Alberta Ingenuity & CMASTE
Laboratory Investigation: Enzyme Activity and Inhibitors (Teacher’s Edition)
CURRICULAR LINKS: Biology 20, Chemistry 30
CLASS TIME: 1-2 periods
Class time depends on whether students perform the quantitative analysis (take a digital photo and use image
processing software to analyze it). The lab is designed so that teachers can opt out of this part if they wish.
Below the recommended omitted sections are identified if you decide to skip the computer analysis. One option
is to provide the students with the photo analysis data provided in this document, if you do not have the
resources and/or the time to have the students perform the analysis themselves.
Sections and questions to omit if you want skip the image processing:
 Evidence/Analysis: Quantitative
 Evaluation (Questions 11, 13
CONNECTIONS TO AICCS SERIES
This activity connects to the following others in the AICCS Series.
 Explore an Issue: Preventable Disease
(Both activities address research into new antibiotics for tuberculosis.)
 Case Study: Using Competitive Inhibitors to Fight Infections Disease
(Both activities address competitive inhibitors as a promising way to develop new antibiotics for
tuberculosis and other bacterial infections.)
 Case Study: Chemical Synthesis to Fight Disease
(Both activities address the study of bacterial cell wall components for developing new treatments for
tuberculosis and other bacterial infections.)
 Laboratory Investigation: Carbohydrate Investigation Using Biotechnology: Making Lactose-Free Milk
(Both investigations study the action of beta-galactosidase on lactose.)
 Career Connections: Carbohydrate Research and,
Career Connections: Professor Ray Lemieux (actual title?)
(Both activities explore the need for carbohydrate research for the purpose of finding new ways to treat
infectious diseases.)
SAFETY CONSIDERATIONS:
Students must wear eye protection, and it is recommended that they wear gloves and aprons. The buffer
solution is mildly acidic and the sodium carbonate solution is basic. pNP-Gal is toxic, so avoid ingestion or
contact with skin or eyes. If contact occurs, wash skin with lots of soapy water and flush eyes with water for at
least 5 minutes. All solutions are dilute and may be disposed of down the drain.
TECHNICAL CONSIDERATIONS:
Acquiring Materials:
The enzyme, substrate and immuno plates can be purchased from Sigma-Aldrich. The plates are reusable.
The enzyme and substrate should be enough for several class set-ups.
Name
p-nitrophenyl-beta-Dgalactopyranoside
β-Galactosidase from Aspergillus oryzae
Nunc-Immuno plates
AICCSLabInvEnzymeActivity
Product #
Amount
Cost
N-1252
250 mg
47.00
-20°C
N-5160
N-9410
25 KU
60 plates
46.50
251.00
-20°C
--
Centre for Carbohydrate Science
(CAD 2007)
Storage
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Alberta Ingenuity & CMASTE
Preparation and Storage of Solutions:
Prepare the NaAc buffer solution in the fume hood to avoid exposure to fumes of acetic acid.
 0.1M NaAc (pH 5.0) buffer solution:
To make 1 L of solution, dissolve 8.204 g of sodium acetate in 1.00 L of distilled water. Add glacial acetic
drop-wise while monitoring the pH until it reaches 5.0. Store at room temperature.
 0.5 M N a2CO3 solution:
To make 1 L of solution, dissolve 50.300 g of sodium carbonate in 1.00 L of distilled water. Store at room
temperature.
 1.0 g/L Beta-galactosidase solution:
To make 100 ml of solution, dissolve 0.1 g of enzyme in 100 ml of NaAc buffer (pH 5.0). Store at 4°C, or
freeze for long-term storage.
 5 mM p-nitrophenyl-beta-galactoside solution:
To make 100 ml of solution, dissolve 0.16 g of p-nitrophenyl-beta-D-galactopyranoside* in 100 ml of
NaAc buffer (pH 5.0). Slow hydrolysis may occur due to contamination from environmental betagalactosidases. Store at 4°C, or freeze for long-term storage.
(* The name of the substrate is shortened in the lab for convenience.)
 125 mM lactose solution: To make 100 ml of solution, dissolve 4.5 g of lactose in 100 ml of NaAc buffer (pH
5.0). Store at room temperature.
