PRACTICAL COURSE

ENZYME ACTIVITY AND INHIBITORS

DR. GILLES TRUAN

SAFETY
Students must wear protection (lab coat). The buffer solution is mildly acidic and the sodium
carbonate solution is basic. ONPG 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

Chemical material
The enzyme, substrates and other molecules can be purchased from Sigma-Aldrich.
Name Product # Storage
Ortho-Nitro-Phenyl-Galactoside
N1127 -20°C
MW = 301.3 g/mol
Beta-galactosidase from Aspergillus oryzae
G5160 -20°C
MW = 109900 Da

Glucose Room
D9434
MW = 180.2 g/mol Temperature
Lactose Room
L3625
MW = 342.3 g/mol Temperature
Galactose Room
G0750
MW = 180.2 g/mol Temperature

Purpose
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.

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The milk industry uses beta-galactosidases on a large scale to produce lactose-free milk. In this lab
work, you will be using beta-galactosidase extracted from a fungus called Aspergillus oryzae.

Figure 1 : Lactose is a disaccharide sugar. In the gut, beta-galactosidase catalyzes a hydrolysis reaction that
yields two monosaccharide products, beta-galactose and glucose.

Problem
1. How does enzyme concentration affect enzyme activity?
2. How does the presence of inhibitors affect enzyme activity?
3. What are the different types of inhibition can you see with the molecules?
The purpose of this set of experiments is to answer all these questions.

Design
In order to measure enzyme activity, an artificial substrate called o-nitrophenyl-beta-galactoside
(ONPG) will be used. This artificially synthesized compound consists of a beta-galactose linked to a
ringed structure called o-nitrophenol. The enzyme catalyzes the hydrolysis of ONPG, breaking the bond
that connects the beta-galactose and the o-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 no longer active and the o-nitrophenol
(ONP) product loses a proton to become an ion that reflects yellow light. The intensity of the yellow
color remaining after the reaction is stopped can be used as an indicator of enzyme activity. A very
intense yellow indicates that relatively more ONPG 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 ONPG. ONP concentration will be measured by using a spectrophotometer to measure
absorbance of ONP.

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Figure 2 : ONPG is used to measure the activity of beta-galactosidase. It is hydrolyzed to yield galactose and o-
nitrophenol. At an alkaline pH, o-nitrophenol donates a proton to become an ion with an intense yellow color.

Materials
 15 ml test tubes
 1.5 ml eppendorf tubes
 Test tube racks
 Graduated cylinders
 Water bath at 37°C
 Pipettes (P1000, P200, P10)
 Timer
 pH-meter
 spectrophotometer
 plastic cuvettes
 0.1 M sodium acetate (NaAc) buffer, pH 5.0, 500 ml
 0.5 M sodium carbonate (Na2CO3), pH 11.0, 500 ml
 5 mM (1.5 mg/ml) o-nitrophenyl-beta-galactoside (ONPG) in buffer, 1 ml
 2 u/ml (0.25 mg/ml) A. oryzae beta-galactosidase (enzyme) in buffer, 4 ml
 10 mM lactose in buffer, 10 ml
 10 mM galactose in buffer, 10 ml
 10 mM glucose in buffer, 10 ml

Procedure A: measurement of product formation against time

1. Prepare a 15 ml test tube containing 4.75 ml of NaAc, 200 µl of ONPG substrate.
2. Prepare 8 tubes containing 0.5 ml of 0.5 M sodium carbonate buffer. They do not need to go in
the water bath.

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 3. Let the 15 ml tube in the water bath to equilibrate the temperature before adding the β-
galactosidase (50 µl in the total reaction).
4. Add the enzyme in the test tube, start the timer. Take immediately a 0.5 ml sample for t=0. Add
it to a prepared eppendorf tube containing 0.5 ml of 0.5 M sodium carbonate buffer (8 tubes).
5. Let the reaction proceed and take a sample at 1, 2, 4, 8, 10, 20, 60, 120 minutes

Procedure B: effect of enzyme concentration on enzymatic hydrolysis rates

1. Prepare a 15 ml test tube containing 4.94 ml of NaAc, 200 µl of ONPG substrate.
2. Prepare 8 identical tubes containing 0.45 µl the above solution and place them in the test tube
rack in numeric order from left to right.
3. Prepare 6 tubes containing 0.05 ml of several dilutions of the enzyme (from the non-diluted
solution, you can dilute by a factor of 2, step by step.

