Biology 107
General Biology
Labs 4, 5 and 6: Enzymes
Now that you have had some experience in the lab, you are ready to use your skills to answer biological questions. First, you will learn how to use a spectrophotometer to measure enzyme activity. The ability to measure
activity will allow you to experiment with some of the factors that affect enzyme reaction rates. Then, you will
plan an experiment of your own to be carried out the following weeks. Your lab group will present your data in
an oral report to the class during the week of October 31 (Lab 9). Your final written report will be due during the
week of November 7.
Objectives
Introduction
When you have successfully completed this lab,
you should be able to:
Enzymes carry out the chemistry of life. Whenever anything happens in the cell, you can be sure
that an enzyme is catalyzing the chemical transformations involved. Each biochemical reaction is carried
out by a specific enzyme. Some of these reactions are
important in food science and industrial settings. For
example, the enzyme amylase breaks carbohydrate
molecules into sugar monomers, and is important in
the production of beer. Microbial proteases are used
to tenderize meat, and lactase can help produce lowlactose dairy products for people with lactose intolerance.
1. Use the spectrophotometer to measure products of
an enzyme reaction.
2. Design and perform an experiment investigating
some aspect of enzyme activity.
3. Analyze your data, draw conclusions from it, and
extend your investigation.
4. Develop and present an oral report with your lab
group.
5. Improve your scientific writing skills.
IMPORTANT: This lab exercise contributes more
than 25% of your lab grade. To help you do well
on this major assignment, your lab manual contains suggestions on how to design and carry out
your independent experiment. Make sure you read
the manual carefully before lab. Then carefully
design your experiment as a group. Each group
member is responsible for typing an abstract and
attending an outside-of-class conference with the
instructor between the first two class meetings for
this lab. Then, in the second lab period, carry out
your experiment, think about your data, and decide
what you need to do differently when you repeat or
extend your work in the third lab period.
© 2005, University of Evansville Biology Department
What are these remarkable enzyme molecules like?
How do they achieve their function so quickly and so
accurately? In this lab, which will continue through
three lab meetings, we will study enzyme action under
several different conditions. These studies can help
you to appreciate some of the properties of these
unique molecules.
Before beginning this lab, you should review course
material related to enzymes. (See Section 8.4 in your
text, pages 150-155.)
Biological processes depend on molecular catalysts
called enzymes to speed up the chemical reactions necessary for cells to function. A catalyst is a chemical
agent that changes the rate of a reaction without being
consumed by the reaction. Enzymes work by lowering
the activation energy for the reaction they catalyze, so
38
39
it’s easier for the reaction to get started. The basic
components of an enzyme-catalyzed reaction are the
substrate(s), the product(s), the solution in which the
reaction takes place, and the enzyme itself.
The substrate is the reactant molecule that is acted
on by the enzyme. Each enzyme has a specific substrate or type of substrate. In this exercise, the substrates catechol and oxygen are acted upon by the
enzyme catecholase. Figure 1 shows that catechol
and oxygen are transformed into benzoquinone and
water, which are the products of this reaction. The
enzyme itself is not changed during the reaction.
work to the class, so be prepared to draw and explain
your graphical predictions on the blackboard.
Graph 1: When substrate is abundant, what should
happen to the reaction rate (amount of product formed
per unit of time) when more enzyme molecules are
added to the reaction mixture? Sketch your prediction on the axes in the following graph. Remember
to place the independent variable (the thing you are
manipulating or testing) on the horizontal axis and the
dependent variable (the thing you are measuring) on
the vertical axis and to label both axes. Which variable is which? Does the enzyme concentration or the
reaction rate belong on the X-axis? What would be a
good caption for this graph?
Figure 1. The Catecholase Reaction
Catecholase, also called tyrosinase and polyphenol
oxidase, is a copper-containing enzyme. This enzyme
is important because one of its reaction products,
benzoquinone, is responsible for the browning of
fruits and vegetables. When cells are disrupted by
cutting or injury, oxygen becomes available, and the
catecholase reaction can occur, causing cut or bruised
fruit to brown. This is an undesirable condition, and it
has been extensively studied by food scientists. Several methods have been successfully used to prevent
browning. Ascorbate retards browning by reducing
the availability of oxygen. Citric and acetic acid
lower the pH and decrease enzyme activity. Sulfite
ions have also been shown to decrease the activity of
this enzyme.
