Alberta Ingenuity & CMASTE
Laboratory Investigation: Enzyme Activity and Inhibitors
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
properly. Figure 1 summarizes
an example of how enzymes
work. They are large proteins that
Figure 1: Enzymes act as catalysts to speed up chemical reactions
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 acts as the “negative control” for enzyme concentration?
2. Explain why two drops of buffer solution are added to the “A” wells.
3. For each of the two problem statements, given earlier in this investigation, identify the manipulated
variable, responding variable, and four relevant controlled variables.
4. Explain how the “A” wells can be considered as a “control group” when testing the effect of lactose on
enzyme activity.
5. Provide a reason for waiting in steps 10 and 12.
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.
2. Referring to your observations, describe:
a. The effect of enzyme concentration on enzyme activity.
b. The effect of the lactose on enzyme activity.
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)
5. For each well in the
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.
Figure 5: Use ImageJ to measure the relative
enzyme activity for each well.
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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.
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.
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.
10. Based on your quantitative analysis, describe:
a. The effect of enzyme concentration on enzyme activity.
b. The effect of the lactose on 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.
12. Explain the effect lactose had on the ability of beta-galactosidase to hydrolyze pNP-Gal. Can lactose be
considered to be a competitive inhibitor?
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.
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.
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)
Author: M. Haak
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