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Laboratory Investigation: Enzyme Activity and Inhibitors
(With Answers and
Sample Evidence)
Purpose
Many important reactions in living
cells require specific enzymes as
catalysts. Catalysts speed up
chemical reactions, but are not
used up in the process. Without
enzymes, reactions occur at rates
far too slow for the organism to function
Figure 1: Enzymes act as catalysts to speed up chemical reactions
properly. Figure 1 summarizes an
example of how enzymes work. They are
large proteins that bind to the reactants, which are called substrates. After binding, the enzyme helps the
reaction along. After the reaction occurs, the enzyme releases the products. In humans, many enzymes are
essential for survival. For example, glycogen storage diseases are caused when one of the enzymes involved a
sequence of reactions that converts glycogen to glucose is missing. Glycogen is a carbohydrate similar to starch
and is stored in the liver and muscles. It must be converted to glucose before it can be delivered to the body’s
cells by the blood stream. Without the enzyme, glycogen builds up in the liver and muscle tissues. Infants with
this disease often suffer from cardiac failure due to the accumulation of glycogen in the cardiac muscle.
Researchers are exploiting the vital importance of enzymes to fight bacterial
infections. Some antibacterial drugs work by inhibiting the function of enzymes
in bacterial cells. For example, penicillin works by inhibiting an enzyme which
helps to build bacterial cell walls. Because human cells do not have cell walls,
inhibiting the bacterial enzyme does not affect them. At the Alberta Ingenuity
Centre for Carbohydrate Science (AICSS), researches are working to design a
molecule that will inhibit an enzyme involved in building a carbohydrate
component of the cell wall of Mycobacterium tuberculosis, which causes the
lung disease tuberculosis. Figure 2 shows how a type of enzyme inhibitor
called a competitive inhibitor works.
Figure 2: Competitive inhibitors impair the function of enzymes.
In order to design effective inhibitors to fight disease, researchers have to develop methods to measure enzyme
activity and how enzymes are affected the presence of inhibitors. In this investigation, you will study an enzyme
called beta-galactosidase, which breaks down the milk sugar lactose into simpler sugars that can be absorbed
into the blood stream. This reaction is shown in Figure 3. A large number of people around the world lack this
enzyme, causing them to be lactose intolerant. The milk industry uses beta-galactosidases on a large scale to
produce lactose-fee milk. In this lab, you will be using beta-galactosidase extracted from a fungus called
Aspergillus oryzae.
Figure 3: Lactose is a disaccharide sugar. In the gut, beta-galactosidase catalyzes a hydrolysis
reaction that yields two monosaccharide products, beta-galactose and glucose.
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Problem
1. How does enzyme concentration affect enzyme activity?
2. How does the presence of a competitive inhibitor affect enzyme activity?
Design
In order to measure enzyme activity, an artificial substrate called p-nitrophenyl-beta-galactoside (pNP-Gal) will
be used. This artificially synthesized compound consists of a beta-galactose linked to a ringed structure called pnitrophenol. The enzyme catalyzes the hydrolysis of pNP-Gal, breaking the bond that connects the betagalactose and the p-nitrophenol. This reaction occurs optimally at pH 5.0. The reaction will be allowed to run
and then stopped by adding sodium carbonate solution increasing the pH to approximately 11. At this alkaline
pH, the enzyme is deactivated and the p-nitrophenol product loses a proton to become an ion that reflects
yellow light. The intensity of the yellow colour remaining after the reaction is stopped can be used as an
indicator of enzyme activity. A very intense yellow indicates that relatively more pNP-Gal has been hydrolyzed.
For the purposes of this investigation, enzyme activity will be defined as the ability of beta-galactosidase to
catalyze the hydrolysis of pNP-Gal. Enzyme activity will be measured by analyzing a digital photo of the reaction
mixtures using an image processing program.
To test the first problem, enzyme solutions will be prepared with varying concentrations and a fixed amount of
pNP-Gal will be added to samples of each solution. To test the second problem, the same process will be
repeated but in the presence of lactose.
Figure 4: pNP-Gal is used to measure the activity of beta-galactosidase. It is hydrolysed in a
yield galactose and p-nitrophenol. At an alkaline pH, p-nitrophenol donates a proton to become
an ion with an intense yellow colour.
