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Objectives

Students should be able to:
 

  1. Define an enzyme and it constituent parts

  2. Describe how an enzyme functions

  3. Describe the effect of temperature; enzyme concentration; substrate concentration; inhibitors; reducing agents; oxidizing agents; denaturation; and pH on enzyme activity.

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Introduction

Enzymes are a class of proteins whose function is that of a biological catalyst. They control the chemical reactions that occur in a cell or organism. The effect of this is the rate at which the chemical reactions take place. Enzymes can cause the reactions to occur thousands to a million times faster than they would without the enzyme being present.

Enzyme names usually end in the suffix, –ase. This may be added to the word of the chemical involved in the reaction such as sucrase, an enzyme which works on the sugar, sucrose. Enzymes are also very specific when it comes to control of a chemical reaction. Each chemical reaction that takes place in a cell is controlled by a different enzyme. This means that a cell contains hundreds to many thousands of different enzymes. The enzyme group that you will use in this lab is called polyphenoloxidase (catecholase or catechol oxidase). These enzymes is found in many varieties of plants. The polyphenoloxidase you will be using is extracted from the cells of the potato plant (Solanum tuberosum) Maybe you have noticed its reaction when cutting fruit such as apples (Malus pumila). The cut surface will darken. This darkening is due to the exposure of the enzyme in the damaged cells with the oxygen in the air.

You will be examining some of the enzyme-catalyzed properties of polyphenoloxidase, and collecting data associated with these properties. The basic reaction is as follows:

                                                                                       
                                                                                                              Polyphenoloxidase                                                   Slow

Catechol  +  ½ O2  ----->  Benzoquinone (orange) + H2O   ----> Melanins (browns)

Melanins are red-brown pigments found in many organisms. The more pigment present the darker the color.
 

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Pre-Lab Activities

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Using any search engine, such as Google.com, your textbook, Learning Resources Center, etc. to perform the following.

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Pre-Lab Activity #1

  1. Review the operation of the Spectrophotometer (found in Laboratory Exercise "Tools" and Measurement - Spectrophotometer)

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Pre-Lab Activity #2

  1. Printout and also save the Word version if you wish to type in your answers:

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    Data Collection Sheet (html version)

                                or

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    Data Collection Sheet (Word version)

  2. Download and save the Spreadsheet.

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Pre-Lab Activity #3

  1. Using any available resources find and make a list of ten biologically important enzymes and their substrates. Attach this list to your Pre-Lab Questions.

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Pre-Lab Questions (html version)      Pre-Lab Questions (Word version)

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The Laboratory Activities and Data Collection

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Lab Activity #1 - Calibration and Settings for Spectrophotometer

Materials:

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Spectrophotometer

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Calibration Cuvette

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Catechol in dropper bottle

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Distilled water (bottle)

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Parafilm squares

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Data Collection Sheet

 Procedures:

  1. Turn on the spectrophotometer and let it warm up for 5 minutes

  2. Prepare the calibration cuvette:

    1. Mark the cuvette at the 2 cm level (measure from the bottom of the cuvette) Place your mark on the side of the cuvette so it will not interfere with the light beam of the spectrophotometer.

    2. Add distilled water up to the 2 cm level

    3. Add 10 drops of catechol, Placing the parafilm over the opening of the cuvette and hold it in place with your finger. Mix the contents by inverting the tube several times.
       

  3. Set the spectrophotometer to 540 nm

  4. Set the spectrophotometer to Absorbance (A)

  5. Open chamber cover

  6. Insert the calibration cuvette into the holder

  7. Close chamber cover

  8. Read Absorbance and record it on your Data Collection Sheet.

  9. Save the calibration cuvette and its contents.

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Lab Activity #2 - The Effect of Enzyme Concentration on the Rate of Reaction

Materials:

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Spectrophotometer

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4 Cuvettes

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Catechol in dropper bottle

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Enzyme (catecholase) mixture with cover (Potato juice / distilled water)

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Dropper (for enzyme mixture)

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Distilled water (bottle)

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Marking Pen

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Plastic ruler

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Parafilm squares

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Spreadsheet

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Data Collection Sheet

Procedures:

  1. Number four cuvettes (1, 2, 3, and 4). Write the numbers at the top of the cuvette. Do not write them very large or they may interfere with the light beam of the spectrophotometer.

  2. Using the small plastic ruler, measure up 2 cm from the bottom of the cuvette and place a mark with the marking pen. Do this for all 4 cuvettes.

