a icc slab in v enzyme activity tr

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Alberta Ingenuity & CMASTE

Laboratory Investigation: Enzyme Activity and Inhibitors(Teachers Edition)

CURRICULAR LINKS: Biology 20, Chemistry 30

CLASSTIME: 1-2 periods

Class time depends on whether students perform the quantitative analysis (take a digitalphoto and use image processing software to analyze it). The lab is designed so thatteachers can opt out of this part if they wish. Below the recommended omitted sectionsare identified if you decide to skip the computer analysis. One option is to provide thestudents with the photo analysis data provided in this document, if you do not have theresources 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 SERIESThis 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 newantibiotics for tuberculosis and other bacterial infections.)

Case Study: Chemical Synthesis to Fight Disease

(Both activities address the study of bacterial cell wall components for developingnew 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 offinding new ways to treat infectious diseases.)

SAFETYCONSIDERATIONS:Students must wear eye protection, and it is recommended that they wear gloves andaprons. 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, washskin with lots of soapy water and flush eyes with water for at least 5 minutes. All solutionsare 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. Theplates are reusable. The enzyme and substrate should be enough for several class set-ups.

AICCSLabInvEnzymeActivity Centre for Carbohydrate Science 1/14


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Name Product # Amount Cost(CAD 2007)

Storage

p-nitrophenyl-beta-D-galactopyranoside

N-1252 250 mg 47.00 -20C

-Galactosidase fromAspergillus oryzae

N-5160 25 KU 46.50 -20C

Nunc-Immuno plates N-9410 60 plates 251.00 --


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Preparation and Storage of Solutions:Prepare the NaAc buffer solution in the fume hood to avoid exposure to fumes of aceticacid.

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 distilledwater. 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 distilledwater. 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 (pH5.0). 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-D-galactopyranoside* in 100 ml of NaAc buffer (pH 5.0). Slow hydrolysis may occurdue to contamination from environmental beta-galactosidases. Store at 4C, orfreeze 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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Figure 1: Enzymes act as catalysts to speed up chemical reacti


Laboratory Investigation: Enzyme Activity and Inhibitors(With Answers andSample Evidence)

PurposeMany important reactions in

living cells require specificenzymes as catalysts.Catalysts speed upchemical reactions, but arenot used up in the process.Without enzymes, reactionsoccur at rates far too slow forthe organism to functionproperly. Figure 1 summarizesan 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 thereaction occurs, the enzyme releases the products. In humans, many enzymes are

essential for survival. For example, glycogen storage diseases are caused when one of theenzymes 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 mustbe 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 thisdisease often suffer from cardiac failure due to the accumulation of glycogen in thecardiac muscle.

Researchers are exploiting the vital importance of enzymes tofight bacterial infections. Some antibacterial drugs work byinhibiting the function of enzymes in bacterial cells. Forexample, penicillin works by inhibiting an enzyme which helpsto build bacterial cell walls. Because human cells do not havecell 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 willinhibit an enzyme involved in building a carbohydratecomponent of the cell wall ofMycobacterium tuberculosis,which causes the lung disease tuberculosis. Figure 2 shows

how a type of enzyme inhibitor called a competitive inhibitorworks.

Figure 2: Competitive inhibitors impair the function of enzymes.

In order to design effective inhibitors to fight disease, researchers have to developmethods to measure enzyme activity and how enzymes are affected the presence ofinhibitors. 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 intothe blood stream. This reaction is shown in Figure 3. A large number of people around theworld 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 usingbeta-galactosidase extracted from a fungus calledAspergillus oryzae.



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Figure 3: Lactose is a disaccharide sugar. In the gut, beta-galactosidasecatalyzes a hydrolysis reaction that yields two monosaccharide products, beta-

galactose and glucose.


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Figure 4: pNP-Gal is used to measure the activity of beta-galactosidase. It ishydrolysed 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.


Problem1. How does enzyme concentration affect enzyme activity?2. How does the presence of a competitive inhibitor affect enzyme activity?