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SAFETY CONSIDERATIONS
The following protective equipment should be worn:
Safety glasses, gloves, apron
Follow your teacher’s instructions concerning the disposal of chemicals
Alberta Ingenuity & CMASTE
Materials
 10 ml test tubes, 6
 Test tube rack
 Pasteur pipettes, 9
 Bulb for Pasteur pipettes
 Immuno plate
 10 ml graduated cylinder
 0.1 M sodium acetate (NaAc) buffer, pH 5.0, 25 ml
 5 mM p-nitrophenyl-beta-galactoside (pNP-Gal) in
buffer, 5 ml
 150 µg/ml A. Oryzae beta-galactosidase (enzyme)
in buffer, 5 ml
 0.5 M sodium carbonate (Na2CO3), 5 ml
 125 mM lactose in buffer, 5 ml
 Digital camera (optional)
 Computer with ImageJ software (optional)
Procedure: Part A: Preparing the enzyme solutions by repeated dilutions
1. Number your test tubes 1-6 and place them in the test tube rack in numeric order from left to right.
2. Use the graduated cylinder to measure 5 ml of the enzyme solution and transfer it to test tube 6.
3. Use the graduated cylinder to measure 0.5 ml of the enzyme solution in test tube 6 and transfer it to
test tube 5. Add 4.5 ml of buffer to test tube 5. Mix the solution by covering and inverting several times.
4. Use the graduated cylinder to measure 0.5 ml of the enzyme solution in test tube 5 and transfer it to
test tube 4. Add 4.5 ml of buffer to test tube 4. Mix the solution by covering and inverting several times.
5. Use the graduated cylinder to measure 0.5 ml of the enzyme solution in test tube 4 and transfer it to
test tube 3. Add 4.5 ml of buffer to test tube 3. Mix the solution by covering and inverting several times.
6. Use the graduated cylinder to measure 0.5 ml of the enzyme solution in test tube 3 and transfer it to
test tube 2. Add 4.5 ml of buffer to test tube.
7. Fill test tube 1 with 5.0 ml of buffer.
Procedure: Part B: Testing enzyme activity
8. Place a clean Pasteur pipette into each test tube and into each solution listed in Table 1. Be careful to
avoid cross-contamination. Table 1 shows the drops to be placed in each well of the immuno plate. Only
use wells in outside rows to allow for a better digital photo later.
Well #
A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6
Enzyme
2
2
2
2
2
2
2
2
2
2
2
2
Solution
Buffer
Lactose
solution
pNP-Gal
solution
Na2CO3
solution
2
2
2
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Photo 1: A good way to label the wells
is to place the immuno plate on a
piece of paper and write the labels on
the paper.
Table 1: the numbers indicate how many drops to place in each well. The
test tube numbers correspond with the well numbers. For example, add
the enzyme solution from test tube 2 to both wells A2 and B2.
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9. Add the enzyme solutions to the immuno plate (as prescribed in Table 1). Next, add the buffer and
lactose solutions.
10. Wait 2 minutes.
11. Add the pNP-Gal solution to the immuno plate.
12. Wait 30 seconds.
13. Add the Na2CO3 solution.
Design/Procedure Questions:
1. Which test tube(s) acts as the “negative control” for enzyme concentration?
Test tubes A1 and B1 act as negative controls for enzyme concentration. They contain no enzyme, and
therefore, can be used to establish a background value for the appearance of the solution if no hydrolysis
occurs.
2. Explain why two drops of buffer solution are added to the “A” wells.
Two drops of buffer must be added to the “A” wells to ensure the same volume of solution, and
therefore, the same concentration of enzyme and substrate compared to the “B” wells, which had 2
drops of lactose solution added.
3. For each of the two problem statements, given earlier in this investigation, identify the manipulated
variable, responding variable, and four relevant controlled variables.
For problem statement 1: “How does enzyme concentration affect enzyme activity?”
Manipulated variable: enzyme concentration
Responding variable: enzyme activity (as measured by the colour of the mixture)
Controlled variables: concentration of substrate, concentration of sodium carbonate, volume of
sodium carbonate added, pH of solution during the reaction, temperature of solution during
reaction, time reaction is allowed to proceed.
