Teacher Notes

Enzyme Optimization—pH and Temperature

Guided-Inquiry Kit

Materials Included In Kit

Albumin, 4 g
Amylase 4 g
Biuret test solutions, 100 mL, 2
Buffer envelope, pH 2
Buffer envelope, pH 7
Buffer envelope, pH 9
Buffer envelope, pH 11
Iodine–potassium iodide solution, 40 mL
Lipase, 4 g
Litmus–buttermilk, 2 g
Pepsin, 4 g
Starch, 1 g
Trypsin, 4 g
Pipets, graduated, 100

Additional Materials Required

Water, distilled or deionized†
Balance, 0.01-g precision†
Beakers, 50-mL, 4*
Erlenmeyer flasks with stoppers or caps, 250-mL, 5†
Erlenmeyer flasks with stoppers or caps, 500-mL, 4†
Graduated cylinder, 50-mL*
Marker*
Stirring rods*
Stopwatch or clock with second hand*
Test tubes, 13 x 100 mm*
Test tube rack*
Weighing paper or wax paper squares†
*for each lab group
†for Prelab Preparation

Temperature Determination
Water, tap
Beakers
Hot plate
Ice
Thermometers
Test tube clamp

pH Determination
Water, tap
Balance, 0.01-g precision (shared)
Erlenmeyer flasks with stopper or cap, 50-mL, 4
Thermometer
Water bath, 40 °C
Weighing paper or wax paper squares

Prelab Preparation

1% albumin (protein) solution: Weigh 2.0 g of albumin and place in a labeled Erlenmeyer flask. Add 40 mL of DI water to the albumin. Allow the albumin to soak for several hours, then dilute up to 200 mL with DI water. Cap the Erlenmeyer flask and shake gently to mix the contents. Refrigerate if prepared in advance. May be prepared several days in advance.

0.5% starch solution: Weigh 1.0 g of starch and place in a labeled Erlenmeyer flask. Add a small amount of very hot DI water to the starch, and mix with a glass stirring rod. Continue to add small amounts of boiling water to the flask, mixing until 200 mL of very hot water has been added. Allow the solution to cool slowly to room temperature and refrigerate. May be prepared several days in advance.

pH buffer envelopes: Add the contents of each envelope into a separate labeled 500-mL Erlenmeyer flask. Add 500 mL of distilled or deionized water to the flask. Stopper the Erlenmeyer flasks and gently shake to mix the contents. May be prepared several days in advance.

1% litmus-milk solution: Transfer the 2 g of litmus-milk powder into a labeled Erlenmeyer flask. Add 200 mL DI water to the solid. Cap the Erlenmeyer flask and shake gently to mix the contents. Refrigerate if prepared in advance. May be prepared two days in advance.

Enzymes solutions: Divide the 4 g of pepsin, trypsin, amylase and lipase into 2 g samples for each student group working with the enzyme. For the pH determination, students will add 0.25 g of enzyme to 50 mL of each buffer solution to make 0.5% solutions. For the temperature determination, students will add 0.25 g of enzyme to 50 mL deionized water to make a 0.5% solution. Amylase and trypsin must be 1% solutions. Note: Enzyme solutions must be prepared the day of the lab.

Prepare a large volume of very hot water for students to use to maintain the temperature of the water baths.

Safety Precautions

Biuret test solution contains copper(II) sulfate and sodium hydroxide and is a corrosive liquid. Biuret test solution is moderately toxic by ingestion and is dangerous to skin and eyes. Iodine–potassium iodide solution is irritating to eyes and skin. The pH 2 buffer is strongly acidic. It is severely toxic by ingestion and is corrosive to skin and eyes. Buffers in the high pH range are strongly alkaline. They are corrosive to skin and eyes. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles and chemical-resistant gloves and apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All of the waste solutions and excess reagents with a neutral pH may be disposed of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #26b. Excess biuret solution and high pH buffers may be neutralized with acid and then disposed of according to Flinn Suggested Disposal Method #10. Excess low pH buffer solution may be disposed of by neutralizing with base and then disposing of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #24b. Excess iodine solution may be reduced with sodium thiosulfate solution and then disposed of according to Flinn Suggested Disposal Method #12a.

