Laboratory
6
Catalase Activity
in Living Yeast
Biology 171L Summer 2017 Lab 6: Catalase Activity in Living Yeast Student Learning Outcomes
1. Demonstrate the use of O2 gas sensor probes in collecting O2 readings.
2. Investigate the decomposition rate of hydrogen peroxide and the role yeast enzymes play.
3. Investigate the effect of concentration and pH on the activity of yeast enzymes.
Relevant Readings
• Campbell Biology, Chapter 8, especially pp. 151-157
• A short Guide to Writing about Biology, Chapter 9, especially pp. 203-211
Homework Synopsis (see pages 6-10 & 6-11 for full description)
• Part I – Mastering Biology
• Part II – Science Communication – Introduction exercises
• Part III – Data Analysis – Short Answer
INTRODUCTION
Proteins are the building blocks of life. They play critical roles in the growth and maintenance of
living cells. They’re so abundant that they make up more than half of a cell’s dry weight.
Proteins are formed by amino acids that are connected by peptide bonds to form chains. There
are just twenty amino acids used by organisms in these polypeptide chains but they form a
multitude of peptide chains. A protein’s shape is dependent up on the interaction (attraction and
repulsion) between amino acids in its polypeptide chain. When a protein loses its normal
configuration, it is said to be denatured.
One important group of proteins is enzymes. Living cells are
able to perform a multitude of chemical reactions very rapidly
because of enzymes. Enzymes are biological catalysts that
lower the activation energy for chemical reactions and speed
up those reactions, without actually being consumed or
changed in the reaction. Therefore, enzymes are extremely
efficient and may be used over and over again. One enzyme
may catalyze thousands of reactions every second. Because
of their ability to act quickly and rapidly, cells often have to
segregate them to protect other parts of the cells or break
down the protein quickly when it is no longer needed.
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Catalase Activity in Living Yeast
Fig 1: Effect of enzymes on chemical reactions
6-2
The polypeptide chains that form enzymes fold up
into globular tertiary structures. A specialized
region in the protein, called the active site, binds
with a substrate. A substrate is a material with
which the enzyme can bond to and react with. The
substrate binds with the active site of the enzyme
and becomes modified to form a new product. The
enzyme releases the modified substrate and moves
on to bind with another substrate molecule.
Enzymes are specific for a given substrate molecule;
they fit together much like a lock and key.
Consequently, enzymes are extremely specialized
and will not react with the wrong material, a
situation that could be devastating to a living cell.
One example of an
beta-D-galactosidase
found in dairy
formed by one
In order to be
cellular respiration,
important enzyme is the enzyme,
(lactase). Lactose is a sugar
products. It is a disaccharide
galactose and one glucose unit.
metabolized and utilized in
lactose must first be broken
down by the enzyme beta-DFig 2: Catalytic cycle of an enzyme sucrose
galactosidase (lactase). The
decomposing sucrose sugar
Fig 3: Specificity of lactase active site
cells lining the intestines produce
this enzyme, which will digest
the lactose sugar as it travels through the gut. The lactase enzyme is able to quickly break down
the lactose sugars, so they can be absorbed into the blood stream and spread to all the cells of the
body for respiration. Some individuals produce drastically less of the lactase enzymes as they
age. The lactose sugars thus passes undigested through the intestines until they become the food
of naturally occurring intestinal bacteria. The bacteria feast on the sugars and produce gas,
which contributes to the uncomfortable and embarrassing symptoms felt by lactose intolerant
people.
Enzyme activity, or the rate at which reactions are modified, is influenced by many factors such
as pH, temperature, substrate concentration, and the enzyme concentration. Some factors
may alter or affect the shape of an enzyme and interfere with the ability to bind well with its
substrate. Other factors simply increase or decrease the frequency that an enzyme and substrate
collide and interact.
In eukaryotic cells, one of the membrane-bound organelles floating in the cell cytoplasm is a
structure called a peroxisome. Peroxisomes contain enzymes responsible for breaking down long
amino acid chains and performing other metabolic reactions. These reactions need to be isolated
from the rest of the cell, because byproducts of these metabolic reactions include peroxides;
hence the name of the organelle. One of the peroxides produced is hydrogen peroxide, H2O2.
