Enzyme Lab -Digestion Introduction As we have learned, enzymes are specialized proteins that catalyze biochemical reactions within and outside the cell. We have learned that enzymes are specific to an individual bond-type and activity is regulated in a variety of ways (i.e. co-enzymes, co-factors, temperature, pH, etc.). In this lab we will discover how specific enzymes act on macromolecules and how changing the conditions of the environment affect enzyme action. Because these enzymes are human enzymes they require an operating temperature of 370C. We will accomplish this with hot water baths. The material an enzyme acts upon is a substrate. The enzyme pepsin facilitates the breakdown of proteins so the enzyme is pepsin and the substrate is protein (actually the peptide bond). We can test for enzyme activity by testing for the substrate concentration or testing for changes in the environment. Lipase acts on fats by breaking them into glycerol and fatty acids. Since fatty acids are acids it is a logical conclusion that as lipase acts on fats the pH of the environment would decrease (i.e. be lower than 7). Indicators like phenolphthalein is pink/deep pink colour in a slightly basic environment (pH=8) which is the conditions within the small intestine. If the pH drops, phenolphthalein becomes clear. Therefore, when we want to test for lipasd activity we must begin with a slightly basic environment. We can accomplish this by adding a “base” like sodium hydroxide to solution. Lipase is stored in the bile salts of your gall bladder but is also secreted by the lining of the stomach to a lesser degree. Amylase acts upon complex carbohydrates (starches) and breaks them into simpler saccharides. When a solution containing iodine or potassium iodide is added to starch it has a deep blue colour. If no starches are present the solution will remain a brown colour. If we allow amylase to work on a starch then when we add the iodine solution (like Lugol’s solution even) there should be a negative result (No starch present) . Pepsin breaks down proteins into smaller peptides and amino acids. Pepsin is released in the stomach which has an acidic environment (low Ph). Therefore, pepsin would be activated/regulated based upon site specific conditions. Biuret reagent is an indicator that detects the presence of protein. Proteins cause Biuret to display a purple/violet colour. When peptides and amino acids are present the indicator takes on more of a pink/lavender colour. We can create an acidic environment by using a dilute solution of hydrochloric acid (the same acid produced in the stomach). Purpose: To observe the breakdown of macromolecules by enzyme catalyzed reactions and observe how changing temperature affects enzyme activity. Materials: Test tubes Test tube rack Starch Solution Enzyme Lab -Digestion Graduated Cylinders Lipase Albumin Solution Pepsin Solution Amylase Solution Rubber stoppers 0.1 M sodium hydroxide 0.1 M Hydrochloric acid solution Olive oil Hot plate Plastic Droppers Biuret Solution Potassium Iodide Solution Procedure: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Create a data table for your rough observations (see Results for the template) Obtain 3 test tubes and matching stoppers. Label them 1 through 3 with the letter A (1 is your control for observations) Add 5 drops of sodium hydroxide solution to all test tubes Add 5 drops of phenolphthalein to all test tubes. Use a dropper to add 10 drops of olive oil to all test tubes. Place test tube one and two in the 37 degree water bath. Place test tube three in the hot water bath. Wait 10 minutes before adding the lipase solution to test tube 2 and 3. Make initial observations. Wait 20 minutes before making final observations. If lipase has broken down the fat the test tube will appear clear. Obtain 3 more test tubes. Label them 1 through 3 with the letter B Use a graduated cylinder to add 4ml of albumin solution to all test tubes. Add 25 drops of 0.1M HCl to all test tubes. Place test tube 1 and 2 in the 37 degree water bath and test tube three in the hot water bath. Wait 10 minutes before adding 1ml of pepsin solution using a graduated cylinder to test tube 2 and 3. Tap the bottom of the test tubes gently to mix the solution. NO SHAKING Make initial observations in the data table. Let incubate for 20 minutes. Add 20 drops of Biuret solution to all three test tubes. If the enzyme has worked the test tube should remain blue. If it has not worked it should appear purple. Obtain 3 more test tubes. Label them 1 through 3 with initials and the letter C Using a graduated cylinder add 4ml of starch solution to all test tubes. Enzyme Lab -Digestion 19. Place test tube 1 and 2 in the 37 degree water bath and test tube three in the hot water bath. Incubate for 10 minutes before adding 5 drops of amylase solution to test tube 2 and 3. Make initial observations. 20. Let the test tubes incubate for 20 minutes before adding 3-4 drops of Lugol’s solution to each of the test tubes. If the enzyme worked then the solution will remain brown. If it did not work the solution will turn black. 21. All solutions can be poured down the sink. Clean all lab equipment and return. Results Table: Enzyme Activity Test Tube 1* (370C) 2(370C) 3 (1000C) Summary Comments Starch Protein Fats Initial Color Final Colour Initial Color Final Colour Initial Color Final Colour *Test Tube one is the control for the experiment. Conclusion This lab requires a self-directed conclusion. See your lab write-up instructions to help formulate this section. Be mindful of the purpose of this lab, your hypothesis and your actual results when composing this conclusion.