Graph 2: If the number of enzyme molecules is
constant and the number of substrate molecules is
low, what will happen to the reaction rate when more
substrate is added to the reaction mixture? Sketch
your prediction below. Remember to add a caption
and label your axes.
Procedures
Section A - Factors that Affect
Enzymatic Reactions
You will examine a number of factors that affect
the reaction rate of an enzymatic reaction. Read
through this section of the laboratory materials and
predict, using graphs of enzyme activity, what you
think the effects of various factors will be on enzyme
reaction rate. Students will be asked to describe their
Most enzymes are protein molecules. As you
remember, proteins are composed of a series of amino
acids linked together with covalent bonds. Amino
acids have various functional groups, some of which
40
attract or repel each other. Each protein molecule
folds into its three dimensional shape as a result of
these attractive and repulsive forces. The particular
part of the enzyme molecule where the substrate fits is
called the active site. Anything that alters the shape of
the enzyme molecule, especially at its active site, may
change the specificity or affinity of the enzyme for its
substrate.
Graph 3: The attractions and repulsions between
chemical groups on the amino acid chain are strongly
affected by pH. Each enzyme has a pH at which it
is optimally shaped and where it functions best. If
the pH is too low or too high, the enzyme molecule
changes its shape and, therefore, its ability to catalyze
reactions. How do you think reaction rate will change
in solutions of different pH? Sketch your idea below,
and don’t forget your caption and labels.
Dissolved ions, such as NaCl, in an enzyme’s environment can also influence its shape by changing
the interactions of the chemical groups on the amino
acids. Just as with pH, there is an optimum salt concentration at which each enzyme is shaped to best
engage the substrate.
Graph 4: Temperature is another important factor
in enzyme reactions. Since the enzyme molecules
must be in actual contact with substrate molecules
for the enzyme to catalyze the reaction, anything that
increases the number of collisions between enzyme
and substrate is expected to increase the reaction
rate. Sketch this relationship between temperature
and reaction rate.
Graph 5: Heat energy has another effect in addition to speeding up the movement of molecules. It can
also disrupt the weak interactions between the chemical side groups of amino acids, causing the enzyme
to lose its shape. When temperature is too high, the
enzyme can’t catalyze its reaction. Therefore, like pH
and salt concentration, enzymes work best in an optimal temperature range. Sketch how temperature is
related to reaction rate on the axes below. (and don’t
forget what?)
Now that you have thought about (and discussed
in class) a number of factors which can affect the
reaction rate of an enzyme, it is time to work with a
specific enzyme.
Section B - The Catecholase Reaction
and Spectrophotometeric Measurement
A potato was placed in a blender with distilled
water and blended for two minutes. The blending
broke the potato cells and released many enzymes and
other molecules. The resulting mixture was filtered
through several thicknesses of cheesecloth to remove
large potato pieces and then centrifuged to provide
a solution containing many enzymes, including one
41
called catecholase. When this enzyme is placed in
contact with catechol, its substrate, the product benzoquinone is produced (Figure 1). Catechol is a colorless substance and benzoquinone is reddish-brown in
color. You have seen this reaction whenever your cut
apple or potato surface turns brown. If a freshly cut
potato turns brown when left standing, why do boiled
and mashed potatoes stay white?
exercise, you diluted solutions of methyl blue and
read the amount of light absorbed at 592 nm (nanometers). In this enzyme experiment, you will use a
wavelength of 540 nm. Check to make sure that your
spectrophotometer is set to 540 nm before you begin
your measurements. Absorbance readings accidentally taken at other wavelengths must be discarded
and your experiments repeated.
The color change produced when benzoquinone
is produced can be measured colorimetrically with a
spectrophotometer. Colorimetric analysis is a method
of determining the concentration of a substance by
measuring the absorbance of a solution at a particular
wavelength of light. For example, the strength of
a cup of coffee may be estimated by simply observing the intensity of color. In reality, one is visually
measuring how much light is being absorbed by the
coffee. The colorimeter, or spectrophotometer (Figure
2), is similar in operation to the observation of the
coffee but it is capable of much greater accuracy than
the eye.
