water added to the liver, and the Boiled liver was put in a bath of
boiling water after distilled water was added to the liver. The liver was left in these conditions
for at least 30 minutes each, before 5 mL of pH 7.0, 3% H
2
O
2
was added to the first test tube.
The stopper was placed over the tube immediately, and just like in Procedure I, the amount of
oxygen gas was measured for a total of six minutes and recorded in one minute increments. This
procedure was repeated for the Room liver, 37C liver, and Boiled liver. The Room liver was
saved, along with the Iced liver (which was returned to room temperature), and the used H
2
O
2
from the Room tube and placed in a tube labeled UP. Any other waste or tubes were discarded
of in the proper receptacle.
Procedure III: The Resiliency of Enzyme Catalase, Are Enzymes Reusable?
A 0.5 gram piece of liver was cut and placed in test tube UP along with the used 5 mL
of pH 7.0, 3% H
2
O
2
. The used liver, saved from Procedure II, was placed in a tube with fresh 5
mL of pH 7.0, 3% H
2
O
2.
For this Procedure, students did not record gas levels, but they watched
for reactions.


Procedure IV: The Effect of Iced Liver Returned to Room Temperature on the
Decomposition of Hydrogen Peroxide with the Enzyme Catalase Using Chicken Liver:
5 mL of pH 7.0, 3% H
2
O
2
was added to a test tube labeled I, along with the Iced liver
that returned to room temperature. The stopper of the gas collection device was placed
immediately on the tube and the amount of O
2
gas was recorded each minute for six minutes.
These results were then compared to the Room liver results of Procedure II. Waste from test tube
I was poured in its container for disposal and the test tube was placed in the waste box designed
for glass.

Procedure V: The Effect of pH Levels on the Decomposition of Hydrogen Peroxide with the
Enzyme Catalase Using Chicken Liver:
Two pieces of 0.5 gram liver were cut and placed in a test tube labeled 3 or 10. 5 mL
of pH 3.0, 3% H
2
O
2
was added to the tube labeled 3. The stopper of the device was placed over
the tube immediately and the amount of O
2
gas was measured each minute for six minutes. This
method was repeated for the tube labeled 10, except 5 mL of pH 10.0, 3% H
2
O
2
was added to
the tube. The waste and test tubes were then disposed of in their appropriate bins.


