1

The Effect of Temperature, pH, and Concentration on Enzyme Activity

By: Lauren Witmer
Introduction:
The objective of this lab was to demonstrate the presence of catalase in living
tissue and observe the conversion of hydrogen peroxide to water and oxygen gas by
the enzyme catalase. The amount of oxygen generated was measured during this
process and the rate at which the reaction occurred was calculated. The effect of pH,
enzyme concentration and temperature on enzyme activity was observed. The
hypothesis for this experiment was that the reaction with a pH of 7, temperature
between 30-35o C, and a concentration of 2.0mL catalase will have the highest, or
fastest, rate of reaction. Enzymes are a type of proteins, and, like all proteins, it can
function best in certain conditions, especially those related to temperature. If the
temperature is too low or too high, the enzyme’s structure can change and will not be
able to activate a reaction because of the change in shape (Nelson). As the temperature
rises, reacting molecules gain more and more kinetic energy, which increases the
chances of successful collisions, therefore the rate increases. Usually, the optimal
temperature is around human body temperature (37.5 0C) because many enzymes are
found in human cells, like catalase, for example, is an enzyme found in the liver. Above
this temperature, the enzyme structure will begin to denature, or break down, since the
intramolecular and intermolecular bonds are being broken as the enzyme molecules
gain even more kinetic energy (Enzymes). Extremely high or low pH values generally
result in complete loss, or denaturation of most enzymes. Each enzyme has a region of
pH optimal stability. According to Table II, catalase’s pH optimum is 7.0 (“Effects of
pH”). The rate of an enzyme-catalyzed reaction depends on the concentrations of
enzyme and substrates. As the concentration of either is increased, the rate of the
reaction will also increase. The reaction rate will increase with an increasing enzyme
concentration up to a point, which would be when there is no more substrate left to fill
active sites. Provided that the substrate concentration is high and that pH and
temperature are held constant, the rate of the reaction is proportional to the enzyme
concentration (Enzymes).
Pre-Lab Questions:
1. What are the general functions of enzymes?
Enzymes are used to speed up reactions by lowering the activation
energy (Ea).
2. What is the relationship between the structure and function of an enzyme?
Structure determines the specificity of the enzyme and the activity of
the enzyme Structure decides how well it will fit its substrate and
catalyzes a reaction.
3. Define initial reaction rate of an enzyme. How does initial reaction rate relate
to this lab?
 2

The initial reaction rate of an enzyme is the slope of the graph during
the period where the enzyme is acting on substrate molecules at
nearly a constant rate. The initial rate of any enzyme catalyzed
reaction is determined by the characteristics of the enzyme molecule
(The College Board).
4. State two conditions that might affect the initial reaction rate of an enzyme.
Temperature and pH
5. Contrast Catalyst and catalase.
Catalase is an enzyme found in the liver that catalyzes the
decomposition of hydrogen peroxide to water and oxygen, also known
as the fastest known enzyme. Meanwhile, a catalyst is a substance
that increases the rate of a chemical reaction without being consumed
in the process.
Methods:
Part I: Obtained all safety equipment and working catalase. Aprons and goggles were
used during this lab.
Part II: Measuring Enzyme Activity with the LabPro
A LabPro was set up and a Vernier gas pressure sensor was connected. A 1 L
beaker was obtained and filled with ice cubes to obtain a 0-5 degree Celsius water bath.
A waterproof marker was used to label each of the four clean test tubes with either “0-
5”, “20-25”, “30-35”, “50-55”. These numbers indicated the temperature range in
degrees Celsius of the water bath in which the enzyme was tested, which was the
independent variable in this trial. Testing began with the test tube labeled “0-5”. Six mL
of 1.5% of H2O2 was dispensed into each of the four test tubes. Using a thermometer,
the temperature of the water bath was found to make sure it was in the desired range
and this value was recorded in Table 2. Next, the test tube with the corresponding
temperature was placed in the correct water bath for 5 minutes, or until the Hydrogen
Peroxide temperature was also in the desired temperature range. This value was also
recorded in Table 2. Once the desired temperature, 1 mL of working catalase was
added to the test tube without letting the solution fall against the side of the test tube
and was swirled to mix the contents. Immediately afterward, the gas pressure sensor
stopper was pushed firmly in the test tube and data collection began on a 180 second
interval with data being recorded every 20 seconds (9 total samples). If the pressure
exceeded 130 kPa, the stopper was removed from the test tube and data collection was
stopped. Once data collection was finished, the contents of the test tube were
discarded. The rate of enzyme activity, or the dependent variable, was found by finding
a linear regression on the data points recorded. Next, a 1 L beaker was filled with 20-25
degree Celsius (room temperature) and using the corresponding test tube labeled “20-
25”, 1 mL of the catalase was inserted and data collection began again. This was done
with all temperatures and their corresponding beakers, getting to the correct water
 3