DesignIn 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 p-nitrophenol. The enzyme catalyzes thehydrolysis of pNP-Gal, breaking the bond that connects the beta-galactose and the p-nitrophenol. This reaction occurs optimally at pH 5.0. The reaction will be allowed to runand then stopped by adding sodium carbonate solution increasing the pH to approximately11. At this alkaline pH, the enzyme is deactivated and the p-nitrophenol product loses aproton to become an ion that reflects yellow light. The intensity of the yellow colourremaining after the reaction is stopped can be used as an indicator of enzyme activity. Avery intense yellow indicates that relatively more pNP-Gal has been hydrolyzed. For thepurposes 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 byanalyzing a digital photo of the reaction mixtures using an image processing program.

To test the first problem, enzyme solutions will be prepared with varying concentrationsand a fixed amount of pNP-Gal will be added to samples of each solution. To test thesecond problem, the same process will be repeated but in the presence of lactose.



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SAFETY CONSIDERATIONSThe following protective equipment should be worn:Safety glasses, gloves, apronFollow your teachers instructions concerning thedisposal of chemicals

SAFETY CONSIDERATIONSThe following protective equipment should be worn:Safety glasses, gloves, apronFollow your teachers instructions concerning thedisposal of chemicals


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/mlA. 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 dilutions1. 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 bycovering and inverting several times.

4. Use the graduated cylinder to measure 0.5 ml of the enzyme solution in test tube 5and transfer it to test tube 4. Add 4.5 ml of buffer to test tube 4. Mix the solution bycovering and inverting several times.

5. Use the graduated cylinder to measure 0.5 ml of the enzyme solution in test tube 4and transfer it to test tube 3. Add 4.5 ml of buffer to test tube 3. Mix the solution bycovering and inverting several times.

6. Use the graduated cylinder to measure 0.5 ml of the enzyme solution in test tube 3and 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 activity8. 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 beplaced in each well of the immuno plate. Only use wells in outside rows to allow fora better digital photo later.



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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 forthe 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 ofsolution, and therefore, the same concentration of enzyme and substrate comparedto the B wells, which had 2 drops of lactose solution added.

3. For each of the two problem statements, given earlier in this investigation, identifythe manipulated variable, responding variable, and four relevant controlledvariables.For problem statement 1: How does enzyme concentration affect enzymeactivity?

Manipulated variable: enzyme concentrationResponding variable: enzyme activity (as measured by the colour of the

mixture)Controlled variables: concentration of substrate, concentration of sodiumcarbonate, volume of sodium carbonate added, pH of solution during thereaction, temperature of solution during reaction, time reaction is allowed to

proceed.For problem statement 2: How does the presence of a competitive inhibitor affectenzyme activity?


Well # A1

A2

A3

A4

A5

A6

B1

B2

B3

B4

B5

B6

EnzymeSolution

2 2 2 2 2 2 2 2 2 2 2 2

Buffer 2 2 2 2 2 2 0 0 0 0 0 0

Lactosesolution

0 0 0 0 0 0 2 2 2 2 2 2

pNP-Galsolution

2 2 2 2 2 2 2 2 2 2 2 2

Na2CO

3

solution

2 2 2 2 2 2 2 2 2 2 2 2

le 1: the numbers indicate how many drops to place inh well. The test tube numbers correspond with the wellbers. For example, add the enzyme solution from test2 to both wells A2 and B2.

Photo 1:A good way to labelthe wells is to place the immuno

plate on a piece of paper andwrite the labels on the paper.


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Manipulated variable: presence or absence of lactoseResponding variable: enzyme activity (as measured by the colour of the

mixture)Controlled variables: concentration of substrate, concentration of sodiumcarbonate, volume of sodium carbonate added, pH of solution during thereaction, 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 sameconcentration of enzyme. The only variable that is altered is the presence orabsence of lactose)

4. Explain how the A wells can be considered as a control group when testing theeffect of lactose on enzyme activity.The A wells can be considered as a control group because they contained nolactose. The B wells were the experimental group, because they contained thelactose.

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 tothe enzyme. The wait time during step 12 was to give the hydrolysis reaction timeto happen.