For problem statement 2: “How does the presence of a competitive inhibitor affect enzyme activity?”
Manipulated variable: presence or absence of lactose
Responding variable: enzyme activity (as measured by the colour of the mixture)
Controlled variables: concentration of substrate, concentration of sodium carbonate, volume of
sodium carbonate added, pH of solution during the reaction, temperature of solution during
reaction, time reaction is allowed to proceed. Concentration of enzyme (e.g. the enzyme activity
in well A6 can be compared to the enzyme activity in well B6, because they have the same
concentration of enzyme. The only variable that is altered is the presence or absence of lactose)
4. Explain how the “A” wells can be considered as a “control group” when testing the effect of lactose on
enzyme activity.
The “A” wells can be considered as a “control group” because they contained no lactose. The “B” wells
were the “experimental group”, because they contained the lactose.
5. Provide a reason for waiting in steps 10 and 12.
The waiting time during step 10 was to allow for the inhibitor (lactose) to bind to the enzyme. The wait
time during step 12 was to give the hydrolysis reaction time to happen.
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Evidence/Analysis: Qualitative
1. In words and/or using a chart, table or diagram, describe the
colour of each reaction mixture after the sodium carbonate
solution has been added. Use words or the following
symbols: (-) for no reaction and (+), (++), (+++), etc, for
varying degrees of yellow.
Answers may vary.
Well
Number
No Lactose
Present
A1
A2
A3
A4
A5
A6
B1
B2
B3
B4
B5
B6
Enzyme Activity
(Colour
Intensity)
++
+++
++++
+++++
+
++
+++
++++
2. Referring to your observations, describe:
a. The effect of enzyme concentration on enzyme
Lactose
activity.
Present
The greater the enzyme concentration is, the greater
the enzyme activity.
b. The effect of the lactose on enzyme activity.
The presence of lactose decreases enzyme activity (the ability of the enzyme to hydrolyze pNPGal).
Evidence/Analysis: Quantitative
Enzyme activity will be measured quantitatively by determining the relative “darkness” (i.e. “value”) of each
solution. A digital photo of the solutions will be analyzed using image processing software. Instructions for using
a free, downloadable program called ImageJ are included, but you may wish to use a different program. ImageJ
is available for free download at the following website:
http://rsb.info.nih.gov/ij/
3. Take a photo of your immuno plate in profile (so that you are looking at the wells side-by-side vertically,
as shown in Figure 5). If possible, use the “macro” setting on your camera to get as clear an image as
possible. Use diffuse light; it is important that all wells receive the same amount of illumination.
4. Upload the photo to your computer, and open it with ImageJ. To do this, right-click on the image file,
select open-with, and then select ImageJ. Two windows will open: one with the ImageJ toolbar and the
other with the image
itself. (See Figure 5)
Figure 5: Use ImageJ to
5. For each well in the
measure the relative enzyme
activity for each well.
photo, select a large
uniform region. From the
toolbar, Select Analyze,
and then Measure. A
Results window will open.
Select and measure all 12
wells. The measurements
will be added one at a
time to the Results
window.
6. The “Mean” values in the
Results window give the average brightness of each measured region of the photo. Copy the Mean
values under the heading of “Average Photo Brightness” in the data table like the one to the right. You
may choose to use a spreadsheet program to do this.
7. Calculate the enzyme concentrations using information presented in the Materials and Procedure
sections. Enter the values into the data table.
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Evidence Table: Enzyme Activity
Enzyme
Average
Relative
Well
Concentration
Photo
Enzyme
Number
(µg/ml)
Brightness
Activity
A1
0
147.009
0
A2
0.015
149.102
-2.093
No
A3
0.15
145.568
1.441
Lactose
A4
1.5
136.612
10.397
Present
A5
15
121.641
25.368
A6
150
106.101
40.908
B1
0
148.526
0
B2
0.015
145.177
3.349
B3
0.15
147.011
1.515
Lactose
Present
B4
1.5
144.718
3.808
B5
15
137.025
11.501
B6
150
120.801
27.725
Calculating Enzyme Concentration:
According the material list the enzyme solution provided had a concentration of 150 µg/ml. Each
of the dilutions in the procedure transferred 10% of the solution to a new test tube, and then
topped it up with buffer to the original volume. This means that the concentration was reduced
to 1/10 of the original concentration each time it was diluted. Wells A1 and B1 contained no
enzyme.