temperature using a heating plate. All results were recorded in Table 3. Enzyme
concentration and pH were held constant in this trial.
Part III: Testing the Effect of Ph
Three clean test tubes were obtained and labeled with 4, 7 or 10, representing
different pH levels. 3 mL of 3% H2O2 was added to each test tube. The test tube with the
label “4” had 3 mL of pH 4 buffer solution added to it, then 1mL of the catalase was
added to the solution without any hitting the sides of the test tube. Immediately
following, the gas pressure sensor was placed the test tube firmly and data collection
began on a 180 second interval with data being recorded every 20 seconds. This was
repeated for every test tube with the corresponding pH buffer solution to that on the test
tube. A linear regression on each graph was made and recorded results in Table 3. The
enzyme concentration and temperature were held constant in this reaction. The
independent variable was the pH of solution that was added which ultimately affected
the dependent variable of the reaction rate.
Part IV: Testing the Effect of Enzyme Concentration
Four clean test tubes and labeled each of them either “0.5”, “1.0”, “1.5”, or “2.0”,
indicating the quantity of catalase, in mL that was put into each test tube. Six mL of
1.5% H2O2 was added to each test tube. 0.5 mL of the catalase into solution test tube
labeled “0.5” and gently swirled to mix. The gas pressure sensor stopper was pressed
firmly into the test tube and data collection started immediately after on a 180 second
interval with data being recorded every 20 seconds. This was repeated with every test
tube with the same amount of catalase corresponding to that written on the test tube. A
linear regression of the graph was found and the rate of reaction recorded in Table 3.
The constants in this segment were temperature and pH. The independent variable was
the enzyme concentration which led to determine the dependent variable, or reaction
rate.
Results and Analysis:
Tables:
Table 1
Activity Observations
Part I A: Presence of Catalase in Liver Working catalase bubbled, rose to top,
and fizzed and turned slightly red
Part I B: Enzyme Activity Bubbles;
Part II B: Effect of Temperature Bubbles; optimum temperature was found
to be 50-550C
Part III: Effect of pH Bubbles; optimum pH was 10
Part IV: Effect of Enzyme Concentration Bubbles; optimum concentration was
1.5mL of catalase
 4

Table 2
Vial Temperature of H2O (in Temperature of H2O2 (in
degrees Celsius) degrees Celsius)
0-5 5.00 4.50
20-25 21.50 22.00
30-35 32.50 32.50
50-55 53.00 52.00

Table 3
Vial Rate (kPa/sec)
0-50C 0.22
20-250C 0.23
30-350C 0.21
50-550C 0.28
pH 4 0.06
pH 7 0.17
pH 10 0.20
0.5mL 0.01
1.0mL 0.13
1.5mL 0.41
2.0mL 0.30
 5

Graphs:

This graph shows the relationship between the time and pressure in a catalyzed reaction with a temperature of 4.5
degrees Celsius. As seen in the graph, the reaction reached 130kPa before the 180 second interval was complete.
The slope of this graph was 0.22 kPa/sec.