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Evidence/Analysis: Qualitative1. 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 thefollowing symbols: (-) for no reaction and (+), (++), (+++), etc, for varying degreesof 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 enzymeto hydrolyze pNP-Gal).

Evidence/Analysis: QuantitativeEnzyme 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 imageprocessing software. Instructions for using a free, downloadable program called ImageJ areincluded, but you may wish to use a different program. ImageJ is available for freedownload 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 wellsside-by-side vertically, as shown in Figure 5). If possible, use the macro settingon your camera to get as clear an image as possible. Use diffuse light; it isimportant 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-clickon the image file, select open-with, and then select ImageJ. Two windows will

open: one with theImageJ toolbar andthe other with theimage itself. (SeeFigure 5)

5. For each well in thephoto, select a largeuniform region. Fromthe toolbar, Select

AICCSLabInvEnzymeActivity Centre for Carbohydrate Science10/14

WellNumb

er

EnzymeActivity(Colour

Intensity)

NoLactosePresent

A1 -A2 -A3 ++A4 +++A5 ++++

A6 +++++

LactosePresent

B1 -B2 -B3 +B4 ++B5 +++B6 ++++

Figure 5: Use ImageJ tomeasure the relativeenzyme activity for eachwell.


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Analyze, and then Measure. A Results window will open. Select and measure all12 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 eachmeasured region of the photo. Copy the Mean values under the heading of AveragePhoto Brightness in the data table like the one to the right. You may choose to usea 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

WellNumb

er

EnzymeConcentration (g/ml)

AveragePhoto

Brightness

RelativeEnzymeActivity

NoLactos

ePresen

t

A1 0 147.009 0A2 0.015 149.102 -2.093

A3 0.15 145.568 1.441A4 1.5 136.612 10.397A5 15 121.641 25.368A6 150 106.101 40.908

Lactose

Present

B1 0 148.526 0B2 0.015 145.177 3.349B3 0.15 147.011 1.515B4 1.5 144.718 3.808B5 15 137.025 11.501B6 150 120.801 27.725

Calculating Enzyme Concentration:According the material list the enzyme solution provided had a concentrationof 150 g/ml. Each of the dilutions in the procedure transferred 10% of thesolution to a new test tube, and then topped it up with buffer to the originalvolume. This means that the concentration was reduced to 1/10 of theoriginal concentration each time it was diluted. Wells A1 and B1 contained noenzyme.

8. Calculate the relative enzyme activity for each A well by subtracting its averagephoto brightness from the average photo brightness of well A1. Do the same for theB 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 = 1.441Well 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 Bdata. 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 directlyproportional, or is it better described by a different mathematical relationship? Inwords, provide an explanation for the general shape of the graphs of enzymeactivity 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 bedue to the limited amount of substrate available to react. As the concentration ofenzyme increases more and more enzyme molecules must compete to bind witha 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 isstopped. So, the presence of lactose can be said to competitively inhibit the abilityof beta-galactosidase to hydrolyze pNP-Gal.

13.Sketch a graph predicting the results of the same experiment if it were conductedwith

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 thanlactose? Explain why pNP-Gal was considered to be the substrate rather that theinhibitor in this investigation.It may be more appropriate to consider pNP-Gal as the competitive inhibitor, ratherthan lactose, because beta-galactosidase hydrolyzes lactose normally in the gut.

pNP-Gal is artificially synthesized. pNP-Gal was considered to be the substratebecause 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 toinsufficient amounts of the hormone insulin. It is possible to measure glucoseconcentration using a glucose meter. Imagine that you are a researcher looking fornew ways to decrease blood glucose concentration in individuals with diabetes. Youwish to test a newly synthesized chemical which may inhibit an enzyme involved inconverting glycogen into glucose. Describe an experiment that would test the effectof the inhibitors concentration on the enzymes activity. Include the following aspart 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?

DesignInhibitor solutions of varying concentrations will be added to solutions

containing fixed concentrations of enzyme and substrate. The reaction will beallowed 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 inhibitorResponding 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 highestcalibre, 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 theGovernment of Alberta to build the capacity for innovation, especially in areas with longlasting social and economic impact.


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