Testing Enzyme Activity Background: Like nonliving matter, all organisms are made of chemicals. No single substance in an organism is alive. But together, all the substances that make up an organism are alive. Organisms are mixtures that contain mostly water, with a pH ranging from 6.5-7.5. Water, having a neutral pH, dissolves many things and makes many of the chemical reactions that occur in our body possible. Enzymes control the chemical activities of living things and also reduce the energy needed to get chemical reactions started. Many factors in a cell’s environment (pH, temperature, salinity, etc) affect the action of an enzyme. Live yeast contain an enzyme, catalase, which breaks down hydrogen peroxide, H2O2, into water and oxygen gas. When H2O2 is formed in cells, bubbles of O2 gas are released. In this laboratory exercise, you will examine the influence of various factors on catalase. Problem: If you are assigned a complete lab write-up, include this in your introduction. Hypothesis: If you are assigned a complete lab write-up, include this in your introduction. Materials: Live & cooked yeast 12 M Hydrochloric Acid (HCl) 1 M Sodium Hydroxide (NaOH) Grease pencil 1 Dropper pipette Salt solution 6 small test tubes Water 3% Hydrogen peroxide solution 0.3% Hydrogen peroxide solution Ruler Caution: solutions in this lab can be irritating to skin and eyes. If you spill any on your skin or clothes, wash it off immediately and tell Mrs. Cardellini. Methods: 1. 2. 3. Mrs. Cardellini will assign your group either the 3% OR 0.3% H2O2 solution to test. Put on your goggles and keep them on until the end of the experiment. Mark 1 ml on the dropper pipette that is to be used for the yeast solution. Obtain 6 small test tubes that are the same size. Fill 4 test tubes (E, A, B, S – see step #4) with 1 ml of yeast solution, 1 test tube with 1 ml of the cooked yeast, and one test tube with 1 ml water. 4. Label each test tube with a grease pencil as follows: E A B S C (put this on the cooked yeast tube) H2O (water tube) 5. Use a grease pencil to mark the volume of the solution on the E, C and H2O test tube. 6. Add 5 drops of the H2O2 solution (3% or 0.3%) into the test tube labeled H2O. Observe the reaction, if any. 7. Enzyme: Add 5 drops of the H2O2 solution into the test tube labeled E. Gently swirl to mix up the solution. 8. Measure (in mm) the length of the head of the oxygen bubbles after the catalase converts the H2O2. You may have to wait a bit until the reaction is finished. 9. Acid test: Mrs. Cardellini will add 5 drops of the 12 M HCL solution into the test tube labeled A. Swirl gently and let sit for 2 minutes and be sure to mark the total volume on the tube. 10. Add 5 drops of the H2O2 solution into the A test tube and gently swirl. 11. Measure (in mm) the length of the head of the oxygen bubbles after the catalase converts the H2O2. 12. Base test: Add 5 drops of the NaOH solution into the test tube labeled B. Gently swirl to mix up the solution and let sit for 2 minutes. Mark the volume line on the tube. 13. Add 5 drops of the H2O2 solution into the B test tube and gently swirl. 14. Measure (in mm) the length of the head of the oxygen bubbles after the catalase converts the H2O2. 15. Salinity test: Add 5 drops of the salt solution into the test tube labeled S. Gently swirl to mix and mark the total volume on the tube. 16. Add 5 drops of the H2O2 solution into the S test tube and gently swirl. 17. Measure (in mm) the length of the head of the oxygen bubbles after the catalase converts the H2O2. 18. Temperature test: Add 5 drops of the H2O2 solution into the C test tube and gently swirl. 19. Measure (in mm) the length of the head of the oxygen bubbles after the catalase converts the H2O2. 20. Dump the contents of the test tubes in the designated plastic container before you rinse them out. 21. Rinse out tubes, wipe off all markings, and place them upside down in the rack. 22. Get the data from your partner group then have Mrs. Cardellini check your station so that you may be excused. Results: Determine the best way to present your lab data in a chart form. Be sure to use a ruler for this on your write-up. Don’t forget to exchange lab data with the group across from you so you have data for both the 3% AND 0.3% H2O2 solution. Discussion questions: 1. What was the enzyme in this experiment? 2. What was the substrate in this experiment? 3. What was the purpose of dropping the H2O2 solution into a test tube with water? 4. Why test two concentrations of H2O2 solution? Compare the data for both trials and explain the significance. 5. In which trial was the enzyme activity the greatest? 6. Water is a major component of cells. What does this tell you about the optimum pH for most enzymes? Explain. Conclusion: Just answer the questions below versus the requirements listed on LabWrite. 1. How does pH, salinity, and temperature affect enzyme activity? 2. Think of at least two more experiments that might be investigated to strengthen or expand the data you have already collected. 3. If you had an increased amount of H+ ions in your blood (increases the acidity) predict what would happen. 4. The stomach produces HCl to help digest food. How would this effect the pH of the stomach. What can you deduce about the enzyme pepsin, which works to digest the protein in the food that you eat?