This graph shows the relationship between time and pressure in a catalyzed reaction with a solution temperature of
22 degrees Celsius. As shown above, the reaction reached 130kPa before the 180 time interval was up. The slope of
this line is 0.23 kPa/sec.
 6

30-35- this graph shows the relationship between time and pressure in a catalyzed solution with a temperature of
around 32.5 degrees Celsius. The slope of this graph was .21 kPa/sec. As seen, there was initially a slower rate of
reaction which then became steeper, therefore increasing the slope (rate).
 7

This graph shows the relationship between time and pressure of a catalyzed reaction with a temperature of 52
degrees Celsius in the hydrogen peroxide solution. This reaction reached a pressure of 130kPa very rapidly,
therefore causing this to be the steepest slope, or largest rate of .28kPa/sec.

Ph 4- This graph shows the relationship between time and pressure of a catalyzed reaction with a buffer with a pH of
4 added. This reaction did not reach 130kPa in the 180 second time interval and had a relatively slow rate of about
0.06kPa/sec.
 8

Ph 7- This graph shows the relationship between time and pressure of a catalyzed reaction with a buffer with a pH of
7 added. The slope of this line was 0.17kPa/sec.

Ph 10- This graph shows the relationship between time and pressure of a catalyzed reaction with a buffer with a pH
of 10 added. The rate of reaction for this pH was 0.20 kPa/sec
 9

0.5 mL- This graph shows the relationship between time and pressure of a catalyzed reaction with only 0.5mL of
catalase being added. The rate of this reaction was .01kPa/sec.

1.0mL- This graph shows the relationship between time and pressure of a catalyzed reaction with a buffer with 1.0mL
of catalase being added to the solution. The rate of this reaction was 0.13kPa/sec.
 10

1.5mL- This graph shows the relationship between time and pressure of a catalyzed reaction with 1.5mL of catalase
being added to the solution. The rate of this reaction was 0.41kPa/sec.

2.0mL- This graph shows the relationship between time and pressure of a catalyzed reaction with 2.0mL of catalase
being added to the solution. The slope for this reaction was around 0.30 kPa/sec.
 11

Discussions/Conclusion:
The conversion of hydrogen peroxide to water and oxygen gas by the enzyme
catalase was observed under different environmental conditions. The amount of oxygen
generated was calculated and the rate at which the reaction occurs was determined for
various reactions. After observing many experiments and determining all of the rates of
reactions, with independent variables of temperature, pH, and enzyme concentration, it
was found that the hypothesis that the reaction with a pH of 7, temperature between 30-
35o C, and the highest concentration of catalase will have the highest, or fastest, rate of
reaction, was incorrect. During the trials with the change in temperature, the highest
rate of reaction was 0.27858 kPa/sec. The highest rate was .4084kPa/sec among the
different amounts of catalase, with 1.5mL of catalase producing this rate. Amongst the
trials dealing with different pH levels, pH of 10 led to the highest reaction rate with .202
kPa/sec. The highest rate of reaction for both pH and temperature were surprising
because it was found in research that the optimum temperature of catalase was 30-35
degrees Celsius, while in the experiment it was found to be 50-55 degrees Celsius.
Research also stated that the optimum Ph for catalase is 7, while in the experiment it
was found to be 10, which was very surprising (Effects on pH). At these high
temperature and pH levels, one would think the enzyme would break down and
denature because the bonds are being broken and the enzyme molecules are gaining
too much kinetic energy. If this lab were to be done again, results should be discussed
amongst the class and a t-test should be used with all of the class data to determine
more exact reaction rates for each independent variable. Some sources of error in this
lab would be not comparing class data, conducting only one trial for each reaction, and
not being able to get the gas pressure sensor stopper in the test tube immediately after
adding the catalase, which would change the initial pressure of the solution which would
impact the slope of the linear regression. There also was no control group in this
reaction, which would have been helpful in determining the effect of an enzyme in
normal conditions.
 12

Questions:
1. Many enzymes end with “ase”. Come up with your own enzyme, then
name and explain what this enzyme does. Draw the enzyme and
substrate in the space provided below.
Strawberryase is an enzyme that speeds up the ripening of a strawberry
by reducing the activation energy of the combining of sugars.

