Lab 6: Enzyme activity and lab report
BIOL&211 – Majors Cellular – Cascadia College
30 pts (including online pre-lab), due: 2 weeks from today2 weeks from today
Purpose:
Translating scientific data into a meaningful form is key to the process of science; the mark of a
successful scientist is someone who can communicate their ideas both to the public as well as
to other scientists. During this lab activity you will engage in the scientific method and
participate in authentic data collection. This will help you develop the transferable skills of
generating hypotheses and making predictions, as well as collecting, analyzing, and interpreting
scientific data. Using results that you generate as well as provided data, you will visualize key
trends in enzyme activity under varying environmental conditions using figures. This will
prepare you to write scientifically on future assignments in the Majors Biology series or in a
future career in science.
Background:
A chemical reaction is the interaction of two or more chemical substances in a way that results
in a chemical change in those substances. In chemical reactions, reactant (or substrate)
molecules are converted to product molecules. For example:
The rate of a chemical reaction is the change in the molecules involved (either the reactants or
the products) divided by the change in time. The rate of reaction is not constant but decreases
with time due to decrease in the concentration of reactants. It can be measured in two ways:
(1) The increase in product molecules over a
specified time interval.
(2) The decrease in reactant molecules over a
specified time interval.
Fig. 6-1. Concentration of the products and reactants in a chemical reaction over time.
As the concentration of the reactants drops, the concentration of the products climbs since they are directly related.
A catalyst is a substance that accelerates the rate of a chemical reaction, but the catalyst itself
is not used up in the reaction. Protein enzymes are responsible for catalyzing virtually all
1
biochemical reactions in cells. Each enzyme works on a specific substrate and their ability to
catalyze particular reactions can be affected by changes in the chemical and physical conditions.
Catalase is the enzyme used in this lab experiment. It has four polypeptide chains, each
composed of more than 500 amino acids. This enzyme is found in essentially all aerobic
organisms. One function of catalase within cells is to prevent the accumulation of toxic levels of
Hydrogen peroxide (H2O2) formed as a by-product of metabolic processes. Catalase also takes
part in some of the many oxidation reactions that occur in the cell.
In the absence of catalase, this reaction will occur spontaneously, but very slowly; if H2O2 is left
in an open container it will slowly decompose, leaving water in the container and giving off
oxygen gas.
Catalase speeds up the reaction considerably! It is one of the fastest enzymes known, with
turnover numbers measured from 30,000–800,000 molecules converted per second! In this
experiment, a rate for this reaction will be determined.
Much can be learned about enzymes by studying the kinetics of enzyme-catalyzed reactions.
For example, it is possible to measure the amount of product formed, or the amount of
substrate used, from the moment the reactants are brought together until the reaction has
stopped. If the amount of product formed is measured at regular intervals and this quantity is
plotted on a graph, a curve like the one that follows is obtained.
Fig. 6-2. Oxygen released
over time.
The volume of oxygen (in
mL) released from the
reaction of catalase
enzyme and hydrogen
peroxide was measured
over a 5-minute time
interval. The initial
reaction rate is 39.5
mL/min, which is much
higher than the average
reaction rate of 20.8
mL/min.
2
Study the solid curved line on Fig. 6-2. As time progresses, the amount of oxygen (product)
being produced increases at a steady rate. This is known as the initial reaction rate. After a
period of time (marked by a dotted line), this rate slows down considerably. Because of this
change, for the purposes of this experiment we will define overall enzyme activity as the
volume of O2 produced over a 5-minute interval, which is proportional to the average rate of
the reaction.
Knowledge – you will
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Discuss the role of enzymes in biological reactions.
Explain a method of measuring the speed of biochemical reactions.
Describe the function of the catalase enzyme.
Discuss how enzyme concentration & temperature affect the rate of enzyme reactions.
Skills – you will be able to
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Follow a laboratory procedure to complete an experiment.
Collect and analyze quantitative (numerical) data.
Write a lab report with results and discussion sections based on self-generated data as well
as data generated collaboratively.
Create scientific figures/graphs using computer software.
Use correct lab report format for captions, citations, and references.
Tasks:
Experimental procedure
In this experiment, you will measure the disappearance of a substrate and the accumulation of
a product to determine the rate of a chemical reaction. The reactant is liquid H 2O2 and the
product is gaseous oxygen (O2). These will be measured by recording the displacement of gas in
a volumetric cylinder over a 5-minue duration (see Fig. 6-3). The initial volume of gas is
recorded at time t=0 (starting point), and the accumulation of gas is measured every 30 seconds
for the first 2 minutes, and then once a minute for the remaining 3 minutes (see table 1 below).
Part 1. Obtaining the Baseline Data for Enzyme Activity
Work in teams of 3 or 4. Assemble the apparatus as demonstrated in class (see Fig. 6-3 below).
Into the clean syringe, first draw 2 mL of 3% liver extract immediately followed by 2 mL H2O
(for 4 mL total). Inject 3 mL of the now diluted liver extract into 10 mL of H2O2 solution (you
should have a little left over in your beaker).
