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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

MW = 301.3 g/mol N1127 -20°C

Beta-galactosidase from Aspergillus oryzae

MW = 109900 Da G5160 -20°C

Glucose

MW = 180.2 g/mol D9434

Room

Temperature

Lactose

MW = 342.3 g/mol L3625

Room

Temperature

Galactose

MW = 180.2 g/mol G0750

Room

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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