Before measuring the absorbance of a sample, the
spectrophotometer must be set to an absorbance of
zero with a blank, which is a solution containing all
of the ingredients of the assay, but for which there is a
zero concentration of the molecule being assayed. In
other words, a tube which contains water and potato
extract but which does not contain the benzoquinone
product will be used as the blank. (Recall that you
used a blank containing plain distilled water when
you measured the absorbance of a distilled water plus
dye solution during Lab #1.)
Figure 2. A Spectrophotometer
White light is passed from a light source through
a monochrometer that selects a single color or wavelength of light. This light then passes through the cell
containing the sample, where some of it is absorbed.
The remaining light intensity is measured by the
detector. The amount of light absorbed is proportional to the concentration of colored molecules in the
sample. Absorbance is measured in absorbance units
and is denoted by the capital “A” on the machine.
Please do not refer to Absorbance as “Absorbancy” in
your lab report.
The wavelength chosen for a particular analysis
usually corresponds to the color at which the molecules of interest absorb maximally. In the first lab
When you are comfortable with the operation of
your spectrophotometer, you are ready to set up an
enzyme-catalyzed reaction. In order to make the
reaction occur, the mixture must contain both substrates and enzyme. Let’s review. The substrates are
____________ and _____________ and the enzyme
is ______________________.
The preliminary experiment outlined below will
establish a relationship between the amount of product
formed in a given amount of time (the reaction rate)
and the amount of enzyme. You will notice that the
potato extract itself has color and therefore absorbs
some light. That amount of color must be subtracted
from the amount of color in the final reaction to give
the amount of color resulting just from the product
(benzoquinone). Your blank contains enzyme from
the potato extract but no substrate, so no reaction will
take place (no product is produced).
In this experiment, you will be using distilled
water, catechol and a catecholase-containing potato
extract suspension. Be careful not to mix up your
pipettes and cross contaminate your solutions. Use 10
ml and 5 ml pipettes for water and catechol, respectively, and a 1-ml pipette for the potato extract. Keep
your pipettes off of the benchtop and do not let them
42
touch each other. Take turns using the pipettes and
the pipette-aids and watch each other to try to prevent
confusion. Catechol, which is light sensitive, has
been placed in a flask covered with aluminum foil.
Do not leave the cork/stopper lying on the countertop.
Because this is a timed reaction, practice will make
you more comfortable and more efficient.
It is especially important to mix the contents of
your tubes when the reaction starts and again before
reading in the spectrophotometer.
1. Mark your eight tubes according to their contents
(see Table 1) using a wax pencil. Do not mark the
white frosted areas of the test tubes with wax pencil
because it can not be removed.
2. Make your blank tubes by pipetting the specified
volume of each reaction component (Table 1). Then,
carefully seal the top of each tube with Parafilm, and
gently invert it several times to mix the contents.
3. Set the wavelength on your spectrophotometer to
540 nm.
4. Wipe your blank tube #1 carefully with a Kimwipe
to remove possible fingerprints, which might interfere with absorbance readings. Place the tube in the
machine, and adjust the absorbance to zero, according
to the directions for your machine.
Now, you will make four reaction tubes. In contrast to the blanks you made above, components must
be added in a particular order, and the reaction must
be timed. Start timing the moment the potato extract
is added, and read your tube at exactly three minutes.
5. Place the proper amount of water in each marked
tube.
6. Place 2 ml catechol in each reaction tube.
7. Add the required amount of potato extract to each
tube, seal with Parafilm, gently invert several times
to mix and place the tube in the rack. Note the time
you started the reaction. Keep track of the time, and
as the three-minute mark approaches, mix again, and
read absorbance in each tube at exactly three minutes.
Record the data in Table 1.
Be sure that you blank the machine before each
reading, using the proper blank for the amount of
extract. Example: Zero the machine with blank #1,
which contains 0.25 ml extract, before you read tube
#1, which also contains 0.25 ml of extract. Use blank
#2 for tube #2, etc.