2. In this lab you use a gas pressure sensor to measure the amount of gas
being evolved. What was the gas being evolved? Suggest a method you
could use to determine this.
Oxygen was the gas that was evolved. A method to determine how much
oxygen was evolved is to use a glowing splint and place it in the gas that’s
being released. If the splint catches fire, its oxygen because oxygen
supports combustion
3. a. Was all of the catalase consumed?
Not all of the catalase was consumed due to the time restraints in our
experiments.
b. Will this reaction continue indefinitely?
No, the reaction will not continue indefinitely because it will run out of
either enzymes or substrates.
c. What are the limiting factors in this experiment?
The limiting factors in this experiment are time, availability of catalase, and
number of trials.
4. Lock and Key Model- an analogy that represents the enzymes and the
substrate in which the enzyme is the lock and the substrate is the key, ultimately
meaning that only the right sized key will fit into the lock. If the substrate is shaped
differently that the active site, it won’t fit in the enzyme
Induced Fit- where an enzyme binds to the substrate and changes its
shape which leads to a tighter grip on the substrate
Competitive Inhibitor- an inhibitor binds to the active site of an enzyme
creating competition with the substrate
 13

5. Recent advances have allowed humans to mass produce certain

enzymes. Research one such enzyme and explain how this enzyme has been
used to benefit society.
E. Coli has been engineered to produce taq polymerase which is important
because without it, PCR and genetic sequencing, which help discover diseases and
sequences in the human genome, would be impossible.
6. Based on your results from this lab, what would be the ideal environment
for catalase?
According to the data, the optimal conditions for catalase reactions to occur
would be a temperature between 50-55oC, a pH of 10, and 1.5mL of Catalase.
7. In the space below, predict what would happen if you were to boil your
catalase prior to performing this experiment, then draw a graph of your
prediction. Be sure to label the graph properly.
If boiled, the enzymes within the catalase would begin to change its shape and
denature due to too high of temperatures which would bring the reaction to a halt. The
graph would begin to increase but quickly level off because enzymes would be
denaturing.

8. Amylase is an enzyme that aids in the digestion of starches and has an

ideal temperature range of 25-40 degrees Celsius (approximately human body
temperature). Predict what would happen to the rate of amylase activity at 25
degrees Celsius.
The rate of the reaction would decrease and therefore the enzyme would not
function as fast and efficiently.
 14

References:
“Effects of PH.” Worthington-Biochem, Worthington Biochemical Corporation, 2017,
www.worthington-biochem.com/introbiochem/effectsph.html.
“Enzymes.” RSC, Chemistry for Biologists.
Nelson, M. Rae. "Enzymes." Experiment Central: Understanding Scientific Principles
Through Projects, edited by Kristine Krapp, 2nd ed., UXL, 2010. Science in
Context,
link.galegroup.com/apps/doc/CV2644200015/SCIC?u=va_s_080_0610&xid=0ed
ed7e2. Accessed 21 Nov. 2017.
The College Board. AP Biology Laboratory Manual for Students. Advanced Placement
Program, the College Board, 2001.
 15

Lab Report Scoring Rubric (130 pts.)

Please attach this rubric to the back of your lab report (-5 if not attached)

Formatting and writing style: (25 pts.)

 Used #12 point Arial font. Yes = 5, No = 0

 Bold Headings / Sections. Yes = 5, Some = 2, No = 0
 Past tense and passive voice. Yes = 5, Some = 2, No = 0
 Writing Style (10 pts.)
o 10 pts. – No obvious typographical or spelling errors. Writing clear, concise and
grammatically correct.
o 5 pts. – Few obvious typographical or spelling orders (2 or so). Writing clear,
concise and almost grammatically correct.
o 0 pts. – Poor sentence structure (incomplete sentences or run on sentences).
Language is unclear or confusing. Spelling errors and grammatical
problems.
TITLE (5 pts.)

Describes the experiment well – The Effect of _______ on ________. Yes = 5, OK = 2, No = 0

INTRODUCTION (20 pts.)