Hydrogen peroxide is a powerful oxidizing agent (reactive with practically any substance and
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Catalase Activity in Living Yeast
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extremely toxic to living cells). Certain cells in our body
take advantage of its antiseptic power. White blood cells
contain a large amount of hydrogen peroxide and other
peroxides that they use to kill any microbes they encounter.
See article reading (link at end of this section).
Still, because hydrogen peroxide poses such a danger to
every living cell, cells must be able to quickly break down
the hydrogen peroxide molecules before damage occurs.
Fig 4: Peroxisome in Yeast cell
Animal cells produce a special enzyme catalyst called
catalase (plant cells produce a similar enzyme called peroxidase) that can break down the
hydrogen peroxide. Hydrogen peroxide is broken down into water and oxygen gas.
2 H2O2 (l) à 2 H2O (l) + O2 (g)
Hydrogen peroxide is very toxic if not broken down.
Laboratory grade hydrogen peroxide is diluted to 30% and it is
still severely corrosive to skin, eyes, and respiratory tract.
Commercially produced hydrogen peroxide is diluted to 3%
before distribution. It’s safe enough for humans but still
powerful enough to kill bacteria, viruses, and fungi. It’s
commonly used as a disinfectant to clean cuts. When hydrogen
Fig 5: Decomposition
peroxide is poured onto a cut or wound, bubbling can occur.
of H O by catalase
Although, hydrogen peroxide is killing bacteria present in the
injury, this is not what is producing the bubbles. Instead, this is a result of your body’s own
catalase enzymes. Your cells contain catalase to counteract the hydrogen peroxide being
produced constantly by cellular metabolism. When cells or tissues become damaged (like in a
cut), the cell contents are released, including the catalase enzyme. As you pour hydrogen
peroxide onto the injury, it is being broken down by the catalase present in the wound and
oxygen gas is formed. Bubbling will occur at the site of the wound. Pouring hydrogen peroxide
on a cut potato will initiate the same results. Likewise, hydrogen peroxide will not bubble when
poured on intact skin because there is no catalase present to break it down.
2
2
This week, you will be studying how quickly catalase
breaks down hydrogen peroxide and how certain factors
like pH, and enzyme concentration affect the enzyme rate
of the catalase. You will be using the catalase enzyme
produced in baker’s yeast.
As part of your lab preparation, read the following
online article “Calling All White Blood Cells” at the
following link:
Fig 6: Potato samples in water (left) and H 2O2 (right)
http://www.sciencemag.org/news/2009/06/calling-all-white-blood-cells
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You can also view the original papers that this article was based on (papers on Laulima):
Martin, P, and Feng, Y., 2009. Wound healing in zebrafish. Nature, 459: 921-922.
Niethammer, P., Grabher, C., Look, A.T. and Mitchison, T.J., 2009. 459: 996-999
And a recent paper that builds on the work described:
Wittmann, C., Chockley, P., Singh, S. K., Pase, L., Lieschke, G. J., and Grabher, C., 2012.
Hydrogen peroxide in inflammation: Messenger, guide and assassin. Advances in Hematology,
2012: 1-6.
Preparation for Lab
1. Read through Introduction
2. Read online article
3. Research topics and terms that you are not familiar with or do not fully understand
a. Enzymes
b. Active Site-Substrate Interaction
c. Decomposition of Hydrogen Peroxide
4. Read through Experiment Procedure
5. Prepare your notebooks (e.g., write out protocols, prepare data tables, etc.)
EXPERIMENT PROCEDURE:
Overview:
In order to investigate how quickly catalase can catalyze hydrogen peroxide, you will be
incubating mixtures of yeast and hydrogen peroxide together and measuring the percent oxygen
produced under a number of different conditions. Each yeast/ hydrogen peroxide treatment will
incubate for 3 minutes. You will use an O2 gas sensor to measure the amount of O2 produced by
the catalase during the incubation time, which will allow you to calculate the reaction rate.
Settng up:
1. You will be sharing a water bath with other lab groups. Your TA will instruct you on how
to use the water baths. Your TA may have already turned on the water bath, but you still
need to monitor that the water bath has adequate water to cover your solution and that the
water has reached the appropriate temperature. Do not use until the water has reached a
temperature of least 38°C. If the bath is not on, turn it on and wait for the water to warm
up.
2. Check often to ensure that the temperature remains in the right range, i.e., 38-40°C. When
using a thermometer to check the water temperature, make sure that the thermometer is NOT
touching the metal sides or bottom of the water bath. This could alter the temperature
reading.