3
Fig. 6-3. Experimental setup.
A graduated cylinder placed upside down in a
water bath is connected via surgical tubing to a
flask containing H2O2. Liver extract containing
diluted catalase enzyme is injected into the flask
to catalyze the decomposition of the reactant.
DO NOT REMOVE THE SYRINGE. As one group member injects the diluted extract, another
member should continuously swirl the flask of H2O2 while O2 produced by the reaction is
collected. In table 1 below, another team member should write down the levels of gas in the
cylinder (in mL) for a total of 5 minutes.
Table 1. Control data.
O2 gas produced at room temperature using 3 mL of 1:1 diluted (1.5%) liver extract. These data serve as a baseline
control.
Time
(min)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Gas level in
cylinder (mL)
Total (cumulative) gas produced (mL)
(gas level at time = t – gas level at
time = 0)
N/A
Room temperature (°C):
Handling of results (to be done later for your Lab Report):
Using Excel or other software, plot the volume of gas produced as a function of time (as shown
in Figure 6-2).
By completing this first experiment, you have just obtained the baseline data for the enzyme
activity in your 1.5% liver extract at room temperature (be sure to record the actual room
temperature). These data will be considered your control. Make sure you understand why this
particular experiment acts as a control.
Your group will be assigned ONE of two the variables listed to investigate further. If your group
is investigating the effect of enzyme concentration on the speed of enzymatic reaction skip to
Part 3 on p. 7 of the lab. Otherwise, if you group is investigating the effect of temperature on
enzyme activity, please continue reading.
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Part 2. Effect of Temperature on Enzyme Activity
Before you move forward with further experimentation make sure to clean your flask (pour
your “used” H2O2 down the drain) and add 10 mL of fresh H2O2 into the flask for the next
injection. Your syringe containing liver extract can be reused since you will be using the same
concentration of liver extract for each experiment. During all experiments, remember to keep
the liver extract (containing catalase) separate from H2O2 until you start timing.
Think about and discuss with your group the following questions before you proceed.
Quickly write your answers in the spaces provided. Although I will not be collecting your
responses, they will help you to write your lab report!
1. Do you expect temperature will affect the rate of an enzymatic reaction?
a. If so, will the reaction rate increase or decrease with increasing temperature?
2. Is there any cutoff point in temperature beyond which the reaction will not occur?
a. If so, how can you explain this?
Three experimental temperatures (4°C, 37°C, & a maximum temperature) have been prepared
for testing the effect of temperature on catalase activity in your liver extract. Max temperature
should be ~ 50°C, but may vary from lab to lab. Temperatures above 55°C are not used because
H2O2 will break down spontaneously and the expansion of air at higher temperatures can
interfere with accurate measurements of O2 production.
Record max temperature used here: __________
Your goal is to regulate the temperature of the reaction. The reaction takes place in the flask
containing H2O2, so that flask must be immersed in a water bath at the desired temperature.
The same must be done for the enzyme solution to prevent the H2O2 mixture from changing
temperature when the enzyme is added. Both the H2O2 and enzyme solutions should sit in the
water bath for at least 2 minutes to thermally equilibrate. Note that your O2 collection cylinders
5
should NOT be in the temperature bath. Record your experimental data (8 data points for each
temperature, for a total of 24 data points) in Table 2 below.
At the end of these experiments, you will have experimental results for 4 different
temperatures (you have already measured enzyme activity at room temperature in Part 1 of
the lab). Once you have finished collecting and recording your data, skip to Part 4 to prepare
your lab report write up.
Table 2. Experimental data (temperature)
O2 gas produced (dependent variable) at various reaction temperatures (independent variable) and 1:1 diluted
(1.5%) liver extract.
For temp=4°C
Time (min)
Gas level in cylinder (mL)
Total (cumulative) gas produced (mL)
N/A
Gas level in cylinder (mL)
Total (cumulative) gas produced (mL)
N/A
Gas level in cylinder (mL)
Total (cumulative) gas produced (mL)
N/A
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
For temp=37°C
Time (min)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
For max temp
Time (min)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Handling of results (to be done later for your Lab Report):
Using Excel or other software, plot the volume of gas produced as a function of time for all four
temperatures as shown in Figure 6-2 (your graph should have four curves).
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Part 3. Effect of Enzyme Concentration on the Speed of Reaction
Before you move forward with further experimentation make sure to clean your flask (pour
your “used” H2O2 down the drain) and add 10 mL of fresh H2O2 into the flask for the next
injection. Your syringe containing liver extract also needs to be rinsed after each run since you
will be using different concentrations of liver extract for each experiment. During all
experiments, remember to keep the liver extract (containing catalase) separate from the H2O2
until you start timing.
Think about and discuss with your group the following questions before you proceed.
Quickly write your answers in the spaces provided. Although I will not be collecting your
responses, they will help you to write your lab report!
1. Does the concentration of catalase have any effect on the rate of the reaction?
a. If so, will the reaction rate increase or decrease with increasing enzyme concentration?