Lab Hints

  • Enough materials are provided in this kit for 24 students working in groups of four or for six groups of students. The laboratory work for this experiment can reasonably be completed in one 50-minute lab period with proper preparation. The most important element for success in an inquiry-based activity is student preparation. Sufficient class time should be allotted before lab to think through the measurements that must be made and how the experiment should be conducted. The Prelab Questions contains leading questions to stimulate class discussion.
  • To ensure a safe lab environment, it is essential that the teacher provide a mechanism for checking the students’ proposed procedures and their understanding of the necessary safety precautions, as recommended in the Procedure.
  • Some teachers tell us that for an inquiry-based lab, they require the students to submit their proposed procedures the day before the lab. The teachers then check the procedures and return the proofed copies to the students before lab. This ensures that students are prepared and that teachers have time to supervise the actual lab activity, not proof the procedures, during lab time.
  • The amylase–starch–iodine and lipase–buttermilk–litmus procedures work very well for optimizing because the indicators are both spectator ions that do not interfere with the enzyme reaction. The indicator’s changing color allows for students to measure the difference in the amount of time required for the reaction at each temperature. The pepsin–albumin–biuret test and trypsin-albumin-biuret test reactions must proceed for a certain amount of time before the biuret is added because the biuret test solution stops the reaction. This limitation makes quantifying the result difficult unless a spectrophotometer or colorimeter is used.
  • Pepsin and trypsin are both very sensitive to pH making them ideal for the pH determination.
  • Pepsin and trypsin digestion of albumin requires a 50 °C water bath for 5 minutes.
  • Biuret test for proteins and peptides—add 1 mL of biuret test solution for every 2 mL of sample. Biuret test solution is bluish-purple in the presence of polypeptides and lavender-pink in the presence of dipeptides and amino acids. Biuret test solution must only be added to solutions that are less than 60 °C. Any hot solutions should be cooled before the biuret test solution is added.
  • Litmus is an acid–base indicator. Litmus appears blue in the basic buttermilk solution. Litmus appears pink in the acidic fatty acid solution that occurs due to digestion of the milk fats by lipase. Students will be unable to determine the optimum pH for the lipase solution because the pH buffer will interfere with any change in the color of the litmus that is due to the digestion of milk into fatty acids.
  • Iodine indicates the presence of starch in a solution. One or two drops of iodine are enough to form a dark-blue complex with starch. Amylase hydrolyzes starch into sugars, leading to a negative starch test—a colorless or pale yellow solution.
  • A buffer is a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. Buffers work by reacting with any added acid or base to control the pH. Students may check the pH of the buffers if you wish for them to practice that lab technique.

Teacher Tips

  • The Digestive Enzymes—Student Laboratory Kit (Flinn Catalog No. FB1862) may be used as a teacher demonstration or introductory lab to ensure that the students understand how to perform the enzyme tests.
  • Extend the lab by having students test the enzymes on various food items. All food-grade items that have been brought into the lab are considered laboratory chemicals and are for lab use only. Do not taste or ingest any materials in the laboratory and do not remove any remaining food items after they have been used in the lab.
  • Make the lab an open-ended inquiry lab by supplying the students with more pH buffer choices such as one each from pH 2 to pH 11 (Flinn Catalog No. B0123) or supply 0.1 M hydrochloric acid (Flinn Catalog No. H0042) and 0.1 M sodium hydroxide (Flinn Catalog No. S0149) to create a wide range of pH solutions by serial dilution. Please review all SDSs and safety guidelines for all chemicals with students before including in the lab.
  • Extend the number of enzyme choices by including invertase (Flinn Catalog No. I0041) and sucrose (Flinn Catalog No. S0134) as a substrate. Benedict’s Quantitative Solution (Flinn Catalog No. B0195) may be used to test for the product—invertase catalyzes the hydrolysis of sucrose, a non-reducing sugar, to glucose and fructose, which are reducing sugars.

Answers to Prelab Questions

  1. Consult biology or physiology reference materials, such as a textbook or the Internet, to complete the following table.
    {10821_PreLabAnswers_Table_3}
  2. Plot the data for the rate of reaction versus pH and use the graph to estimate the optimum pH for the digestion of maltose by maltase at 40 °C.
    {10821_PreLabAnswers_Figure_3}
  3. The stomach has a pH of 1.5–2.5. Discuss in detail what would happen if maltase were secreted into the stomach rather than into the duodenum. Explain your prediction in terms of the lock and key theory of enzyme action.

    If maltase was accidentally secreted into the acid environment of the stomach the acid would cause alterations in the shape of the enzyme. The high concentrations of H+ affect the charges of certain areas of the enzyme, altering the enzymes ionic bonds and causing structural changes.