3. If water bath is too warm, bring the temperature down by adding a small amount of
distilled water and/or adjusting the temperature controls.
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Probe and software:
1. Set up laptop, Vernier interface, and O2 gas sensor probe.
a. Under “Data Collection…,” set the experiment duration
to 3 minutes and the sampling rate to 60 samples per
minute.
b. If necessary, change units to “ %.”
c. Use a utility clamp to fasten an O2 Gas Sensor to a ring
stand (Fig. 1). DO NOT calibrate O2 Gas Sensors!
d. Place a stir bar into the bottle.
e. Position a magnetic stirrer on the base of the ring stand.
Testing Effect of Enzyme Amount on Hydrogen Peroxide
Decomposition:
2. There should be nine (9) 15 mL falcon tubes, already prelabeled at your station. Label if needed.
Figure 7
3. Locate the falcon tubes labeled “5 drops” “10 drops” and “15 drops.” You will be using
these to compare the effect of enzyme amount on the breakdown of hydrogen peroxide.
Tube Label
Tube Color
5 Drops
5 Drops
White
10 Drops
10 Drops
White
15 Drops
15 Drops
White
4. Hydrogen peroxide is already on your lab bench in a 50 mL falcon tube with a white
“1.5%” label.
5. Fill the tubes with 6 mL of 1.5% hydrogen peroxide. Do this by using the plastic transfer
pipette on your lab bench to transfer hydrogen peroxide to the three 15 mL plastic falcon
tubes. Use the markings on the side of the plastic test tube to approximate 6 mL.
1.5% Hydrogen peroxide and the plastic transfer pipette are both labeled white
6. Your TA has prepared an enzyme suspension of catalase using live yeast.
7. Use one of the glass test tubes (or other container designated for this purpose) and the
plastic transfer pipette to bring back a small aliquot (~1.5 mL) of enzyme solution to your
lab bench. This will save you a lot of time in walking back and forth. You will only
need a little, as you will be using about 100 drops total today. Be sure to mix the
suspension well before you take drops for each experiment.
8. Begin the first test by adding 5 drops of enzyme suspension to the corresponding falcon
tube. Make sure that the pipet you use to measure drops of enzyme suspension
doesn’t touch anything so it doesn’t become contaminated and you can continue to
use it.
9. Cover the opening with a gloved finger and gently invert 2-3 times to mix.
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10. Swirl the contents of the bottle for 2–3 seconds to ensure thorough mixing.
11. Pour the contents into a clean respiration chamber (250 mL Nalgene bottle).
12. Place the bottle onto the O2 Gas Sensor as shown in Figure 1. Gently push the bottle up
onto the sensor until it stops. The sensor is designed to seal the bottle without the need for
unnecessary force.
13. Position the O2 Gas Sensor and Nalgene bottle assembly on the magnetic stirrer.
14. Start the magnetic stirrer, and adjust it to a medium speed.
15. Wait about 10 seconds and then click COLLECT to begin data collection.
16. When data collection has finished,
17. Highlight the graph of your results where you see a steady increase in O2 production and
click on Linear Fit [Rx icon] and click OK. A best-fit linear regression line will be
shown for the run. Record the value of the slope m, the rate (%/time).
18. Remove the O2 gas sensor from the respiration chamber.
19. Empty the bottle in the sink with running water and rinse the chamber with water. Dry
well with paper towel.
20. Repeat the steps above for 10 drops test tube using 10 drops of the enzyme suspension.
21. Repeat the steps above for 15 drops test tube using 15 drops of the enzyme suspension.
Data Table
Enzyme Amount
Rate of oxygen production (%O2/ min)
5 drops enzyme
10 drops enzyme
15 drops enzyme
Testing Effect of pH on Catalase Activity
Tube Label
Tube Color
pH 4
pH 4
Orange
pH 7
pH 7
Green
pH 10
pH 10
Blue
22. Add 6 mL of 1.5% H2O2 (it’s been diluted in different buffers to achieve different pH
environments) to the corresponding tube.
23. Start testing with the test tube labeled pH 4 by adding 10 drops of enzyme solution.
24. Collect data for oxygen gas production.
25. Repeat the steps to test the pH 7 solution by adding 10 drops of enzyme solution and
collecting oxygen gas data.