In sequence you will conduct four separate experimental runs by injecting different dilutions of
the liver extract into H2O2 (do not forget to replace your H2O2 and to rinse your syringe after
each run). For each run, prepare 4 mL of the diluted enzyme solution, of which you will use only
3.0 mL total volume per reaction (see the table below when preparing each dilution). The liver
extract and distilled water can be measured using your syringe.
Table 3. Dilution recipes
Prepare 4 mL for each concentration of enzyme. Of this, you will only be using 3 mL of the prepared enzyme
solution for each run, so you will have a little extra in your beakers after each injection.
Enzyme concentration
0% liver extract
1.5% liver extract*
3.0% liver extract
0.75% liver extract
0.375% liver extract
Dilution recipe
4.0 mL distilled water
2.0 mL 3% extract + 2.0 mL of water
*you have already completed this: see Part 1
4.0 mL of undiluted extract, no water is added
1.0 mL of 3% liver extract + 3.0 mL distilled water
0.5 mL of 3% liver extract + 3.5 mL distilled water
For lower strength enzyme concentrations (0%, 0.75% and 0.375%), measure the O2 in a 100 mL
graduated cylinder (if available), rather than a 250 mL cylinder. Record your data in the
corresponding tables below.
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Table 4. Experimental data (concentration)
O2 gas produced (dependent variable) at various enzyme concentrations (independent variable) at room
temperature.
Enzyme concentration = 0%
Time (min)
Gas level in cylinder (mL)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Enzyme concentration = 3.0%
Time (min)
Gas level in cylinder (mL)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Enzyme concentration = 0.75%
Time (min)
Gas level in cylinder (mL)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Enzyme concentration = 0.375%
Time (min)
Gas level in cylinder (mL)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Total (cumulative) gas produced (mL)
N/A
Total (cumulative) gas produced (mL)
N/A
Total (cumulative) gas produced (mL)
N/A
Total (cumulative) gas produced (mL)
N/A
Handling of results (to be done later for your Lab Report):
Using Excel or other software, plot the volume of gas produced as a function of time for all five
enzyme concentrations as shown in Figure 6-2 (your graph should have five curves).
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Part 4. Lab Report write up.
Working individually, you will craft a lab report using 1) your groups’ data for your group
variable (the variable your group tested in lab), and 2) provided class data that has been
compiled across multiple class sections, for the class variable (the variable your group did not
test). You will generate two separate hypotheses: the first will be about your groups’ variable
(either temperature or concentration), and the second will be about the variable you did not
investigate.
Your goal should be to write your report as if was a real scientific journal article, rather than just
an in-class assignment. A standard scientific paper includes the following sections: Introduction,
Methods, Results, Discussion/Conclusions, and References. However, for this assignment, your
lab report will only have Hypotheses, Results, Discussion, and References sections. Note that
you may not use quotations anywhere in your writeup; all material should be in your own
words.
Criteria for success:
This assignment is to be submitted online on CANVAS. Paper copies will not be accepted.
Required elements for your writing assignment:
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A title for the report (and your name, class, and section)
Hypotheses (covering both variables) that are specific and have been actually tested by the
methods you used.
T written Results section that presents 1) data from your group tests, and 2) the provided
class data for the other variable you did not test.
o Minimum of 2 figures total: one for your variable and one for the class data.
A Discussion section that utilizes at least 2 unique and appropriate references (see below
for specific guidelines). Each variable should be addressed in a separate paragraph, each
with its own unique reference.
A References section at the end of the report in APA format.
Proper grammar and spelling throughout.
Required formatting elements:
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All text (except that found in captions and the References Section) should be neatly typed
using grammatically correct sentences and correct spelling (this is VERY important in this
particular assignment)
Each section except the title should have its own heading
Figures and captions must follow the guidelines outlined later in this handout.
References and in-text citations must follow the format and style outlined later in this
handout.
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Length requirements:
There is no minimum length requirement. However, keep in mind that it would be impossible to
receive a good grade if your report is very short (one page is not enough space to satisfactorily
analyze and discuss your results). I will enforce a maximum length requirement—your report
must not include more than four pages of text (excluding figures and references). I am looking
for quality of writing, not quantity. With technical writing the goal is to make your writing
precise and concise. Important considerations (this information is relevant to any type of
scientific report) are as follows:
Title
Purpose: to accurately and efficiently convey to your reader the nature of your experiment.
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Your title should not be “Enzyme Activity lab.” It should be professional in tone and should
describe to your reader the scope and nature of your experiment.
Here is an example of a title recently published in the journal Science: “Revealing the
Angular Symmetry of Chemical Bonds by Atomic Force Microscopy.” This title is concise
and conveys to the reader the exact nature of the experiment. Try to achieve the same level
of sophistication and clarity.
Results section
Purpose: to summarize and present meaningful results of the experiment to your reader.
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You will create two parts to Results: Part I, which addresses your groups’ variable only; and
Part II, which addresses the class variable only (the one your group did not test). Label the
Results section AND Parts I and II clearly.
The Results Section should contain only results—resist the urge to make conclusions or to
interpret your data – save that for the Discussion section.