Sample Data

Optimal Temperature for Amylase

Control Samples

  • 1 mL of 0.5% starch solution plus 2 drops of iodine–potassium iodide solution were equilibrated in the water bath.
  • In a separate test tube, 2 mL of deionized water was equilibrated in the water bath.
  • After 1 minute, the contents of the two test tubes were combined and the stopwatch was started.
Test Samples
  • 1 mL of 0.5% starch solution plus 2 drops of iodine–potassium iodide solution were equilibrated in the water bath.
  • In a separate test tube, 2 mL of 1% amylase solution in deionized water was equilibrated in the water bath.
  • After 1 minute, the contents of the two test tubes were combined and the stopwatch was started. The time needed for the sample to turn colorless was measured.
    {10821_Data_Table_4}

Optimal Temperature for Lipase

Control Samples

  • 1 mL of 1% litmus–buttermilk solution was equilibrated in the water bath.
  • In a separate test tube, 1 mL of deionized water was equilibrated in the water bath.
  • After 1 minute, the contents of the two test tubes were combined and the stopwatch was started.

Test samples

  • 1 mL of 1% litmus–buttermilk solution was equilibrated in the water bath.
  • In a separate test tube, 1 mL of 0.5% lipase solution was equilibrated in the water bath.
  • After 1 minute, the contents of the two test tubes were combined and the stopwatch was started. The time needed for the litmus solution to turn hot pink was measured.
    {10821_Data_Table_5}

Optimal pH for Amylase

Control samples

  • 1 mL of 1% starch solution plus 2 drops of iodine–potassium iodide solution were equilibrated in a 54 °C water bath.
  • In a separate test tube, 1 mL of buffer was equilibrated in a 54 °C water bath.
  • After 1 minute, the contents of the two test tubes were combined and the stopwatch was started.

Test Samples

  • 1 mL of 1% starch solution plus 2 drops of iodine–potassium iodide solution were equilibrated in a 54 °C water bath.
  • In a separate test tube, 1 mL of 1% amylase solution in buffer was equilibrated in a 54 °C water bath.
  • After 1 minute, the contents of the two test tubes were combined and the stopwatch was started. The time needed for the solution to turn colorless was measured.
    {10821_Data_Table_6}

Optimal pH for Pepsin

Control Samples

  • 1 mL of 1% albumin (protein) solution was equilibrated in a 54 °C water bath.
  • In a separate test tube, 1 mL of buffer was equilibrated in a 54 °C water bath.
  • After 1 minute, the contents of the two test tubes were combined and the stopwatch was started.
  • After 5 minutes, 1 mL of biuret test solution was added to the control samples and the color was compared to that of the enzyme samples.

Test Samples

  • 1 mL of 1% albumin (protein) solution was equilibrated in a 54 °C water bath.
  • In a separate test tube, 1 mL of 0.5% pepsin solution in buffer was equilibrated in a 54 °C water bath.
  • After 1 minute, the contents of the two test tubes were combined and the stopwatch was started.
  • After 5 minutes, 1 mL of biuret test solution was added to the enzyme samples and the color was compared to that of the control samples. The color change from the blue Biuret test solution was noted.
    {10821_Data_Table_7}

Optimal pH for Trypsin

Control Samples

  • 1 mL of 1% albumin (protein) solution was equilibrated in a 54 °C water bath.
  • In a separate test tube, 1 mL of buffer was equilibrated in a 54 °C water bath.
  • After 1 minute, the contents of the two test tubes were combined and the stopwatch was started.
  • After 5 minutes, 1 mL of biuret test solution was added to the control samples and the color was compared to that of the enzyme samples.

Test Samples

  • 1 mL of 1% albumin (protein) solution was equilibrated in a 54 °C water bath.
  • In a separate test tube, 1 mL of 1% trypsin solution in buffer was equilibrated in a 54 °C water bath.
  • After 1 minute, the contents of the two test tubes were combined and the stopwatch was started.
  • After 5 minutes, 1 mL of biuret test solution was added to the enzyme samples and the color was compared to that of the control samples.
    {10821_Data_Table_8}

Answers to Questions

  1. In the following data table, summarize the class data obtained for the pH and temperature optimization of different enzymes.
    {10821_Answers_Table_9}
  2. Compare the optimization results determined by testing with those discovered in the Pre-Lab Question research. Discuss any possible discrepancies between the predicted and experimental results.

    Many enzymes are obtained from microbial sources—such as amylase from Aspergillus oryzae—which function at higher temperatures. Although the typical internal body temperature is 37 °C, not all proteins denature above this temperature. A few critical proteins denature above 42 °C. These enzymes denature during a high fever and cause significant harm or death.

