Vous êtes sur la page 1sur 14

Alberta Ingenuity & CMASTE

Laboratory Investigation: Enzyme Activity and Inhibitors (Teachers 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 setups.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science 1/14

Alberta Ingenuity & CMASTE Name p-nitrophenyl-beta-Dgalactopyranoside -Galactosidase from Aspergillus oryzae Nunc-Immuno plates Product # N-1252 N-5160 N-9410 Amount 250 mg 25 KU 60 plates Cost
(CAD 2007)

Storage -20C -20C --

47.00 46.50 251.00

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science 2/14

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 4C, 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-Dgalactopyranoside* in 100 ml of NaAc buffer (pH 5.0). Slow hydrolysis may occur due to contamination from environmental beta-galactosidases. Store at 4C, 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.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science 3/14

Alberta Ingenuity & CMASTE

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 Figure 1: Enzymes act as catalysts to speed up chemical reactions the organism to function 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 bodys 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 betagalactosidases 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.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science 4/14

Alberta Ingenuity & CMASTE

Figure 3: Lactose is a disaccharide sugar. In the gut, beta-galactosidase catalyzes a hydrolysis reaction that yields two monosaccharide products, betagalactose and glucose.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science 5/14

Alberta Ingenuity & CMASTE 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-betagalactoside (pNP-Gal) will be used. This artificially synthesized compound consists of a beta-galactose linked to a ringed structure called p-nitrophenol. The enzyme catalyzes the hydrolysis of pNP-Gal, breaking the bond that connects the beta-galactose and the pnitrophenol. 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 betagalactosidase 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, pnitrophenol donates a proton to become an ion with an intense yellow colour.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science 6/14

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 g/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)

SAFETY SAFETY CONSIDERATIONS CONSIDERATIONS The The following following protective protective equipment equipment should should be be worn: worn: Safety glasses, gloves, apron Safety glasses, gloves, apron Follow Follow your your teachers teachers instructions instructions concerning concerning the the disposal disposal of of chemicals chemicals

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.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science 7/14

Alberta Ingenuity & CMASTE


Photo 1: A good way to label the wells is to place the immuno Well #piece A of Apaper A and A A plate on a 1 2 3 4 5 write the labels on the paper.

A 6

B 1

B 2

B 3 2 0 2

B 4 2 0 2

B 5 2 0 2

B 6 2 0 2

Enzym Table 1: the numbers indicate how many drops to place in e tube numbers correspond with the well each well. The test 2 2 2 2 2 2 2 2 Soluti add the enzyme solution from test numbers. For example, on A2 and B2. tube 2 to both wells Buffer Lactos e solutio n pNPGal solutio n Na2CO
3

2 0

2 0

2 0

2 0

2 0

2 0

0 2

0 2

2 2 2 2 2 2 2 2 2 2 2 2 solutio n 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?

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science 8/14

Alberta Ingenuity & CMASTE 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.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science 9/14

Alberta Ingenuity & CMASTE

Well Numb er No Lactose Present A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6

Enzyme Activity (Colour Intensity) ++ +++ ++++ +++++ + ++ +++ ++++

Lactose Present

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. 2. Referring to your observations, describe: a. The effect of enzyme concentration on enzyme activity. 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 pNP-Gal). 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 Figure 5: Use ImageJ to ImageJ toolbar and measure the relative the other with the enzyme activity for each image itself. (See well. Figure 5) 5. For each well in the photo, select a large uniform region. From the toolbar, Select

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science10/14

Alberta Ingenuity & CMASTE 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.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science11/14

Alberta Ingenuity & CMASTE Evidence Table: Enzyme Activity Average Well Enzyme Relative Photo Numb Concentrat Enzyme Brightnes er ion (g/ml) Activity s A1 0 147.009 0 No A2 0.015 149.102 -2.093 Lactos A3 0.15 145.568 1.441 e A4 1.5 136.612 10.397 Presen A5 15 121.641 25.368 t A6 150 106.101 40.908 B1 0 148.526 0 B2 0.015 145.177 3.349 Lactos B3 0.15 147.011 1.515 e Presen B4 1.5 144.718 3.808 t 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 g/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.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science12/14

Alberta Ingenuity & CMASTE 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 pNPGal. 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.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science13/14

Alberta Ingenuity & CMASTE 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 inhibitors concentration on the enzymes 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.

AICCSLabInvEnzymeActivity

Centre for Carbohydrate Science14/14