Discussion:
This lab proved that enzymes are affected by changes in surface area, temperature, and
pH. Procedure I demonstrated that a high surface area to volume ratio was preferred in enzymatic
reactions. The more surface area an enzyme has, the more substrate it can reach, and the more
enzyme activity can occur. Data from Graph and Table #1 supports this finding, as the mashed
banana continues to react at a steady pace after the whole banana slows down.
Procedure II exhibited the effect of various temperatures on the rate in which reactions
occur. The more extreme the temperature change was, the less oxygen gas there was released.
Harsh changes to environmental temperature caused denaturation, making the enzyme less useful
or perhaps useless in producing the desired reaction (University of Illinois at Chicago, nd.)
Boiling temperatures completely denatured catalase, 37C temperatures slightly decreased the
rate of reaction, room temperature showed a steady increase of enzyme reaction, and iced liver
dramatically reduced the enzymes ability. I believe there was an error in this procedure because
the enzyme should have shown the most activity at 37C because it is the temperature of the
human body and where many enzymes are found.
Procedures III and VI demonstrated an enzymes resiliency to certain factors. Procedure
III revealed that hydrogen peroxide was not reusable, but already used catalase is. This was most
likely because the hydrogen peroxide had already used its hydrogen to initiate a different
reaction, so there was a lack of water in the substrate. However, a previously used enzyme did
create a reaction when paired with fresh hydrogen peroxide, demonstrating the capacity of an
enzyme to be reused. This proves that cold temperatures did not denature the enzyme, but just
slow down their reactions. With cold temperatures comes decreased molecular activity, meaning
that the enzyme and substrate would be unable to collide together as rapidly. Procedure VI
measured the enzymes ability to react after temperature change. The experiment concluded that
activation site of most catalase will return to their original form, but will be less effective in
inducing a reaction.
Lastly, Procedure V measured the effect of severe pH change on an enzymes
configuration, or ability to trigger a reaction. The results showed that extremely basic or acidic
environments greatly impact the effectiveness of an enzyme, with its most favorable pH to be a
perfectly neutral set of surroundings. Any inflow of outflow of hydrogen ions (with pH increase
or decrease) presents the possibility of structure change, which may lead to activation site shape
change or denaturation (University of Illinois at Chicago, n.d.). pH specificity plays a
tremendous role in the body. Simply put, enzymes are generally buoyant to most environmental
change, as they usually return to their shape, but extreme change will denature an enzyme,
causing irreversible damage to the lock and key model (University of Illinois at Chicago, n.d.).
In every lab, there are several inherent sources of error. In Procedures I-V, an imbalanced
or inaccurate scale would lead to ununiformed pieces of banana or liver. This would affect the
enzymes ability to speed the reaction; the more of an enzyme present in the H
2
O
2
, the quicker the
reaction will occur. Although there is not much that can be done about inherent discrepancies
such as faulty equipment, the student can always strive to be more careful in selecting tools and
noticing abnormal variations in data. A second inherent source of error could be simply
inaccuracies in O
2
readings. Once an inaccuracy was recorded by the student, they can be very
tough to distinguish. This would certainly lead the student to draw invalid conclusions. To help
improve this source of error, it was important for the person measuring to be familiar with lab
materials, as well as be prepared to analyze the readings immediately after the timer alerts.
Lastly, an error found in most experiments was issues with timing. If a measurement was taken
even seconds before or after it should have been, there would most likely be a discrepancy in the
data. The longer catalase had to act upon hydrogen peroxide, the more oxygen would be leaked,
especially in favorable conditions (Hershey et al., 2012). A useful strategy to help reduce error in
this sense is to assign each member with a responsibility they can focus solely on.
Particularly in this lab, a major source of error was the time it takes for the student to
plug the stopper in the test tubes after the substrate had been poured over the enzyme. It can be
assumed that some O
2
would be released prior to the placement of the stopper, forming an
imprecise reading. A more elaborate contraption may be able to fix this error. Perhaps using a
stopper with a syringe or compartment attachment to hold the hydrogen peroxide in the stopper
would be beneficial. Another source of error in the lab could be the failure to pour deionized
water onto the boiling liver sample and the 37C liver sample. This may have caused both
samples of liver to dehydrate in the temperature change process, which would change variables
we were not measuring for. Due to extreme dehydration, some enzymes may have denatured
prematurely, leaving us with faulty results. It is critical for each group member to familiarize
themselves and plan out the lab before they begin. With more than one person directing the
group, it is more likely for attention to be drawn to a missed step or an additional step. A final
specific source of error in this lab can be found in Procedure IV. The instructions in the manual
direct students to return the previously iced liver to room temperature but they do not specify
what room temperature is. It is also difficult for the student to determine whether or not the
sample has returned to room temperature, due to lack of equipment (thermometers). If a
sample was not restored to room temperature, the enzyme catalase would be at a disadvantage,
as it was not given a fair amount of time to return to its previous state. This error could be
eliminated with the addition of definitive room temperatures, or even ranges. If a thermometer
were to be provided, students could also compare the first room temperature reading to last,
eliminating the possibility of the liver to still be iced.





















Works Cited
Energy. (n.d.). University of Illinois at Chicago - UIC. Retrieved October 22, 2013, from
http://www.uic.edu/classes/bios/bios100/lecturesf04am/lect04.htm
Hershey, J., Baugh, J., Thorndill, D., ReValle, P., DeStefano, C., & Artes, L. (2012). Biology
110 Laboratory Textbook (Thirteenth Edition.). Boston, Massachusetts: Pearson.
Kaiser, Gary. "Enzymes." <i>Enzymes</i>. N.p., 27 June 2001. Web. 1 Nov. 2013.
&lt;http://student.ccbcmd.edu/~gkaiser/biotutorials/proteins/enzyme.html&gt;.
Solomon, E., Berg, L., & Martin, D. (2012). Energy and Metabolism. Biology: Community
College of Baltimore County Edition (Ninth ed., pp. 154-171). Mason, Ohio: Cengage
Learning.