This section should contain a written description of your results addressing your groups’
data and the class data for the other variable.
Use the topic sentence of each paragraph to summarize the main trend or a key pattern
that you’ve found in the data. [If you don’t know what a topic sentence is, please look it up]
Very important: In your text, focus on describing the overall trends and patterns that are
apparent in your results. Only highlight individual results that are meaningful. Quantitatively
compare overall oxygen production between the different treatments (see next bullet point
for details). Your text should not be a long list of all the individual data points that you
obtained.
At least once in each paragraph, make quantitative comparisons between treatments to
highlight the trends in your data. For example, “the average volume of oxygen produced at
0°C was X % greater than at 25°C.” This type of analysis can drastically improve the quality
of your Results Section.
Figures
Purpose: to convey important information to your reader visually.
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Any graph, map, photograph, or drawing is a figure.
Label the axes carefully with titles and specify the correct units.
Unprocessed, raw data are usually not included in the body of a scientific paper. Instead,
process and present the data in a way that makes your main findings readily interpretable
by someone who was not in lab.
o If you must include numerical results, use summary statistics like mean and standard
deviation.
For assistance with graphing, see: 211 Graphs for Lab 6 (https://youtu.be/ryt6QO0xOzQ).
Number figures and tables separately and consecutively in the order to which you refer to
them in the text.
Titles/captions/figure legends should orient your reader to your figure and to explain any
information needed for them to fully understand it.
Each figure should have its own title and legend and have all axes labeled.
Captions are descriptive statements related to your figure, while titles convey the essence
of the graph. They should be written in sentence form and use normal grammar.
Figure captions are normally positioned below the figure, while titles are positioned above
the figure.
This figure is a good size to embed
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in your write up (it’s ~2” x 3”).
Corn Yield of Various Farm Sizes
As it should be possible to
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understand the main point made
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by each figure without reading the
results narrative, figure captions
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(required) must include all of the
information needed to interpret
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the data presented; however you
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should not include a discussion of
your findings in figure legends or
0
table headings. In addition, make
0
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10
15
20
sure you substantially reduce the
Farmland area (acres)
description of the data collection
(compared to the level of detail
Figure 1. The average yield of corn by farm size in southern
Kentucky during the 2009–2011 growing seasons
found in your methods section).
Discussion section
Purpose: to interpret your results and discuss their meaning within a scientific context.
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Think of your paper as an argument. Your data and references to outside sources are the
evidence, and your discussion section is where you bring them together to make your case
that your hypothesis is definitively supported or definitely rejected. At the end of the day, it
doesn’t matter which side of the argument you are on so long as your evidence is good!
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It is imperative that your Discussion section offers biological/scientific explanations for the
results you obtained. Connect the results of your experiment to topics discussed in class or
to broader areas of research in biology and human health.
o Regardless of what variable you were assigned, you must do this for both variables
tested (concentration & temperature)!
Discussion sections should include two paragraphs: one for your groups’ variable, and one
for the other class variable. Within each paragraph you need at least one unique reference.
You need to cite a minimum of two different reliable sources within this section (one of
which can be your textbook) for each variable.
o Reliable sources can include: articles from peer-reviewed journals, books, and websites
with .edu or .gov.
Make sure that you include in-text citations anytime you include research results or
scientific knowledge that you didn’t discover on your own. Failure to do this is a form of
plagiarism.
Just to be clear, you need to scientifically explain your experimental results, and in doing so,
provide citations support your conclusions.
• In the discussion of your results, make sure to address what you found was the optimal
temperature and concentration of catalase. Is this what you expected? Why or why not?
References section
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References should be written in APA format and should be listed in alphabetical order by
first author’s last name.
o For example:
Button, K., You, H., Kirkland, J. I., & Zanno, L. (2017). Incremental growth of therizinosaurian
dental tissues: implications for dietary transitions in Theropoda. PeerJ, 5. doi:
10.7717/peerj.4129
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In-text citations should be placed at the end of the sentence (before the period) that they
are citing and follow the following format: (author’s last name year of publication)
o For example: (Cohen, 2013). Or if two authors: (Cohen & Stewart, 2013). Or if there are
3 or more authors: (Cohen et al., 2013).
Every external source that is used in the report must be cited in-text as well as in the full
references at the end. Do not include a source in the references list if it is not also cited in
text somewhere in the report.
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Grading rubric
30 points max (3 points come from the prelab):
Section
Content/points each
Points possible
Pre-lab
All questions answered correctly
3
Title
Complete content
1
12
Hypotheses
2/1 pt each
2
Results
2 correct figures/2 pt each
4
.
2 correct variables addressed/1 pt each
2
.
Initial rates addressed/2 pts each
4
Discussion
Full analysis of both variables/3 pts each
6
.