  3. Many supplement manufacturers claim that ingesting powdered enzymes will increase the ability of the body to digest food. Using the optimization results obtained by the class, defend or criticize the marketing position of the supplement manufactures.

    Enzymes ingested as supplements are proteins and therefore are probably denatured in the pepsin–acid environment of the stomach before they can digest their intended substrate. The exception may be pepsin, however, if pepsin is ingested instead of its inactive precursor pepsinogen, it may digest the lining of the mouth and esophagus causing significant damage. In defense of the supplements, students may discuss the fact that enzymes are often ingested inside a capsule so the contents are delivered to the small intestine without exposure to the upper digestive tract.

References

http://www.bbc.co.uk/education/asguru/biology/02biologicalmolecules/01proteins/11enzymes/index.shtml (accessed March 2007).

http://www.chemsoc.org/networks/LearnNet/cfb/enzymes.htm (accessed March 2007).

Campbell, N. A., Reece, J. B., Biology, 7th edition; Pearson Education, Inc: San Francisco, CA; 2005.

Hole, J. W., Human Anatomy and Physiology, 5th Edition; Wm. C. Brown Publishers: Dubuque, IA; 1990

Recommended Products

Item No. Description
FB1867 Enzyme Optimization—pH and Temperature Guided-Inquiry Kit

Student Pages

Enzyme Optimization—pH and Temperature

Introduction

Organisms that do not make their own food must break down large macromolecules to generate “the building blocks of life.” How does the digestive system of a complex animal like a human break down plant and animal tissues into nucleic acids, amino acids, fatty acids and glucose?

Concepts

  • Enzymes
  • Lock and key theory
  • Induced fit theory

Background

A catalyst is any substance that speeds up the rate of a chemical reaction by providing an alternative reaction pathway which requires a lower activation energy. Catalysts are not permanently altered during the reaction and are therefore reused repeatedly. Enzymes are selective biological catalysts. Most enzymes are composed of a globular, three-dimensional protein and a nonprotein cofactor. Like all proteins, enzymes have unique, characteristic shapes that are produced by attractive forces between the amino acid side chains, which create the secondary and tertiary structures of the protein. One section of the protein structure contains the active site, which has the right shape and functional groups to bind to the substrate. The overall shape of an enzyme may be distorted by a change of pH or temperature that affects the secondary and tertiary structures. The special shape of an enzyme is often compared to a lock into which the substrate will fit (bind) like a key. The lock and key theory was proposed in 1894 by Emil Fischer (see Figure 1). Each type of substrate (key) has a different shape that needs a different enzyme (lock). This exclusive nature of enzyme/substrate binding means that humans contain thousands of different enzymes to catalyze all the different biochemical reactions that must occur.

{10821_Background_Figure_1}
After many years of careful scientific study, a change was proposed to the lock and key model by Daniel Koshland of the University of California–Berkeley. In Dr. Koshland’s induced fit theory, the active site of the enzyme is not a perfect fit for the substrate. The enzyme’s active site is reshaped (induced to fit) by the substrate (see Figure 1). The induced fit model explains why small molecules with the same shape as part of the substrate may not react with the enzyme but other chemicals, which fit into a separate blocking active site on the enzyme, may inhibit the enzyme from binding to the substrate.

Experiment Overview

The purpose of this inquiry-based experiment is to design and carry out a procedure to determine the optimum pH or temperature for one of several digestive enzymes.

Materials

Albumin solution, 1%, 50 mL**
Amylase, 2 g used to make a 1% solution*
Biuret test solution, 50 mL†
Iodine–potassium iodide solution, 10 mL†
Lipase, 1 g used to make a 0.5% solution*
Litmus–buttermilk solution, 1%, 50 mL**
Pepsin, 1 g used to make a 0.5% solution*
Starch solution, 0.5%, 50 mL**
Trypsin, 2 g used to make a 1% solution*
*Enzyme
**Substrate
†Test solution
 
Temperature Determination
Water, tap‡
Beakers, 100-mL, several
Beakers, 250-mL, several‡
Graduated cylinder, 100-mL
Hot plate or Bunsen burner‡
Ice‡
Marker
Pipets, graduated, several
Stirring rods
Stopwatch or clock with second hand
Test tube rack
Thermometers‡
Test tubes, 13 x 100 mm
Test tube clamp‡
‡for water baths

pH Determination
pH 2 buffer solution, 100 mL
pH 7 buffer solution, 100 mL
pH 9 buffer solution, 100 mL
pH 11 buffer solution, 100 mL
Water, tap
Balance, 0.01-g precision (shared)
Beakers, 100-mL, 4
Graduated cylinder, 100-mL
Marker
Pipets, graduated
Stirring rods
Stopwatch or clock with second hand
Test tubes, 13 x 100 mm
Test tube rack
Thermometer
Water bath, 40°C

Prelab Questions

  1. Consult biology or physiology reference materials, such as a textbook or the Internet, to complete the following table.
    {10821_PreLab_Table_1}

    Questions 2 and 3 relate to the following information—

    Maltase is secreted by the villi of the small intestine to hydrolyze maltose, a disaccharide sugar, into two molecules of body-usable monosaccharide glucose (see Figure 2).