While you are waiting for other groups to gather
their data, decide among yourselves if you are satisfied with your experiment. You have been given
enough tubes to run the reaction again if you are
interested or if you feel that you made mistakes the
Table 1. Catecholase Reaction Using Varying Amounts of Potato Extract
43
first time. Perhaps you had trouble using the pipetting
devices or forgot to invert and mix one of your tubes.
Feel free to repeat the experiment if you would like.
Before you discard the contents of your test tubes,
note how much time has passed since you mixed
your substrate and enzyme together. Run your tubes
through the spectrophotometer again and don’t forget
to use the proper blank with each tube. Did your
absorbance readings change over time? Why or why
not? How many minutes elapsed since your previous
readings? Could timing errors affect your data?
Each lab group has its own estimate of the amount
of product produced over a three minute time interval.
Since everyone in the class is trying to measure the
same thing, it would be helpful to calculate a class
mean absorbance for each amount of extract. Would
a standard deviation be meaningful for these data? If
so, what would it tell us?
Your instructor will construct a plot of enzyme
activity as a function of concentration. On this graph,
the mean will be plotted as a point and the standard
deviation as y-error bars like those on page 17 of
your laboratory manual. Remember that the dependent variable, for which you determine the standard
dcviation, is plotted on the y-axis. To add y-error
bars to your own graphs, right click on a point in the
data series, and then choose y error bars from the
format data series window. Tell Excel to show error
bars both above and below the point, and then select
custom values for the y-error values. You want the
size of your y-error bars to be your standard deviation, so use the data range icon to indicate the cells
containing your standard deviation for both the plus
and minus categories. When you click OK to close
this window, your data series will have y-error bars.
Collect the data for all groups in Table 2, below,
calculate mean and standard deviation, and use these
data and the plot constructed by your instructor to
think about the following questions.
1. Why is it necessary to add the potato extract to
each tube last, after the water and catechol are already
measured? Did all groups do this correctly?
2. Why was a different amount of water added to
each tube?
3. What is the relationship between enzyme concentration and reaction rate?
4. If you missed the three minute mark, and one of
your tubes was allowed to react for four minutes,
would you have to repeat the experiment, or could
you correct your reading?
Now that you have seen what happens when you
vary the amount of enzyme in a reaction, what do
you think might happen if you change any of the
Table 2. Class Results for Catecholase Reaction Using Varying Amounts of Potato Extract
44
factors discussed at the beginning of class? These
might include (a) concentration of substrate, (b) temperature, (c) pH, (d) salt concentration, (e) source of
enzyme containing extract.
Can you think of any other factors that you could
vary? What about changing the polarity of the solution with isopropanol or a detergent? Could you try
to limit the presence of oxygen in the tube? Are there
any other molecules shaped like the substrate that you
think might react with the enzyme?
As you consider these possible future experiments,
think about which ones might be technically feasable.
You know how to set up reaction tubes and how to
use the spectrophotometers. How many tubes do
you think you could measure in two hours? Did your
group have any technical problems that you need to
practice? Handling the pipettes? Using the Parafilm?
Zeroing the machine? Do you know why different
blanks were used?
Section C - Designing Your Experiment
During the second week and third weeks of this
lab exercise, your group will carry out an independent
experiment to investigate some aspect of enzyme
activity. Before the end of the first lab period, you
must discuss your plans with your instructor and make
a list of the materials that you will need. Think about
how long it might take to set up and run your experiment and be sure to include enough time to make at
least four trials of each condition. Four trials will
allow you to calculate a standard deviation even if the
data from one trial is not usable.
You should also consider alternative experimental
topics when your group discusses this experiment
since no two groups will be allowed to select the same
topic. It is possible, though, for two groups to cooperate, dividing between them a project that would be
too large for a single group. If you choose to do this,
remember that you will be writing your lab report
using data generated by another group.
Think about which elements in your experiment
can be controlled or manipulated by the investigator.
Would your absorbance readings change if you read
them after two minutes instead of after three minutes?