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26. Repeat the steps to test the pH 10 solution by adding 10 drops of enzyme solution and
collecting oxygen gas data.
Data Table
pH of
environment
Rate of oxygen production (%O2/ min)
4.0
7.0
10.0
Testing effect of temperature on catalase activity
Cold
cold
blue
Tube Label
Tube Color
Room
room
white
Warm
warm
pink
Hot
hot
red
27. For this part of the experiment, run each trial completely through before starting on the
next one.
28. Add 6 mL of 1.5% (cold) H2O2 (get from your TA or TI) to the correct tube.
Temp: _________
29. Quickly add 10 drops of the enzyme solution.
30. Collect data for oxygen gas production.
31. Repeat the steps to test the same reaction using room temperature 1.5% H2O2.
Temp: ___________________
32. Repeat the steps to test the same reaction using warm 1.5% H2O2 (warming in the warm
water bath). Temp: _______
Temperature
Biol 171L - Summer 2017
Data Table
Rate of oxygen production (%O2/ min)
Catalase Activity in Living Yeast
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Clean-up
33. Empty unused enzyme suspension into class waste container. Rinse out the plastic
beaker and pipette and leave to dry.
34. Empty all 15 mL plastic falcon tubes into class waste.
a. Rinse out and leave tubes to dry on lab bench
35. Rinse out respiration chamber and leave to dry for next class.
36. Color-labeled transfer pipettes on lab bench for transferring hydrogen peroxide should be
left on lab bench.
37. Refill H2O2 (white label) tube on lab bench, using hydrogen peroxide on class counter
38. There’s no need to refill the supply of buffered H2O2 (pH 4,7 &10) on your lab bench but
let your TA know if it has less than 10 mL of solution left.
39. Wipe down lab bench area and desk area.
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Homework Due Tuesday, June 13, 2017 Please Note!!! You have two assignments due this day!
I. Using the Scientific Literature
II. Lab 6 Homework - Format is important. All written homework should be typed, doublespaced, Times New Roman 12-pt font, with 1-inch margins. Remember to include your name,
section and the name of your TA.
Read about best practices when writing the Introduction (pp. 203-209) sections of a lab report
in “A Short Guide to Writing About Biology”, and any other resources you find helpful (e.g.,
http://writingcenter.unc.edu/handouts/scientific-reports/ or https://labwrite.ncsu.edu/).
Part 1 – Mastering Biology (43 points): A. Answer the questions in the assignment entitled “7. Photosynthesis” on the Mastering
Biology site. You have until the night before lab at 11:59pm to complete these
questions.
Part 2 – Science Communication (30 points): A. Writing the Introduction Section of a Lab Report (10 pts)
a. Describe of the elements of a well-written ‘Introduction’ section in one or two
paragraphs. Include such elements as the purpose, correct amount of detail, proper
tense and voice, reference to past studies, and any other information you feel is
important.
b. Why is the Introduction section important? How does it contribute to the construction
of knowledge in biology?
B. Writing the Introduction Section of the Catalase Activity Lab (15 pts)
Write the Introduction section for this lab. Use the guidelines you developed in your
answer above, and the information you reviewed in both the Introduction section in “A
Short Guide to Writing About Biology”, pages 196-203, and
http://writingcenter.unc.edu/handouts/scientific-reports/ or https://labwrite.ncsu.edu/.
Find one article from the primary literature to use in your introduction to support the
rationale for doing the experiment. Be sure to cite this article appropriately. Grades will
be based on the Universal Grading Rubric that can be found at the end of this manual and
on Laulima.
C. Reflection (5 pts)
Evaluate your Introduction section for the respiration lab (question 2.B.). Describe how
well your Introduction section conforms to the guidelines you outlined in question 2.A.
Use the rubric provided at the end of this manual to help you. Do you need to make any
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changes to improve your Introduction section? In your answer, outline the steps you will
take. Make these changes now to get maximum points for your answer in part 2.B.
