Salivary Amylase Activity PDF

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SALIVARY AMYLASE ACTIVITY Enzymes are proteins that facilitate chemical reactions in cells. These are reactions that would happen spontaneously but at a rate much too slow to support life. Enzymes allow biochemical reactions to proceed at rates of hundreds of times per second. Because most are proteins, enzymes are sensitive to fluctuations in temperature and pH. Generally speaking, chemical reactions are faster as temperature increases. However, boiling and extreme pH values denature (unravel) enzymes which deactivates them. Chemical reactions will proceed outside of the body and can be monitored using tests that illustrate the presence or absence of specific molecules by changes in color. Temperature and pH environments can be altered to determine what conditions are ideal for enzyme activity.

Starch digestion Most carbohydrate in our diet is provided by starchy foods. Enzymes called amylases break apart the sugar units from starch. The first amylase to encounter food is salivary amylase, which will break starch (the substrate) down into the disaccharide maltose (the product) in our mouths. This explains why starchy foods begin to taste sweet when you chew on them for a few minutes. Tests for enzyme activity The following demonstrations will evaluate the activity of salivary amylase on starch. This is accomplished by monitoring the disappearance of starch and appearance of sugar. An iodine compound called Lugol’s reagent (IKI) can be added to a substance to test for the presence of starch. When starch is present, it interacts with the iodine, which is a golden color, to become a blue-black color. As starch is digested, it disappears so that an iodine test would produce a golden color.

Benedict’s reagent tests for the presence of sugars. When heated, these sugars react with Benedict’s reagent to produce colors ranging from yellows to oranges and reds. A bluish green color indicates that there might only be a trace of sugar present. This generally means that the glassware is contaminated from previous use.

Experimental results may vary among saliva samples. Following meals, for example, the concentration of amylase in saliva would be lowered. Additionally, there are differences in the effectiveness of enzymes among individuals due to genetic variability.

Lab Activity What you will need Contents of Kit Bottle of starch solution. Bottle of Lugol’s solution. Bottle of Benedict’s reagent. Disposable pipets. These have a hash mark at 1 ml for measuring. Test tubes. Test tube clamp. What you provide from home Heat resistant mug to boil test tubes in. A stove and small pot. A clear glass to put test tubes in for photographs. Tap water. Waterproof marker or crayon. Samples of several starchy foods such as bread, crackers, chips. Samples of several light-colored fruits, such as apple, pear, pineapple. Samples of a few light-colored liquids such as ginger ale, apple juice, corn syrup, green tea. These are the types of arrangements you’ll need to support and boil test tubes:

I.

Demonstrate Reagent Activity

(A) Use of Lugol’s solution to determine if starch is present 1. Use a pipet to add 2 ml. of the starch solution to a test tube. Keep that pipet for the starch solution to be used for it, and nothing else, again. Always shake the starch bottle before sampling from it. 2. Place your small food samples on a plate or paper towel. 3. Add a drop of Lugol’s solution to the test tube and to each of your food samples. 4. Photograph your results. All photographs must have a legend below them that describes what’s in the picture. Points will be lost if the image isn’t clear or large enough to be seen. Remember to have your ID next to the test tubes in the photos. 5. Take notes for the analysis in your lab report. Which samples contained starch? (B) Use of Benedict’s Reagent to determine if sugar is present 1. Use a pipet to add 2 ml. of tap water to each of three test tubes. Mark a line at the top of the solution and discard the water from three tubes. This allows you to approximate the amount of liquid in the tube without measuring in the future. 2. Using new pipets, add approximately 2 ml. of each of your test beverages to two separate test tubes. The third test tube should contain 2 ml. of water.

3. Use a new pipet to add 1 ml. of Benedict’s reagent to each of the three test tubes. Keep that pipet for the Benedict’s reagent to be used for it, and nothing else, again. 4. Place the test tubes in a heat resistant mug containing approximately three inches of water. 5. Boil the test tubes for five minutes. Use a small pot on a stovetop. Add a few inches of water to the pot and place the mug with water and test tubes into it. Time the five minutes from when the water begins to boil. 6. Carefully remove the mug from the heat and, using the clamp, transfer the test tubes to the rack or clear container. 7. Photograph the test tubes. All photographs must have a legend below them that describes what’s in the picture. Points will be lost if the image isn’t clear or large enough to be seen. 8. Take notes for the analysis in your lab report. Which samples contained sugar? THOROUGHLY WASH THE TEST TUBES USING SOAP AND RINSING WELL.