2 unique citations/1 pt each
2
Refs
2 minimum, in APA format
3
Formatting
All correct
3
Total points
30
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BIOL&211 – Majors Cellular – Cascadia College
30 pts (including online pre-lab), due: 2 weeks from today2 weeks from today
Purpose:
Translating scientific data into a meaningful form is key to the process of science; the mark of a
successful scientist is someone who can communicate their ideas both to the public as well as
to other scientists. During this lab activity you will engage in the scientific method and
participate in authentic data collection. This will help you develop the transferable skills of
generating hypotheses and making predictions, as well as collecting, analyzing, and interpreting
scientific data. Using results that you generate as well as provided data, you will visualize key
trends in enzyme activity under varying environmental conditions using figures. This will
prepare you to write scientifically on future assignments in the Majors Biology series or in a
future career in science.
Background:
A chemical reaction is the interaction of two or more chemical substances in a way that results
in a chemical change in those substances. In chemical reactions, reactant (or substrate)
molecules are converted to product molecules. For example:
The rate of a chemical reaction is the change in the molecules involved (either the reactants or
the products) divided by the change in time. The rate of reaction is not constant but decreases
with time due to decrease in the concentration of reactants. It can be measured in two ways:
(1) The increase in product molecules over a
specified time interval.
(2) The decrease in reactant molecules over a
specified time interval.
Fig. 6-1. Concentration of the products and reactants in a chemical reaction over time.
As the concentration of the reactants drops, the concentration of the products climbs since they are directly related.
A catalyst is a substance that accelerates the rate of a chemical reaction, but the catalyst itself
is not used up in the reaction. Protein enzymes are responsible for catalyzing virtually all
1
biochemical reactions in cells. Each enzyme works on a specific substrate and their ability to
catalyze particular reactions can be affected by changes in the chemical and physical conditions.
Catalase is the enzyme used in this lab experiment. It has four polypeptide chains, each
composed of more than 500 amino acids. This enzyme is found in essentially all aerobic
organisms. One function of catalase within cells is to prevent the accumulation of toxic levels of
Hydrogen peroxide (H2O2) formed as a by-product of metabolic processes. Catalase also takes
part in some of the many oxidation reactions that occur in the cell.
In the absence of catalase, this reaction will occur spontaneously, but very slowly; if H2O2 is left
in an open container it will slowly decompose, leaving water in the container and giving off
oxygen gas.
Catalase speeds up the reaction considerably! It is one of the fastest enzymes known, with
turnover numbers measured from 30,000–800,000 molecules converted per second! In this
experiment, a rate for this reaction will be determined.
Much can be learned about enzymes by studying the kinetics of enzyme-catalyzed reactions.
For example, it is possible to measure the amount of product formed, or the amount of
substrate used, from the moment the reactants are brought together until the reaction has
stopped. If the amount of product formed is measured at regular intervals and this quantity is
plotted on a graph, a curve like the one that follows is obtained.
Fig. 6-2. Oxygen released
over time.
The volume of oxygen (in
mL) released from the
reaction of catalase
enzyme and hydrogen
peroxide was measured
over a 5-minute time
interval. The initial
reaction rate is 39.5
mL/min, which is much
higher than the average
reaction rate of 20.8
mL/min.
2
Study the solid curved line on Fig. 6-2. As time progresses, the amount of oxygen (product)
being produced increases at a steady rate. This is known as the initial reaction rate. After a
period of time (marked by a dotted line), this rate slows down considerably. Because of this
change, for the purposes of this experiment we will define overall enzyme activity as the
volume of O2 produced over a 5-minute interval, which is proportional to the average rate of
the reaction.
Knowledge – you will
•
•
•
•
Discuss the role of enzymes in biological reactions.
Explain a method of measuring the speed of biochemical reactions.
Describe the function of the catalase enzyme.
Discuss how enzyme concentration & temperature affect the rate of enzyme reactions.
Skills – you will be able to
•
•
•
•
•
Follow a laboratory procedure to complete an experiment.
Collect and analyze quantitative (numerical) data.
Write a lab report with results and discussion sections based on self-generated data as well
as data generated collaboratively.
Create scientific figures/graphs using computer software.
Use correct lab report format for captions, citations, and references.
Tasks:
Experimental procedure
In this experiment, you will measure the disappearance of a substrate and the accumulation of
a product to determine the rate of a chemical reaction. The reactant is liquid H 2O2 and the
product is gaseous oxygen (O2). These will be measured by recording the displacement of gas in
a volumetric cylinder over a 5-minue duration (see Fig. 6-3). The initial volume of gas is
recorded at time t=0 (starting point), and the accumulation of gas is measured every 30 seconds
for the first 2 minutes, and then once a minute for the remaining 3 minutes (see table 1 below).
Part 1. Obtaining the Baseline Data for Enzyme Activity
Work in teams of 3 or 4. Assemble the apparatus as demonstrated in class (see Fig. 6-3 below).
Into the clean syringe, first draw 2 mL of 3% liver extract immediately followed by 2 mL H2O
(for 4 mL total). Inject 3 mL of the now diluted liver extract into 10 mL of H2O2 solution (you
should have a little left over in your beaker).
3
Fig. 6-3. Experimental setup.
A graduated cylinder placed upside down in a
water bath is connected via surgical tubing to a
flask containing H2O2. Liver extract containing
diluted catalase enzyme is injected into the flask
to catalyze the decomposition of the reactant.
