Materials Included In Kit

Additional Materials Required

Prelab Preparation

• 0.5% Amylase Solution: Weigh 1.0 g of amylase. Add 200 mL of distilled or deionized water to the amylase. Mix well. Must be prepared the day of the laboratory.
• 1% Starch Solution: Weigh 2.0 g of starch. Add a small amount of very hot distilled or deionized 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.

Safety Precautions

Iodine is a permanent stain and will stain skin and clothing. Hydrochloric acid solution and sodium hydroxide solution are toxic by ingestion or inhalation and corrosive to skin or eyes. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please consult Safety Data Sheets for additional safety and handling procedures.

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 materials used in the reaction plates can be disposed of according to Flinn Suggested Disposal Method #26b.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. Parts I–IV are likely to be completed in one class period with careful organization. More than likely, however, a second day will be required and Part V can be started at that time. Additional day(s) can be spent on Part V depending upon your goals and curriculum schedule. Note: Be sure to test the activity of your amylase solution prior to a second day of laboratory work.

• Many factors can affect the reaction time in Part I of the laboratory. The freshness of the amylase solution, the homogeneity of the starch solution, and the evenness of drop size can be very significant. The relative trend for enzyme concentration is more important than the actual times. The first part of the lab should demonstrate that a massive increase in the concentration of amylase does not have a correspondingly great effect on the rate of digestion. Once the concentration is doubled from 1 drop/10 to 2 drops/10, the time difference is hard to detect. A five times concentration of amylase does not make the reaction go five times faster.
• The pH and temperature experiments are crude at best and only demonstrate the extremes. They are purposefully preliminary in nature so that students might “refine” them in Part V of the laboratory. The problem students might grapple with if they choose temperature is how to keep the cold water from warming and the warm water from cooling.

Sample Data

Part I. The Tests

Part II. Enzyme Concentration

Part III. pH

Part IV. Temperature

Answers to Questions

Part I. The Tests

1. Describe a positive test for sugar with Benedict’s solution.

   After heating, the initial blue solution turns a yellow/orange color.

2. Describe a positive test for starch using iodine solution.

   Starch turns dark purple in presence of iodine.

3. Which test tubes served as controls in your tests? Explain.

Benedicts:

Tube 1 To show heating Benedicts doesn’t turn it yellow/orange.
Tube 2 To show heating starch doesn’t turn it to sugar.

Iodine:

Tube 5 To show sugar doesn’t turn iodine purple.
Tube 6 To show starch does turn solution purple.

4. Use your results to defend the statement—Amylase digests starch into sugar.

Positive results in Tubes 4 and 7 both indicate that amylase digests starch.

Part II. Enzyme Concentration

Graph your results and write a statement about the relationship your graph depicts.

Increased concentration of enzyme does not increase rate of reaction once a critical concentration is reached.

Part III. pH

What can be stated about pH and amylase activity?

Extreme pHs seem to inhibit amylase ability to digest starch.

Part IV. Temperature

Does temperature seem to influence amylase activity?

Extreme temperature conditions seem to inhibit amylase ability to digest starch.

Introduction

Living systems are a sea of chemical reactions. At moderate temperatures (like the temperature of most living systems) chemical reactions occur rather slowly. How can chemical reactions be made to go fast enough to keep something alive? The answer lies in understanding enzymes, the catalysts of life.

Concepts

• Catalysts

• Enzymes

Background

A living organism is teeming with many interrelated chemical reactions, many occurring simultaneously. Like all chemical reactions, those in living organisms are affected by changes in conditions such as temperature or pH. The same things that might speed up a chemical reaction in a test tube (such as intense heat or cold) might be deadly to a living organism! How can chemical reactions occur in a living organism without killing it? The answer lies in an understanding of catalysts. Catalysts are chemicals that affect the rate of a chemical reaction without itself changing during the reaction. Biological catalysts are called enzymes. Enzymes speed up most of the chemical reactions in living things. In this laboratory you can discover what influences the activity of the digestive enzyme, amylase.

Molecules are “digested” in living cells and in complex processes energy is released for use by the cell. Starch molecules are a common food source but must be digested into a more soluble form (sugar) before it can be used effectively by cells. In cells, one enzyme that catalyzes the breakdown of starch into sugar is amylase. A generalized formula for the digestion process is:

Materials

Safety Precautions

Iodine is a permanent stain and will stain skin and clothing. Hydrochloric acid solution and sodium hydroxide solution are toxic by ingestion or inhalation and corrosive to skin and eyes. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron.