4. Let the tubes in the water bath to equilibrate the temperature.

5. Start the reaction by mixing the enzyme with the equilibrated buffer containing substrate.
6. Stop the reaction after 2 minutes by adding the reaction to 0.5 ml of 0.5 M sodium carbonate
buffer (8 tubes).

Procedure C: measuring Km, Vmax

1. Prepare a 15 ml test tube containing 4.94 ml of NaAc, 5 µl of enzyme.
2. Prepare 8 identical tubes containing 450 µl the above solution and place them in the test tube
rack in numeric order from left to right.
3. Prepare 6 tubes containing 0.05 ml of several dilutions of ONPG (starting from 500 µM, you can
dilute by a factor of 2, step by step). You can further dilute by a factor of 2 like in the above
procedure. Each concentration of substrate will be half of the previous one.
4. Let the tubes in the water bath to equilibrate the temperature.
5. Start the reaction by mixing the substrate with the equilibrated buffer containing enzyme.
6. Stop the reaction after 2 minutes by adding the reaction to 0.5 ml of 0.5 M sodium carbonate
buffer (8 tubes).

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Procedure D: Inhibition with lactose at 100 µM
1. Prepare a 15 ml test tube containing 4.94 ml of NaAc, 5 µl of enzyme.
2. Prepare 8 identical tubes containing 450 µl the above solution and place them in the test tube
rack in numeric order from left to right.
3. Prepare 6 tubes containing 0.05 ml the same dilutions of ONPG BUT with the inhibitor present
in each (adding 5 µl of lactose in each substrate tube).
4. Let the tubes in the water bath to equilibrate the temperature.
5. Start the reaction by mixing the substrate with the equilibrated buffer containing enzyme.
6. Stop the reaction after 2 minutes by adding the reaction to 0.5 ml of 0.5 M sodium carbonate
buffer (8 tubes).

Procedure E: Inhibition with galactose at 100 µM

1. Prepare a 15 ml test tube containing 4.94 ml of NaAc, 5 µl of enzyme.
2. Prepare 8 identical tubes containing 450 µl the above solution and place them in the test tube
rack in numeric order from left to right.
3. Prepare 6 tubes containing 0.05 ml the same dilutions of ONPG BUT with the inhibitor present
in each (adding 5 µl of galactose in each substrate tube).
4. Let the tubes in the water bath to equilibrate the temperature.
5. Start the reaction by mixing the substrate with the equilibrated buffer containing enzyme.
6. Stop the reaction after 2 minutes by adding the reaction to 0.5 ml of 0.5 M sodium carbonate
buffer (8 tubes).

Procedure F: Inhibition with glucose at 100 µM

1. Prepare a 15 ml test tube containing 4.94 ml of NaAc, 5 µl of enzyme.
2. Prepare 8 identical tubes containing 450 µl the above solution and place them in the test tube
rack in numeric order from left to right.
3. Prepare 6 tubes containing 0.05 ml of the same dilutions of ONPG BUT with the inhibitor
present in each (adding 5 µl of glucose in each substrate tube).
4. Let the tubes in the water bath to equilibrate the temperature.
5. Start the reaction by mixing the substrate with the equilibrated buffer containing enzyme.
6. Stop the reaction by adding the reaction to 0.5 ml of 0.5 M sodium carbonate buffer (8 tubes).

Reading the data

1. Prepare a solution of 250 µM ONPG, no enzyme, in 500 µl NaAc buffer. Mix it with 500 µl of
NaCO3 buffer. This will be the “zero” for the spectrophotometer (reference).
2. Take the solution containing ONP formed and put it in the cuvette. Read at 410 nm the value.

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 3. This value represents the ONP absorbance; hence, you can now calculate the ONP
concentration and therefore have a good estimate of the concentration of product formed
within the time of the reaction.

Report
Your report is PERSONAL. All of you will have the same set of data. Please interpret your data
alone, as it will help you preparing for the exam. In this report I want you to make plots of rates etc.
They can be either done in excel or on a sheet of paper that you will bring to me on Thursday. Interpret
the data that you see. Calculate the different values that you will have (Vm, Km, Vmapp, Kmapp, Ki etc.). A
typical report will describe the purpose of the experiment at the beginning, then give the raw data,
plot the data and then make calculations. This applies for all the different procedures. So be prepared
to answer all these questions for EACH PROCEDURE:
1. What is the purpose of the experiment?
2. What are we measuring in this reaction?
3. What are the controls?
4. Based on the data you have, what are your conclusions?
5. Interpret the entire experiment, would you consider that the goal is achieved? Why?

Further information

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