DO NOT REMOVE THE SYRINGE. As one group member injects the diluted extract, another
member should continuously swirl the flask of H2O2 while O2 produced by the reaction is
collected. In table 1 below, another team member should write down the levels of gas in the
cylinder (in mL) for a total of 5 minutes.
Table 1. Control data.
O2 gas produced at room temperature using 3 mL of 1:1 diluted (1.5%) liver extract. These data serve as a baseline
control.
Time
(min)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Gas level in
cylinder (mL)
Total (cumulative) gas produced (mL)
(gas level at time = t – gas level at
time = 0)
N/A
Room temperature (°C):
Handling of results (to be done later for your Lab Report):
Using Excel or other software, plot the volume of gas produced as a function of time (as shown
in Figure 6-2).
By completing this first experiment, you have just obtained the baseline data for the enzyme
activity in your 1.5% liver extract at room temperature (be sure to record the actual room
temperature). These data will be considered your control. Make sure you understand why this
particular experiment acts as a control.
Your group will be assigned ONE of two the variables listed to investigate further. If your group
is investigating the effect of enzyme concentration on the speed of enzymatic reaction skip to
Part 3 on p. 7 of the lab. Otherwise, if you group is investigating the effect of temperature on
enzyme activity, please continue reading.
4
Part 2. Effect of Temperature on Enzyme Activity
Before you move forward with further experimentation make sure to clean your flask (pour
your “used” H2O2 down the drain) and add 10 mL of fresh H2O2 into the flask for the next
injection. Your syringe containing liver extract can be reused since you will be using the same
concentration of liver extract for each experiment. During all experiments, remember to keep
the liver extract (containing catalase) separate from H2O2 until you start timing.
Think about and discuss with your group the following questions before you proceed.
Quickly write your answers in the spaces provided. Although I will not be collecting your
responses, they will help you to write your lab report!
1. Do you expect temperature will affect the rate of an enzymatic reaction?
a. If so, will the reaction rate increase or decrease with increasing temperature?
2. Is there any cutoff point in temperature beyond which the reaction will not occur?
a. If so, how can you explain this?
Three experimental temperatures (4°C, 37°C, & a maximum temperature) have been prepared
for testing the effect of temperature on catalase activity in your liver extract. Max temperature
should be ~ 50°C, but may vary from lab to lab. Temperatures above 55°C are not used because
H2O2 will break down spontaneously and the expansion of air at higher temperatures can
interfere with accurate measurements of O2 production.
Record max temperature used here: __________
Your goal is to regulate the temperature of the reaction. The reaction takes place in the flask
containing H2O2, so that flask must be immersed in a water bath at the desired temperature.
The same must be done for the enzyme solution to prevent the H2O2 mixture from changing
temperature when the enzyme is added. Both the H2O2 and enzyme solutions should sit in the
water bath for at least 2 minutes to thermally equilibrate. Note that your O2 collection cylinders
5
should NOT be in the temperature bath. Record your experimental data (8 data points for each
temperature, for a total of 24 data points) in Table 2 below.
At the end of these experiments, you will have experimental results for 4 different
temperatures (you have already measured enzyme activity at room temperature in Part 1 of
the lab). Once you have finished collecting and recording your data, skip to Part 4 to prepare
your lab report write up.
Table 2. Experimental data (temperature)
O2 gas produced (dependent variable) at various reaction temperatures (independent variable) and 1:1 diluted
(1.5%) liver extract.
For temp=4°C
Time (min)
Gas level in cylinder (mL)
Total (cumulative) gas produced (mL)
N/A
Gas level in cylinder (mL)
Total (cumulative) gas produced (mL)
N/A
Gas level in cylinder (mL)
Total (cumulative) gas produced (mL)
N/A
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
For temp=37°C
Time (min)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
For max temp
Time (min)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Handling of results (to be done later for your Lab Report):
Using Excel or other software, plot the volume of gas produced as a function of time for all four
temperatures as shown in Figure 6-2 (your graph should have four curves).
6
Part 3. Effect of Enzyme Concentration on the Speed of Reaction
Before you move forward with further experimentation make sure to clean your flask (pour
your “used” H2O2 down the drain) and add 10 mL of fresh H2O2 into the flask for the next
injection. Your syringe containing liver extract also needs to be rinsed after each run since you
will be using different concentrations of liver extract for each experiment. During all
experiments, remember to keep the liver extract (containing catalase) separate from the H2O2
until you start timing.
Think about and discuss with your group the following questions before you proceed.
Quickly write your answers in the spaces provided. Although I will not be collecting your
responses, they will help you to write your lab report!
1. Does the concentration of catalase have any effect on the rate of the reaction?
a. If so, will the reaction rate increase or decrease with increasing enzyme concentration?
In sequence you will conduct four separate experimental runs by injecting different dilutions of
the liver extract into H2O2 (do not forget to replace your H2O2 and to rinse your syringe after
each run). For each run, prepare 4 mL of the diluted enzyme solution, of which you will use only
3.0 mL total volume per reaction (see the table below when preparing each dilution). The liver
extract and distilled water can be measured using your syringe.