What might happen if you read half of them after two
minutes and the other half after three minutes? What
would happen after twenty or thirty minutes? Many
successful groups choose to increase the accuracy
of their data by keeping careful track of the time for
their reaction and reporting a rate (Absorbance per
minute). This simple calculation can vastly simplify
your experiment. Instead of discarding a tube that ran
for three and a half minutes, divide all Absorbances by
the time of the reaction in minutes.
Each group will present their proposed experiment to their instructor in an out-of class conference.
Decide what you want to do and make a list of the
other supplies you will need. Download the proposal
form from the website, and fill it out with all the
details of your proposed experiment.
Schedule a conference with your instructor to
thoroughly discuss the plans for your experiment.
When can all the members of your lab group meet?
Select your conference time with your instructor and
don’t forget to show up.
The conference time for this group is:
When you come to this conference, each group
should bring one completed proposal form, and each
student should bring her or his own independently
written experimental abstract. This abstract is limited
to a single typed page, and must completely describe
the background, experimental design, and methods.
Include on the same page a computer-generated data
table like the one on page 43, including the components and volumes for each tube. Include a column
in your table to record the time for each reaction, in
case it is different. Any student who does not independently complete the experimental abstract will not
be allowed to take part in the independent experiment
until they finish this important preliminary step. A
45
Figure 3. Opossum extract decreases catecholase activity.
sample abstract for our opossum extract experiment
is shown on page 47. Your abstract must be included
with your lab report.
Section D - Interpreting Your Results,
Repeating or Extending your Experiment
Collect your time and absorbance data carefully.
Look at your procedures and data with a critical eye
while you are collecting it. Are you really measuring
what you thought you were measuring? It is important to think about your data as you collect it so that
problems can be fixed during the lab period. Remember your osmosis experiment - did you discover errors
and correct them in lab, or did you graph your data
later only to discover that your experiment did not
work correctly?
After you have collected your data, analyze and
graph it the same day. Write your preliminary
results section, including properly constructed
graphs and tables (due at the beginning of the third
enzyme lab period). Do you need to repeat your
experiment during the third week? How might
you refine and extend your experiments? Type
a paragraph explaining what you would like to
do during the third week, print a new blank data
table, and discuss your plans with your instructor
before lab.
Think about the following example. A lab group
hypothesized that adding opossum extract to their
enzyme reaction will slow down the reaction (opossums are slow, after all). They run four trials of each
of the following conditions: 0 ml, 0.5 ml, 1 ml, and 2
ml of opossum extract. They find the following mean
reaction rates (in abs/min).
ml opossum extract
0
0.5
1.0
2.0
rate (abs/min)
+/- S.D.
0.130
0.122
0.086
0.037
.020
.018
.006
.004
What can they say about these data? It is not correct to say that the reaction rate decreased when opossum extract was added. The rates for zero and 0.5 ml
of opossum extract seem to be different at first, but
note that the standard deviations indicate that there is
enough uncertainty in these means to make it impossible to say for certain that they are different. Only the
1.0 and 2.0 ml additions caused significant decrease in
the enzyme rate of reaction. These data are graphed
in Figure 3, above. Notice how it is much easier to
tell that the first two conditions are not significantly
different.
Note that perhaps these experimenters could have
demonstrated a significant difference between the first
46
two conditions if they had controlled their fixed variables more carefully. These data are probably good
enough to write a lab report, if they are interpreted
correctly in the discussion section. If, on the other
hand, the means were the same as the ones in this
experiment but all of the standard deviations were so
large that they overlapped, then these students would
not be able to say anything conclusive about the effects
of opossum extract. If this happened, those students
would want to return to the laboratory to repeat their
experiment.
Another important use of standard deviation in
experiments like these is to see which data points
might be incorrectly calculated. Think about data like
those in Table 2. If, for instance, the class mean for 1.0
ml of enzyme were 0.239 +/- .026 abs units per three
minutes and your value for this data point was 0.984
abs units per three minutes, you would know that you
must have made a mistake somewhere. In general, if
an experimental value you measure or calculate is far
outside the range indicated by the mean and standard
deviation of other lab groups, then it is likely that your
group has made an experimental or calculation error.