    {10821_PreLab_Figure_2}

    The optimal pH for maltase in the body (in vivo) is 6.1–6.8. An experiment was conducted to determine the optimal pH for maltase in a test tube (in vitro) at 40 °C. The independent variable was pH. The dependent variable was the amount of glucose produced. The enzyme and substrate concentrations were held constant. The reaction was allowed to proceed for 5 minutes before the reaction was stopped by plunging the test tubes into a 0 °C water bath. pH was tested from 5 to 8. Quantitative testing yielded the following rates of reaction (in mg/of glucose produced per minute).

    {10821_PreLab_Table_2}
  2. Plot the data for the rate of reaction versus pH and use the graph to estimate the optimum pH for the digestion of maltose by maltase at 40 °C.
  3. The stomach has a pH of 1.5–2.5. Discuss in detail what would happen if maltase were secreted into the stomach rather than into the duodenum. Explain your prediction in terms of the lock and key theory of enzyme action. 
  4. From the list of Materials, determine which enzyme you wish to test. Three of the enzymes—pepsin, trypsin, and amylase—will be used to test the optimal pH for the enzyme. Two of the enzymes—amylase and lipase—will be used to test the optimal temperature for the enzyme. Form a laboratory group of four based on the enzyme and parameter to be tested. Design an experiment to determine the optimal temperature or pH for the enzyme. Consider the following questions:
    1. Which substrate will react with the enzyme?
    2. How will you be able to determine whether the enzyme produces any product?
    3. What measurements must be made to determine the optimal pH or optimal temperature for the enzyme?
    4. The independent variable in an experiment is the variable that is changed by the experimenter, while the dependent variable responds to (depends on) changes in the independent variable. Choose the dependent and independent variables for your proposed experiment.
    5. What other variables will affect the results in this experiment? How can these variables be controlled?
    6. Read the Materials section and the recommended Safety Precautions. Write a step-by-step procedure for the experiment, including the specific safety precautions that must be followed.

Safety Precautions

Biuret test solution contains copper(II) sulfate and sodium hydroxide and is a corrosive liquid. Biuret test solution is moderately toxic by ingestion and is dangerous to skin and eyes. Iodine-potassium iodide solution is irritating to eyes and skin. The pH 2 buffer is strongly acidic. It is severely toxic by ingestion and is corrosive to skin and eyes. The pH 11 buffer is strongly alkaline (basic). It is corrosive to skin and eyes. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles and chemical-resistant gloves and apron. Wash hands thoroughly with soap and water before leaving the laboratory. Follow all laboratory safety guidelines.

Procedure

Solution Preparation and Calculations

Mass (weight)–volume percent solutions will be used in this laboratory procedure. A 0.5% solution is prepared by dissolving 0.5 g of solid in 100 mL of solvent. For example, 0.5 g of pepsin in 100 mL of pH 5 buffer is a 0.5 w/v% pepsin solution. If 100 mL of solution will not be needed, multiply the volume needed by the desired percent solution to determine the mass of solid. If 50 mL of a 0.5% pepsin solution is needed—50 mL × 0.5% = 0.25 g of pepsin should be added to 50 mL of pH 5 buffer.

The rate of hydrolysis of starch can be determined visually using iodine by measuring the time for the blue color to disappear. Biuret test solution gives a pink or purple color change with peptides and proteins, respectively.

Procedure

  1. Verify the procedure (see the Prelab Questions) with your instructor and review all safety precautions.
  2. Carry out the procedure and record all data in a suitable data table such as time to complete the reaction of the substrate, concentration of the substrate, concentration of the enzyme, and pH or temperature of the reaction.
  3. Graph the data to determine the mathematical relationship, if any, between the rate of the reaction and temperature or pH.
  4. Write a paragraph describing how temperature or pH affects the reaction of an enzyme. Include in this paragraph a discussion of the possible errors involved in the experiment and their effect on the results.
  5. Answer the Post-Lab Questions.

Student Worksheet PDF

10821_Student1.pdf

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