Table 3. Dilution recipes
Prepare 4 mL for each concentration of enzyme. Of this, you will only be using 3 mL of the prepared enzyme
solution for each run, so you will have a little extra in your beakers after each injection.
Enzyme concentration
0% liver extract
1.5% liver extract*
3.0% liver extract
0.75% liver extract
0.375% liver extract
Dilution recipe
4.0 mL distilled water
2.0 mL 3% extract + 2.0 mL of water
*you have already completed this: see Part 1
4.0 mL of undiluted extract, no water is added
1.0 mL of 3% liver extract + 3.0 mL distilled water
0.5 mL of 3% liver extract + 3.5 mL distilled water
For lower strength enzyme concentrations (0%, 0.75% and 0.375%), measure the O2 in a 100 mL
graduated cylinder (if available), rather than a 250 mL cylinder. Record your data in the
corresponding tables below.
7
Table 4. Experimental data (concentration)
O2 gas produced (dependent variable) at various enzyme concentrations (independent variable) at room
temperature.
Enzyme concentration = 0%
Time (min)
Gas level in cylinder (mL)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Enzyme concentration = 3.0%
Time (min)
Gas level in cylinder (mL)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Enzyme concentration = 0.75%
Time (min)
Gas level in cylinder (mL)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Enzyme concentration = 0.375%
Time (min)
Gas level in cylinder (mL)
t=0
t=0.5
t=1.0
t=1.5
t=2.0
t=3.0
t=4.0
t=5.0
Total (cumulative) gas produced (mL)
N/A
Total (cumulative) gas produced (mL)
N/A
Total (cumulative) gas produced (mL)
N/A
Total (cumulative) gas produced (mL)
N/A
Handling of results (to be done later for your Lab Report):
Using Excel or other software, plot the volume of gas produced as a function of time for all five
enzyme concentrations as shown in Figure 6-2 (your graph should have five curves).
8
Part 4. Lab Report write up.
Working individually, you will craft a lab report using 1) your groups’ data for your group
variable (the variable your group tested in lab), and 2) provided class data that has been
compiled across multiple class sections, for the class variable (the variable your group did not
test). You will generate two separate hypotheses: the first will be about your groups’ variable
(either temperature or concentration), and the second will be about the variable you did not
investigate.
Your goal should be to write your report as if was a real scientific journal article, rather than just
an in-class assignment. A standard scientific paper includes the following sections: Introduction,
Methods, Results, Discussion/Conclusions, and References. However, for this assignment, your
lab report will only have Hypotheses, Results, Discussion, and References sections. Note that
you may not use quotations anywhere in your writeup; all material should be in your own
words.
Criteria for success:
This assignment is to be submitted online on CANVAS. Paper copies will not be accepted.
Required elements for your writing assignment:
•
•
•
•
•
•
A title for the report (and your name, class, and section)
Hypotheses (covering both variables) that are specific and have been actually tested by the
methods you used.
T written Results section that presents 1) data from your group tests, and 2) the provided
class data for the other variable you did not test.
o Minimum of 2 figures total: one for your variable and one for the class data.
A Discussion section that utilizes at least 2 unique and appropriate references (see below
for specific guidelines). Each variable should be addressed in a separate paragraph, each
with its own unique reference.
A References section at the end of the report in APA format.
Proper grammar and spelling throughout.
Required formatting elements:
•
•
•
•
All text (except that found in captions and the References Section) should be neatly typed
using grammatically correct sentences and correct spelling (this is VERY important in this
particular assignment)
Each section except the title should have its own heading
Figures and captions must follow the guidelines outlined later in this handout.
References and in-text citations must follow the format and style outlined later in this
handout.
9
Length requirements:
There is no minimum length requirement. However, keep in mind that it would be impossible to
receive a good grade if your report is very short (one page is not enough space to satisfactorily
analyze and discuss your results). I will enforce a maximum length requirement—your report
must not include more than four pages of text (excluding figures and references). I am looking
for quality of writing, not quantity. With technical writing the goal is to make your writing
precise and concise. Important considerations (this information is relevant to any type of
scientific report) are as follows:
Title
Purpose: to accurately and efficiently convey to your reader the nature of your experiment.
•
•
Your title should not be “Enzyme Activity lab.” It should be professional in tone and should
describe to your reader the scope and nature of your experiment.
Here is an example of a title recently published in the journal Science: “Revealing the
Angular Symmetry of Chemical Bonds by Atomic Force Microscopy.” This title is concise
and conveys to the reader the exact nature of the experiment. Try to achieve the same level
of sophistication and clarity.
Results section
Purpose: to summarize and present meaningful results of the experiment to your reader.
•
•
•
•
•
•
You will create two parts to Results: Part I, which addresses your groups’ variable only; and
Part II, which addresses the class variable only (the one your group did not test). Label the
Results section AND Parts I and II clearly.