If this happens, you should find the source of the error,
repeat the experiment or calculation without error, and
report the correct value to other lab groups in your
section as soon as possible. This will be particularly
important in the bacterial transformation lab.
47
Enzyme Lab: Sample Abstract
Mara Supial. Lab Group A1
Our lab group proposes to investigate the effects of adding opossum extract to the catecholase reaction.
We will use the potato extract as our source of catecholase. The catecholase reaction requires oxygen, and we
hypothesize that if opossum extract also contains enzymes that consume oxygen, adding opossum extract to the
catecholase reaction will consume oxygen and slow down the catecholase reaction. If this hypothesis is correct,
then adding increasing concentrations of opossum extract should further decrease the rate of catecholase activity.
We propose to measure the rate of enzyme activity (our dependent variable) in the presence of zero, 0.5 , 1.0, and
2.0 ml of opossum extract (our independent variable). The amount of water in each tube will be adjusted so that
the total volume of each reaction is the same (this is a fixed variable). Other important fixed variables are the
volume of catecholase-containing potato extract, and the reaction temperature. We will try to run each reaction
for three to five minutes, and will record the exact time for each time in the table. Dividing the absorbance by the
time in minutes will give reaction rate in abs/min. We will carry out four trials for each concentration of opossum extract, and calculate the mean and standard deviation for each dataset. The means and standard deviations
will be used to compare the activity in the presence of different amounts of opossum extract. Mrs. Akrabawi
has agreed to prepare opossum extract for us. We will not need any other special equipment or supplies for this
experiment.
Tube
Blank
A
Blank
B
Blank
C
Blank
D
A1
A2
A3
A4
B1
B2
B3
B4
C1
C2
C3
C4
D1
D2
D3
D4
Water,
ml
Catechol,
ml
Potato
Extract,
ml
Opossum
Extract,
ml
5
0
1
0
4.5
0
1
0.5
4
0
1
1
3
0
1
2
3
3
3
3
2.5
2.5
2.5
2.5
2
2
2
2
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0.5
0.5
0.5
0.5
1
1
1
1
2
2
2
2
Start
Time
End
Time
Abs
at 540
nm at
End
Time
Elapsed
Time
(min)
Abs/
min
48
Lab Assignment - Enzyme Labs
Experiments should be designed, carried out, and presented to the class by the whole group, but
each student must write the lab report, including tables and graphs, independently.
A. Design your independent enzyme experiment. Remember that it will be much easier to write a good lab report
if you have designed your experiment carefully and understand what you are doing and why. Be prepared to collect your data carefully and thoroughly.
B. Explain your experimental design to your instructor. Before your conference, complete your experiment
abstract so that your instructor can go over it with you. No student can move on to the independent experiment
without an approved abstract.
C. Carry out your experiment. Collect your data, analyze it, and write a preliminary results section (due at the
beginnning of the third lab period.) Decide which direction you would like to take during the next lab.
D. Repeat or extend your experiment during the third lab period. If you need additional lab time for further
experiments, your group can schedule a time with your instructor.
E. Present your experimental design, results, and conclusions to the class. A group oral report on your enzyme
experiment will be presented the week of October 31. This is the same week as the third lecture exam. This should
not present a problem if you prepare your presentation ahead of time. You will finish the lab work in early October and have several weeks to think about your data. The oral report is designed to give you feedback on your
work, and to help you finish your report. Each student in the lab group should be prepared to present the entire
experiment. In class, your instructor will choose one student to present the introduction, one student to present
the methods, one student to present the results, and one student to present the discussion from each group.
F. Write a complete lab report describing your experiment. Your report is due the week of November 7, 2005,
the week following the 3rd exam. Make sure to use the feedback from your group presentation to improve your
written report. Turn in your group planning sheet and experiment abstract along with your report. Review the
requirements for writing a lab report listed and explained previously. Be sure you submit your final draft to
www.turnitin.com and place your lab report AND previous assignments (including all quizzes, corrections, and
error analyses) in your portfolio. This lab report is worth 80 lab points – plan ahead and get help as soon as you
need it.