  3. Fill each of the four cuvettes to the 2 cm mark with distilled water.
     

  4. Preparation of cuvette contents: NOTE: Aerate the tubes every minute over the 5 minutes by covering the cuvettes with parafilm and shaking them.

    1. Add 5 drops of enzyme (potato juice) to cuvette 2, 15 drops to cuvette 3 and 45 drops to cuvette 4.

    2. Add 10 drops of catechol to each of the 4 tubes

    3. Let the cuvettes set for 5 minutes.
       

  5. At the end of 5 minutes place cuvette 1 in the spectrophotometer and measure its absorbance with the previously calibrated spectrophotometer.

  6. Repeat Step 5 with cuvettes 2, 3, and 4.

  7. Record the results on your Data Collection Sheet and Spreadsheet.

  8. Empty the cuvette's contents into the waste container provided.

  9. Rinse out the cuvettes and dry them. They can be used in subsequent lab activities for this lab.

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Lab Activity #3 - The Effect of Substrate Concentration on the Rate of Reaction

Materials:

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Spectrophotometer

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7 Cuvettes

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Catechol in dropper bottle

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Enzyme (catecholase) mixture with cover (Potato juice / distilled water)

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Dropper (for enzyme mixture)

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Distilled water (bottle)

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Marking Pen

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Plastic ruler

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Parafilm squares

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Spreadsheet

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Data Collection Sheet

Procedures:

  1. Number seven cuvettes (1, 2, 3, 4, 5, 6, and 7). They may be already numbered 1 - 4 from Lab Activity #2, or you may have gotten some that were already numbered (5, 6, or 7)

  2. Using the small plastic ruler, measure up 2 cm from the bottom of the cuvette and place a mark with the marking pen. Do this for all 7 cuvettes. They may already be marked at the 2 cm level.

  3. Fill each cuvette to the 2 cm mark with distilled water.
     

  4. Preparation of cuvette contents:  NOTE: Aerate the cuvettes every minute over the 10 minute incubation time by covering the cuvettes with parafilm and shaking them.

    1. Add 1 drop of catechol to cuvette 1, 3 drops to cuvette 2, 5 drops to cuvette 3, 10 drops to cuvette 4, 20 drops to cuvette 5, 40 drops to cuvette 6 and 60 drops to cuvette 7. Note:  cuvettes 6 and 7 may not hold all of this material. If you notice that this could occur, then dump the contents into a large test tube for the incubation time (10 minutes) an then pour the contents into a cuvette filling it approximately 3/4 full.

    2. Add 10 drops of enzyme to each of the 7 cuvettes

    3. Let the cuvettes set for 10 minutes.
       

  5. At the end of 10 minutes place cuvette 1 in the spectrophotometer and measure its absorbance with the previously calibrated spectrophotometer.

  6. Repeat Step 5 with cuvettes 2, 3, 4, 5, 6, and 7.

  7. Record the results on your Data Collection Sheet and Spreadsheet.

  8. Empty the cuvette's contents into the waste container provided.

  9. Rinse out the cuvettes and dry them. They can be used in subsequent lab activities for this lab.

 

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Lab Activity #4 - The Effect of External Factors on Enzyme Activity

Materials:

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Spectrophotometer

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6 Cuvettes

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Catechol in dropper bottle

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Enzyme (catecholase)  mixture with cover (Potato juice / distilled water)

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Boiled Enzyme mixture (from instructor)

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Phenylthiourea in dropper bottle

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Ascorbic acid in dropper bottle

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Dropper (for enzyme mixture)

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Distilled water (bottle)

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Marking Pen

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Plastic ruler

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Parafilm squares

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Spreadsheet

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Data Collection Sheet

Procedures:

  1. Number six cuvettes (1, 2, 3, 4, 5, and 6). They may be already numbered from Lab Activity #2 and 3.

  2. Using the small plastic ruler, measure up 2 cm from the bottom of the cuvette and place a mark with the marking pen. Do this for all 6 cuvettes. They may already be marked at the 2 cm level.

  3. Fill each tube to the 2 cm mark with distilled water.
     

  4. Preparation of cuvette contents:  NOTE: Aerate the tubes every minute over the 5 minutes by covering the cuvettes with parafilm and shaking them.

    Add all materials, except catechol, to the 6 cuvettes first then add the catechol to the 6 cuvettes at the same time

    1. Add 10 drops of enzyme to cuvette 2, 10 drops of enzyme to cuvette 3, 10 drops of boiled enzyme to cuvette 4, 10 drops of enzyme and 10 drops of phenylthiourea to cuvette 5, 10 drops of enzyme to cuvette 6.