The Results Section should contain only results—resist the urge to make conclusions or to
interpret your data – save that for the Discussion section.
This section should contain a written description of your results addressing your groups’
data and the class data for the other variable.
Use the topic sentence of each paragraph to summarize the main trend or a key pattern
that you’ve found in the data. [If you don’t know what a topic sentence is, please look it up]
Very important: In your text, focus on describing the overall trends and patterns that are
apparent in your results. Only highlight individual results that are meaningful. Quantitatively
compare overall oxygen production between the different treatments (see next bullet point
for details). Your text should not be a long list of all the individual data points that you
obtained.
At least once in each paragraph, make quantitative comparisons between treatments to
highlight the trends in your data. For example, “the average volume of oxygen produced at
0°C was X % greater than at 25°C.” This type of analysis can drastically improve the quality
of your Results Section.
Figures
Purpose: to convey important information to your reader visually.
10
•
•
•
•
•
•
•
•
•
•
•
Any graph, map, photograph, or drawing is a figure.
Label the axes carefully with titles and specify the correct units.
Unprocessed, raw data are usually not included in the body of a scientific paper. Instead,
process and present the data in a way that makes your main findings readily interpretable
by someone who was not in lab.
o If you must include numerical results, use summary statistics like mean and standard
deviation.
For assistance with graphing, see: 211 Graphs for Lab 6 (https://youtu.be/ryt6QO0xOzQ).
Number figures and tables separately and consecutively in the order to which you refer to
them in the text.
Titles/captions/figure legends should orient your reader to your figure and to explain any
information needed for them to fully understand it.
Each figure should have its own title and legend and have all axes labeled.
Captions are descriptive statements related to your figure, while titles convey the essence
of the graph. They should be written in sentence form and use normal grammar.
Figure captions are normally positioned below the figure, while titles are positioned above
the figure.
This figure is a good size to embed
30
in your write up (it’s ~2” x 3”).
Corn Yield of Various Farm Sizes
As it should be possible to
25
understand the main point made
20
by each figure without reading the
results narrative, figure captions
15
(required) must include all of the
information needed to interpret
10
the data presented; however you
5
should not include a discussion of
your findings in figure legends or
0
table headings. In addition, make
0
5
10
15
20
sure you substantially reduce the
Farmland area (acres)
description of the data collection
(compared to the level of detail
Figure 1. The average yield of corn by farm size in southern
Kentucky during the 2009–2011 growing seasons
found in your methods section).
Discussion section
Purpose: to interpret your results and discuss their meaning within a scientific context.
•
Think of your paper as an argument. Your data and references to outside sources are the
evidence, and your discussion section is where you bring them together to make your case
that your hypothesis is definitively supported or definitely rejected. At the end of the day, it
doesn’t matter which side of the argument you are on so long as your evidence is good!
11
•
•
•
•
It is imperative that your Discussion section offers biological/scientific explanations for the
results you obtained. Connect the results of your experiment to topics discussed in class or
to broader areas of research in biology and human health.
o Regardless of what variable you were assigned, you must do this for both variables
tested (concentration & temperature)!
Discussion sections should include two paragraphs: one for your groups’ variable, and one
for the other class variable. Within each paragraph you need at least one unique reference.
You need to cite a minimum of two different reliable sources within this section (one of
which can be your textbook) for each variable.
o Reliable sources can include: articles from peer-reviewed journals, books, and websites
with .edu or .gov.
Make sure that you include in-text citations anytime you include research results or
scientific knowledge that you didn’t discover on your own. Failure to do this is a form of
plagiarism.
Just to be clear, you need to scientifically explain your experimental results, and in doing so,
provide citations support your conclusions.
• In the discussion of your results, make sure to address what you found was the optimal
temperature and concentration of catalase. Is this what you expected? Why or why not?
References section
•
References should be written in APA format and should be listed in alphabetical order by
first author’s last name.
o For example:
Button, K., You, H., Kirkland, J. I., & Zanno, L. (2017). Incremental growth of therizinosaurian
dental tissues: implications for dietary transitions in Theropoda. PeerJ, 5. doi:
10.7717/peerj.4129
•
In-text citations should be placed at the end of the sentence (before the period) that they
are citing and follow the following format: (author’s last name year of publication)
o For example: (Cohen, 2013). Or if two authors: (Cohen & Stewart, 2013). Or if there are
3 or more authors: (Cohen et al., 2013).
Every external source that is used in the report must be cited in-text as well as in the full
references at the end. Do not include a source in the references list if it is not also cited in
text somewhere in the report.
•
Grading rubric
30 points max (3 points come from the prelab):
Section
Content/points each
Points possible
Pre-lab
All questions answered correctly
3
Title
Complete content
1
12
Hypotheses
2/1 pt each
2
Results
2 correct figures/2 pt each
4
.
2 correct variables addressed/1 pt each
2
.
Initial rates addressed/2 pts each
4
Discussion
Full analysis of both variables/3 pts each
6
.
2 unique citations/1 pt each
2
Refs
2 minimum, in APA format
3
Formatting
All correct
3
Total points
30
13
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