Part 3 – Data Analysis (25 points) Answer the following questions:
1. Figures and Tables are used to summarize data. Choose the most appropriate method to
summarize your data and submit any relevant statistics, charts, etc. Be sure to include
appropriate captions. (6 pts)
2. What were your hypotheses with respect to temperature, pH and enzyme concentration?
Write out each hypothesis separately. Do your results support your hypotheses? (3 pts)
3. Explain possible inconsistencies or problems with the experimental design around measuring
the effect of temperature. If there were no problems, explain how this was achieved. (3 pts)
4. How does changing the pH affect the rate of enzyme activity? Which pH was best for
catalase activity? Why? (3 pts)
5. How does changing the concentration of enzyme affect the rate of hydrogen peroxide
decomposition? Which concentration was best for catalase activity? Why? (3 pts)
6. How does changing the temperature of the reaction affect the rate of hydrogen peroxide
decomposition? Which temperature was best for catalase activity? Why? (3 pts)
7. Why is it important that cells contain catalase? (2 pt)
8. List at least one researchable question concerning catalase activity (other than one you did
today). (2 pts)
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Criteria
Introduction:
Demonstrates
a
Context
clear
understanding of
the big picture;
Why is this
question
important/
interesting in the
field of biology?
•
•
Not addressed
Novice
Intermediate
Expert
0-2
3
4
5
The
importance of
the question
is not
addressed
How the
question
relates within
the broader
context of
biology is not
addressed.
• The writer
•
provides a
generic or vague
rationale for the
importance of
the question.
The writer
provides vague
or generic
references to
the broader
context of
biology.
The writer
provides one
explanation
of why
others would
find the topic
interesting.
The writer
provides some
relevant
context for the
research
question(s).
•
•
•
•
The writer
provides a
clear sense of
why this
knowledge
may be of
interest to a
broad
audience
The writer
describes the
current gaps in
our
understanding
of this field
and explains
how this
research will
help fill those
gaps
Introduction: Accuracy and relevance
0-2
Content
knowledge is
accurate, relevant
and provides
appropriate
background for
reader including
defining critical
terms.
•
•
•
3
Background
information is
missing or
contains major
inaccuracies.
Background
information
is accurate,
but irrelevant
or too
disjointed to
make
relevance
clear
Primary
literature
references are
absent or
irrelevant. May
contain website
or secondary
references
websites or review
papers are not
primary
Biol 171L - Summer 2017
•
•
•
Background
omits
information or
contains
inaccuracies
which detract
from the
major point of
the paper.
Background
information is
overly narrow
or overly
general (only
partially
relevant).
Primary
literature
references,
if present,
are
inadequately
explained.
4
• Background
•
•
information
may contain
minor
omissions or
inaccuracies
that do not
detract from
the major
point of the
paper.
Background
information
has the
appropriate
level of
specificity to
provide
relevant
context.
Primary
literature
references are
relevant and
adequately
explained but
few.
Catalase Activity in Living Yeast
5
•
•
•
Background
information
is completely
accurate
Background
information
has the
appropriate
level of
specificity to
provide
concise and
useful context
to aid the
reader’s
understanding.
Primary
literature
references are
relevant,
adequately
explained, and
indicate a
reasonable
literature
search.
6-12
Use of Primary Literature
0-2
Relevant and
reasonably
complete
discussion of how
this research
project relates to
others’ work in the
field (scientific
context provided).
•
Primary
literature
references
are not
included.
Primary literature
is defined as:
- peer reviewed
- reports original
data
- authors are the
people who
collected the
data.
- published by
a noncommercial
publisher.
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3
•
•
•
Primary
literature
references are
limited (only
one or two
primary
references in
the whole
paper)
References to
the textbook,
lab manual, or
websites may
occur.
Citations are at
least partially
correctly
formatted.
Note that proper
format includes a
one-to-one
correspondence
between in-text and
end of text references
(no references at end
that are not in text
and vice versa) as
well as any citation
style currently in use
by a relevant biology
journal.
4
•
•
•
•
Catalase Activity in Living Yeast
Primary
literature
references are
more
extensive (at
least one
citation for
each major
concept)
Literature
cited is
predomina
ntly (>
90%)
primary
literatures.
Primary
literature
references are
used
primarily to
provide
background
information
and context
for
conclusions
Primary
literature
references
5
•
•
•
•
Primary
literature
references
indicate an
extensive
literature
search was
performed.
Primary
literature
references
frame the
question in the
introduction by
indicating the
gaps in current
knowledge of
the field.
Primary
literature
references are
used in the
discussion to
make the
connections
between the
writer’s work
and other
research in the
field clear
Primary
literature
references
are
properly
and
accurately
cited
6-13