Austin Peay State University Department of Chemistry CHEM 1021 BREAKING DOWN STARCH USING SALIVARY AMYLASE Caution: You will be using a Bunsen burner and glassware to create your own constant water bath. Appropriate caution should be exercised when dealing with the Bunsen burner, hot water, and glassware. Purpose: Many plants store their energy in the form of starch, a polysaccharide made from repeating units of the monosaccharide glucose. Our bodies break down starch into the individual glucose units, which are further metabolized into CO2 and water through the process of glycolysis—this is the process we commonly call digestion. The enzyme amylase is present in our saliva and begins the process of digestion when starch containing foods enter our mouths. In this lab you will measure the amount of amylase present in your saliva by monitoring the breakdown of starch. Introduction: You can probably name a variety of foods that are referred to as “starchy”. Such foods include potatoes, rice, wheat, and corn. In essence, the starch is a long biopolymer made from thousands of repeating glucose units. When these glucose units are attached in a straight line, the type of starch is called amylose. When the units are branched, the type of starch is called amylopectin. Natural starch contains both forms of the polymer (Figure 1). The long chains of glucose that make amylose form a tight helix. If we mix an iodine solution with the amylose, the iodine will lodge itself in the interior of the helix. This causes the solution to turn a dark blue color. If the enzyme amylase is added to the solution first, it will chemically remove one glucose monomer at a time from the ends. Addition of iodine to this solution does not give a blue color but will be colorless (Distinguish between clear and colorless; colorless means without color, whereas clear means “see through”. Kool‐aid is clear, but is not colorless. Water is clear and colorless. Non‐
flavored gelatin is colorless but not always clear). All enzymes speed up a reaction to a time scale useful for our bodies. Starch can be broken down over thousands of years without aid of other chemicals, or it can be broken down in less than an hour with the enzyme amylase (Figure 2). Not everyone’s amylase works at the same rate. This is called the “activity” of the enzyme. In this lab you will be determining how well your (or your lab partner’s) amylase works by how fast it breaks down a starch solution. You will report the activity in terms of the amylase Revision SP11 Page 1 of 7 Austin Peay State University Department of Chemistry CHEM 1021 BREAKING DOWN STARCH USING SALIVARY AMYLASE number, which is the number of milliliters of a 1% starch solution that can be digested in 30 minutes by 1 mL of saliva. Materials: 11 4‐in test tubes and rack Ring stand Iron ring Bunsen burner 10, 25, and 100‐mL graduated cylinder Procedure: 50, 150, and 400‐mL beakers wax pencil 110 oC thermometer 2% starch solution 0.01 M iodine solution 1. Prepare a 37 oC water bath by heating water over a Bunsen burner in a 400‐mL beaker. Use the thermometer to monitor the reaction. Too hot or too cold, and the enzymes won’t work. Go on to Step 2 while you are waiting for your water bath to stabilize. 2. Making 3 amylase solutions: Fill a clean test tube with about 10 mL of saliva. You will be making two dilutions of this solution so you will have a 1%, 10%, and 100% (undiluted) solution. In a 10‐
mL graduated cylinder, add 1 mL of saliva and 9 mL of distilled water for the 10% solution. Pour this into a clean 50‐mL beaker to mix. In a 100‐mL graduated cylinder, put in 1 mL of saliva and 99 mL of water for the 1% solution. Pour this into a clean 150‐mL beaker to mix. 3. Set up 10 reaction tubes (label with a wax pencil) of varying saliva concentrations in a test tube rack as follows: Tube # Saliva Distilled water Final saliva %
1 3 mL 100%
0 mL 100% 2 2 mL 100%
1 mL 66.7% 3 1 mL 100%