Procedure

Part I. The Tests

1. Add 150 mL of water to a 250-mL beaker. Heat to boiling.
2. Label seven test tubes 1–7 and use a graduated cylinder to measure out solutions in each tube as shown in Table 1. Let all test tubes set for 2–3 minutes.

   (Note: Be sure to rinse the graduated cylinder in between each solution.)

   {10259_Procedure_Table_1_Solutions for Part I}
3. Add 1 mL of Benedict’s solution to test tubes 1–4 and then place the test tubes in a boiling water bath for 2–3 minutes. Note all color changes on the Enzyme Studies Worksheet.
4. Add one drop of iodine to test tubes 5, 6 and 7. Note any color change on the Enzyme Studies Worksheet.
5. Answer the questions on Part I of the Enzyme Studies Worksheet.

1. Locate a stopwatch or other timing device with a second hand.
2. Use clean Beral-type pipets for each solution and add nine drops of distilled water and one drop of amylase solution to 12 successive wells on a reaction plate as shown in Figure 1. Use separate pipets for the various solutions and hold the pipets straight up when dropping to create uniform drops.
   {10259_Procedure_Figure_1_Reaction plate solutions}
3. Use a clean pipet to add two drops of starch solution to each of the twelve wells. Note the color of the solution.
4. Quickly add one drop of iodine to well 1 and start timing. Note any color change on the Enzyme Studies Worksheet.
5. Every 15 seconds add 1 drop of iodine to the next successive well. Use a (+) to indicate a positive starch test and a (–) to indicate a negative starch test. Continue timing and testing with iodine until you are convinced the chemical reaction is complete. (Note: You may not have to test all 12 wells.)
6. Thoroughly wash and dry your reaction plate.
7. Repeat steps 2–6. This time, however, use 2 drops of amylase and 8 drops of distilled water at step 2. Record your results on the Enzyme Studies Worksheet.
8. Repeat steps 2–6 using 3 drops amylase and 7 drops distilled water, 4 drops amylase and 6 drops distilled water, etc. until you feel the rate of the reaction is no longer affected by adding a higher concentration of amylase.
9. Graph your results on the Enzyme Studies Worksheet. Then write a statement about the relationship between the concentration of amylase and its ability to digest starch.

1. Using a clean reaction plate, add 2 drops of starch solution to each of three successive wells.
2. To well 1 add 1 drop of 0.1 M hydrochloric acid solution.

   To well 2 add 1 drop of distilled water.

   To well 3 add 1 drop of 0.1 M sodium hydroxide solution.

3. Add 8 drops of distilled water to each of the three wells.
4. Use small strips of pH paper to determine the pH of the solution in each well.
5. Add 2 drops of amylase solution to each well.
6. Wait three minutes and add a drop of iodine to all three wells.
7. Record the results on Part III of the Enzyme Studies Worksheet.
8. Answer the question for Part III on the worksheet.

1. Using a clean reaction plate, add 2 drops of starch solution to each of three successive wells.
2. To well 1 add 8 drops of boiling water.

   To well 2 add 8 drops of room temperature water.

   To well 3 add 8 drops of ice water.

3. Quickly add 2 drops of amylase to each of the three wells.
4. After three minutes test for the presence of starch by adding one drop of iodine to each well.
5. Record results on Part IV of the Enzyme Studies Worksheet.
6. Answer the question for Part IV on the worksheet.

1. What is the optimum temperature, pH or concentration of amylase for the digestion of starch? Form a research group as directed by your teacher. Brainstorm hypotheses about possible variables and discuss in detail how a controlled experiment might be used to test each hypothesis.
2. Choose the hypothesis that seems to be the most viable and interesting to your group. Design an experiment to test the hypothesis. Write up the experimental design in detail and discuss it with your teacher.
3. Conduct your experiment. Create appropriate data tables and record the results from your experimental work.
4. Write a complete laboratory report for your experiment. Be sure to include
   1. A statement of your hypothesis.
   2. A description of your experimental design. (Be sure to include the controls you used.)
   3. The results.
   4. An interpretation of the results.
   5. A confidence level relative to your original hypothesis.
   6. Suggestions for further experimentation.
   7. Conduct a class seminar and share all the experimental designs and results.
   8. Consult your instructor for appropriate disposal procedures.
5. Conduct a class seminar and share all the experimental designs and results.
6. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10259_Student1.pdf