8. Calculate the relative enzyme activity for each “A” well by subtracting its average photo brightness from
the average photo brightness of well A1. Do the same for the “B” wells, except subtract each value from
the average photo brightness of well B1. Enter the values into the data table.
Sample Calculations:
Well A3: Relative enzyme activity = 147.009 - 145.568 = 1.441
Well B3: Relative enzyme activity = 148.526 - 147.011 = 1.515
9. Plot two graphs on the same set of axes – one for the “A” data the one for the “B” data. On the
horizontal axis, plot enzyme concentration, and on the vertical axis, plot enzyme activity. For each series
of data points draw a curve of best fit.
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10. Based on your quantitative analysis, describe:
a. The effect of enzyme concentration on enzyme activity.
As enzyme concentration increases, enzyme activity increases.
b. The effect of the lactose on enzyme activity.
The presence of lactose decreases enzyme activity.
Evaluation
11. Is the relationship between enzyme concentration and enzyme activity directly proportional, or is it
better described by a different mathematical relationship? In words, provide an explanation for the
general shape of the graphs of enzyme activity versus enzyme concentration plotted in this
investigation.
The relationship between enzyme concentration and enzyme activity is not directly proportional. The
general shape shows a “plateau” in enzyme activity. This could be due to the limited amount of substrate
available to react. As the concentration of enzyme increases more and more enzyme molecules must
“compete” to bind with a fixed number of substrate molecules.
12. Explain the effect lactose had on the ability of beta-galactosidase to hydrolyze pNP-Gal. Can lactose be
considered to a competitive inhibitor?
Lactose is recognized by the enzyme and therefore “competes” with pNP-Gal to bind with the enzyme.
Any lactose that bind with the enzyme and are hydrolyzed, do not produce the p-nitrophenyl that shows
up as yellow after the reaction is stopped. So, the presence of lactose can be said to competitively inhibit
the ability of beta-galactosidase to hydrolyze pNP-Gal.
13. Sketch a graph predicting the results of the same experiment if it were conducted with
a. A greater concentration of lactose.
b. A lesser concentration of lactose.
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14. Is it more appropriate to consider pNP-Gal as a competitive inhibitor, rather than lactose? Explain why
pNP-Gal was considered to be the substrate rather that the inhibitor in this investigation.
It may be more appropriate to consider pNP-Gal as the competitive inhibitor, rather than lactose,
because beta-galactosidase hydrolyzes lactose normally in the gut. pNP-Gal is artificially synthesized.
pNP-Gal was considered to be the substrate because it is convenient to measure, due to the colour
change, and the experimental design still adequately investigates the problems posed.
15. People with diabetes suffer from high concentrations of glucose in the blood due to insufficient amounts
of the hormone insulin. It is possible to measure glucose concentration using a glucose meter. Imagine
that you are a researcher looking for new ways to decrease blood glucose concentration in individuals
with diabetes. You wish to test a newly synthesized chemical which may inhibit an enzyme involved in
converting glycogen into glucose. Describe an experiment that would test the effect of the inhibitor’s
concentration on the enzyme’s activity. Include the following as part of your response:
 Problem
 Design (including identification of the manipulated variable, responding variable and controlled
variables)
Problem:
What is the effect of inhibitor concentration on enzyme activity?
Design
Inhibitor solutions of varying concentrations will be added to solutions containing fixed
concentrations of enzyme and substrate. The reaction will be allowed to run for a fixed amount of
time. The concentration of the product, glucose, will be measured using a glucose meter.
Manipulated variable: concentration of inhibitor
Responding variable: concentration of glucose after reaction has occurred
Controlled variables: Time for reaction to occur, concentration of substrate, concentration of enzyme,
temperature, pH
Author: M. Haak
The Alberta Ingenuity Fund supports science and engineering research of the highest calibre, to create a
prosperous future for the province. This support includes programs designed to engage the public and encourage
young people to pursue careers in science. It draws funding from a $1 billion endowment established and
managed by the Government of Alberta to build the capacity for innovation, especially in areas with long lasting
social and economic impact.
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