2 mL 33.3% 4 3 mL 10% 0 mL 10% 5 2 mL 10% 1 mL 6.7% 6 1 mL 10% 2 mL 3.3% 7 3 mL 1% 0 mL 1% 8 2 mL 1% 1 mL 0.67% 9 1 mL 1% 2 mL 0.33 % 10 0 mL 3 mL 0% 4. Start the reactions: Add 3 mL of a 2% starch solution to the tubes. Mix, and then simultaneously add all 10 tubes to the water bath. After exactly 30 minutes, add 2 drops of the iodine solution to the tubes and record the resultant color. A blue solution means starch still remains, and a colorless solution means all starch has been broken down into glucose. Revision SP11 Page 2 of 7 Austin Peay State University Department of Chemistry CHEM 1021 BREAKING DOWN STARCH USING SALIVARY AMYLASE 5. Determine the amylase number: a. Find the most dilute solution of saliva that still digested the starch (it didn’t turn blue upon iodine addition). This is the least amount of saliva that could digest the starch in the half‐hour time. Example: If you look at the test tubes and find that tubes 1 through 7 remained colorless, and 8‐10 were still colored (10 should always be colored since it is without any enzyme), then we know tube 7, the 1% saliva solution, was the limit on amount of saliva needed to digest the amylose. This is the tube we will use for the amylase number calculations (determination of activity). b. Determine the number of mL of saliva in the test tube of interest. Example: For tube 7, there was 3 mL of a 1% saliva solution. To find the mL of saliva in the sample, multiply the percent solution by 3 mL: 3
100
1% 3
100
0.03
c. Determine the amylase number. The amylase number is the number of mL of a 1% starch solution digested per mL of saliva in 30 minutes. Because we used 3 mL of a 2% starch solution instead of a 1% solution, we must account for that in our calculations. A 2% solution of 3 mL is equivalent (in starch content) to 6 mL of a 1% solution. The higher the amylase number is, the faster (better) the enzyme works. Example: For tube 7: 1%
6
0.3
200 Waste Disposal and Cleanup: Dispose of solutions according to instructions provided by your lab instructor. Wash all glassware thoroughly, rinse with de‐ionized water and return to their original places. Revision SP11 Page 3 of 7 Austin Peay State University Department of Chemistry CHEM 1021 BREAKING DOWN STARCH USING SALIVARY AMYLASE THIS PAGE INTENTIONALLY LEFT BLANK Revision SP11 Page 4 of 7 Austin Peay State University Department of Chemistry CHEM 1021 BREAKING DOWN STARCH USING SALIVARY AMYLASE DATA SHEET Name:__________________________________ Partner: ________________________________ 1. Fill in the following table (space for calculations provided on the back): Tube # Saliva Distilled water Final saliva % Color after iodine Total mL saliva
1 3 mL 100% 0 mL 100% 2 2 mL 100% 1 mL 66.7% 3 1 mL 100% 2 mL 33.3% 4 3 mL 10% 0 mL 10% 5 2 mL 10% 1 mL 6.7% 6 1 mL 10% 2 mL 3.3% 7 3 mL 1% 0 mL 1% 8 2 mL 1% 1 mL 0.67% 9 1 mL 1% 2 mL 0.33 % 10 0 mL 3 mL 0% 2. Which tube will you use for calculating the amylase number? ____________________________ 3. Amylase number (show your calculations): Calculations: Revision SP11 Page 5 of 7 Austin Peay State University Department of Chemistry CHEM 1021 BREAKING DOWN STARCH USING SALIVARY AMYLASE POST‐LAB QUESTIONS Name: _________________________________________ Partner:__________________________________________ 1. Why did you use a 2% solution, when the activity definition relies on a 1% solution? ______________________________________________________________________________
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______________________________________________________________________________ 2. Why does a cracker taste sweet after it has been in your mouth for a while? 3. What is the structural difference between amylose and amylopectin? What is the structural similarity of amylase and amylopectin? 4. If one person’s amylase number is 200 and another person’s amylase number is 150, which person can digest larger amounts of starch more effectively? Revision SP11 Page 6 of 7 Austin Peay State University Department of Chemistry CHEM 1021 BREAKING DOWN STARCH USING SALIVARY AMYLASE PRE‐LAB ASSIGNMENT Name:_____________________________________ Partner: ___________________________________ 1. What is the purpose of an enzyme (generically, not just amylase)? ______________________________________________________________________________
______________________________________________________________________________ 2. Why does the water bath need to be at 37 oC? ______________________________________________________________________________
______________________________________________________________________________ 3. If tube 3 is the most dilute solution that remains colorless upon iodine addition, what is the amylase number? Show your work. 4. What monosaccharide will be present in the test tube after enzymatic digestion? _________________________ 5. In this lab we assume that the amount of amylase in saliva is the same from person to person. Why is this important to our calculations? _____________________________________________________________________________________
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