    2. Add 10 drops of catechol to cuvettes 1, 3, 4, 5, and 6. (Do NOT add catechol to cuvette 2). Do this rapidly

    3. Let the cuvettes set for 5 minutes.
       

  5. At the end of 5 minutes measure the absorbance of cuvette 6 with the previously calibrated spectrophotometer.

  6. Remove cuvette 6 from the spectrophotometer and add 10 drops of ascorbic acid. Let the cuvette stand for another 5 minutes, and then determine its absorbance as part of Step 11.

  7. At the end of 5 minutes place cuvette 1 in the spectrophotometer and measure its absorbance with the previously calibrated spectrophotometer.

  8. Repeat step 7 with cuvettes 2, 3, 4, 5, and 6.

  9. Record the results on your Data Collection Sheet and Spreadsheet.

  10. Empty the cuvette contents into the waste container provided.

  11. Rinse out the cuvettes and dry them. They can be used in subsequent lab activities for this lab.

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Lab Activity #5 - The Effect of pH on Enzyme Activity

Materials:

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Spectrophotometer

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6 Cuvettes

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pH Buffers 3, 5, 7, 9, and 11

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Catechol in dropper bottle

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Enzyme (catecholase) mixture with cover (Potato juice / distilled water)

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Dropper (for enzyme mixture)

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Distilled water (bottle)

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Marking Pen

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Plastic ruler

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Parafilm squares

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Spreadsheet

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Data Collection Sheet

Procedures:

  1. Number six cuvettes (1, 2, 3, 4, 5, and 6). They may be already numbered from Lab Activity #2 and 3.

  2. Using the small plastic ruler, measure up 2 cm from the bottom of the cuvette and place a mark with the marking pen. Do this for all 6 cuvettes. They may already be marked at the 2 cm level.
     

  3. Preparation of cuvette contents:  NOTE: Aerate the cuvettes every minute over the 5 minutes by covering the cuvettes with parafilm and shaking them.

    1. Fill cuvette 1 with up to the 2 cm mark with pH 3 buffer solution and 10 drops of enzyme. 

    2. Fill cuvette 2 with up to the 2 cm mark with pH 5 buffer solution and 10 drops of enzyme. 

    3. Fill cuvette 3 with up to the 2 cm mark with pH 7 buffer solution and 10 drops of enzyme. 

    4. Fill cuvette 4 with up to the 2 cm mark with pH 9 buffer solution and 10 drops of enzyme. 

    5. Fill cuvette 5 with up to the 2 cm mark with pH 11 buffer solution and 10 drops of enzyme. 

    6. Fill cuvette 6 with up to the 2 cm mark with distilled water and 10 drops of enzyme. 

    7. Add 10 drops of catechol to cuvettes (1, 2, 3, 4, 5 and 6)

    8. Let the cuvettes stand for 5 minutes

     

  4. At the end of 5 minutes place cuvette 1 in the spectrophotometer and measure its absorbance with the previously calibrated spectrophotometer.

  5. Repeat Step 4 with cuvettes 2, 3, 4, 5, and 6.

  6. Record the results on your Data Collection Sheet and Spreadsheet.

  7. Empty the cuvette contents into the waste container provided.

  8. Rinse out the cuvettes and dry them. They can be used in subsequent lab activities for this lab.

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Post-Lab Activity and Data Analysis

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Results & Analysis:

  1. Return all materials to their proper location. Place your waste container in a location indicated by the instructor.

  2. Clean up your lab area.

  3. Graph the data. You need to make 3 separate graphs. The titles for each graph are listed below. Make sure you do line graphs, and that their is a proper legend (place it at the bottom of the graph), and a properly labeled X and Y axes.

    1. Graph #1 - The Effect of Enzyme Concentration on the Rate of Reaction

    2. Graph #2 - The Effect of Substrate Concentration on the Rate of Reaction

    3. Graph #3 - The Effect of pH on Enzyme Activity

  4. Use your graphs to help answer the Post-Lab Questions 

  5. Items to turn in before or at the beginning of the next lab period.

    1. Your three separate graphs

    2. Your completed Spreadsheet

    3. Your completed Data Collection Sheet

    4. Your completed Post-Lab Questions

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Post-Lab Questions (html version)    Post-Lab Questions (Word version)

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