II.

Demonstrate Activity of Salivary Amylase

1. Collect your saliva in a test tube. Fill the test tube approximately halfway to the top. Keep this test tube until the completion of all activities. This is your stock supply of enzyme. (A) Testing for the Disappearance of Starch 2. Use the starch pipet to add 2 ml. of starch to another test tube. 3. Use the saliva pipet to add ten drops of the saliva to the starch in the test tube. Leave that pipet in the saliva test tube for future use. 4. After ten minutes have passed, use a new pipet to transfer 1 ml. from the bottom of the starch/ saliva test tube to a clean test tube. (The starch will always settle to the bottom; if you collect from the top you’ll miss it) 5. Add a drop of Lugol’s solution to the test tube with 1 ml. of the starch/ saliva mixture. You will need the original tube with the starch/ saliva mixture for the sugar test. 6. Photograph the test tube with the Lugol’s test. All photographs must have a legend below them that describes what’s in the picture. Points will be lost if the image isn’t clear or large enough to be seen.

7. Take notes for the analysis in your lab report. Is starch still present? If yes, repeat the procedure with 15-20 drops of saliva. Continue to the test for sugar only after starch has disappeared. (B) Testing for the Appearance of Sugar 8. Add 1 ml. Benedict’s reagent to the original test tube containing the starch/ saliva mixture. 9. Following previous instructions for the Benedict’s test, boil the tube for five minutes. 10.Carefully remove the test tube with the clamp and return to the rack or container. 11. Photograph the test tube. All photographs must have a legend below them that describes what’s in the picture. Points will be lost if the image isn’t clear or large enough to be seen. 12.Take notes for the analysis in your lab report. Was there sugar in this test tube? THOROUGHLY WASH THE TEST TUBES USING SOAP AND RINSING WELL.

III.

Demonstrate the Effects of boiling on Enzyme Activity

1. Boil the test tube with the remaining saliva in it for five minutes. Start the timing from when the water in the mug begins to boil. Using the boiled saliva, repeat steps from “Demonstrate Activity of Salivary Amylase”: (A) Testing for Disappearance of Starch 2. Use the starch pipet to add 2 ml. of starch to a clean test tube. 3. Add ten drops (or the same number used previously) of the boiled saliva to the starch in the test tube. 4. After ten minutes have passed, use a new pipet to transfer 1 ml. from the bottom of the starch/ saliva test tube to a clean test tube. (The starch will always settle to the bottom.) 5. Add a drop of Lugol’s solution to the test tube with 1 ml. of the starch/ saliva mixture. You will need the original tube with the starch/ saliva mixture for the sugar test.

6. Photograph the test tube with the Lugol’s test. All photographs must have a legend below them that describes what’s in the picture. Points will be lost if the image isn’t clear or large enough to be seen. 7. Take notes for the analysis in your lab report. Is starch still present? (B) Testing for the Appearance of Sugar 8. Add 1 ml. Benedict’s reagent to the original test tube containing the starch/ saliva mixture. 9. Following previous instructions for the Benedict’s test, boil the tube for five minutes. 10. Carefully remove the test tube with clamp and return to the rack or container. 11. Photograph the test tube. All photographs must have a legend below them that describes what’s in the picture. Points will be lost if the image isn’t clear or large enough to be seen. 12. Take notes for the analysis in your lab report. Was there sugar in this test tube?

Lab Report Instructions Your lab report should contain the following: Photographs of the results from each section, demonstrating activity of Lugol’s solution, Benedict’s reagent, the enzyme and the enzyme after it has been boiled. There will be a minimum of six photographs. It may be necessary to include more than that if sufficient detail can’t be seen without separating tubes or samples into multiple pictures. All photographs must have a legend below them that describes what’s in the picture. Points will be lost if the image isn’t clear or large enough to be seen. All photographs should be taken with your photo ID placed beside the materials being photographed. This is how you demonstrate to me that these are your own original images and not “borrowed”. A brief explanation should accompany each photograph. Your analysis will include comments regarding: (1) The initial use of the reagents (which samples contained starch and which contained sugar).

(2) The action of salivary amylase on starch before and after the saliva was boiled. Your lab report should end with a paragraph summarizing your findings and explaining any problems you encountered as you preformed the activities. The discussion needs to focus on Salivary Amylase activity. What have you learned about the enzyme and how it works? It is expected that your sentences will be grammatically correct and that your thoughts will be clearly presented. Points will be lost if you do not clearly articulate your thoughts....