 Background
o 10 pts. - concise, references used and cited, provides foundation of study. Why
is any of this important? What have others found? Includes answers to
pre-lab questions (if applicable).
o 5 pts. – somewhat concise, outside references used but too few, wrong type of
references or references used inadequately.
o 0 pts. – background overall lacking, no references, poorly written
 Objectives
o 5 pts. – clearly stated
o 3 pts. – not clearly stated
o 0 pts. – not stated
 Hypothesis
o 5 pts. – clearly stated and supported by background research
o 3 pts. – not clearly stated and/or not supported by background research
o 0 pts. – not stated
METHODS AND MATERIALS (20 pts.)

 20 pts. – Detailed (but not overly, can be distracting) instruction regarding materials
used (not listed) and methods performed. Past tense and passive voice used.
Someone could actually repeat the experiment. Variables, controls, and
constants included. Statistical methods, replications/sample size and safety
included.
 15 pts. – Some details regarding materials used and methods performed but some
important details missing or incorrect and/or statistical methods,
replications/sample size, and/or safety incomplete. Variables, controls,
constants included.
 10 pts. – Some details regarding materials used and methods performed but some
important details missing or incorrect. Statistical methods, replications/sample
size and/or safety incomplete. Variables, controls, constants missing or incomplete.
 16

 5 pts. – Overall incomplete, unclear, could not repeat the experiment, many incorrect or
missing details (statistics, replication/sample size, safety, variables, constants,
control).
 0 pts. – not included
RESULTS (20 pts.)

Visual:

 10 pts. – Visually describes overall results. Proper graph / table legends and labels.
Properly represented data. Numbers to the tenths position. Gives proper p
value and / or other statistics.
 5 pts. – Visually describes overall results. Graph / table legends and labels incorrect or
lacking. Somewhat properly represented data. Numbers to the tenths position.
Gives proper p values and / or other statistics.
 0 pts. – No graphs or tables or extremely poorly constructed.
Written:

 10 pts. – Verbally describes overall results (for each graph / figure). NO discussion,
conclusion or methods. Gives averages or trends important to the experiment.
Numbers to the tenths position. Gives p values and / or other statistics.
Correctly identifies table or graph / figure in text.
 5 pts. – Verbally describes overall results but too much information giving details which
should be in a table or figure. Some information is discussion, conclusion or
methods. Lacks concise averages or trends. Numbers inappropriately
represented. Gives p values but not correctly. Poorly identifies table or graph /
figure in text.
 0 pts. – No written results or extremely poorly constructed
DISCUSSION / CONCLUSIONS (20 pts.)

 20 pts. – Clear, concise conclusion. Relates actual results to the background which is
concise. Relates results to concepts learned in class. Relates results to
objective(s) and hypothesis and explains unexpected or contradictory data.
Outside references used. Discusses what could be corrected or done
differently.
 15 pts. – Clear, concise conclusion. Relation of results to background and / or concepts
learned in class is lacking. Relates results to objective(s) and hypothesis and
explains unexpected or contradictory data. Outside references used.
Discusses what could be corrected or done differently.
 10 pts. – Conclusion unclear. Relation of results to background and / or concepts
learned in class is lacking; no outside references. Relates results to
objective(s) and hypothesis and explains unexpected or contradictory data.
Discusses what could be corrected or done differently.
 5 pts. – Conclusion unclear. Relation of results to background and / or concepts learned
in class is lacking or nonexistent; no outside references. Inadequately relates
results to objective(s) and hypothesis and / or fails to explain unexpected or
contradictory data. Inadequately discusses what could be corrected or done
differently.
 0 pts. – No discussion / conclusion
QUESTIONS (10 pts.)

REFERENCES (10 pts.)

 17

 10 pts. – Number and type (appropriate to study? Very old? New references? NO labs
used as references) of references appropriate. All are used in text correctly.
Correct format.
 5 pts. – Lacking proper references and / or incorrectly used in text.
 0 pts. – No references listed and / or not used in text.