Name:___________________________________________ Date: ____________ Block:_______ Lab Partners:_______________________________________________________________________ CATALYTIC ACTIVITY OF ENZYMES Background: A catalyst is defined as something that precipitates a process or event, without being involved in or changed by the consequences. In chemistry, a catalyst is a chemical that facilitates the reaction of other chemicals without reacting itself. The thousands of chemical reactions in living systems could all take place by themselves, but they would generally happen too slowly to sustain life. So cells have evolved to make catalysts to speed up the reactions of cellular metabolism. The catalysts in living systems are protein molecules and, based on their function, are called enzymes. Hydrogen peroxide is a highly reactive chemical, often used for bleaching and for cleaning minor wounds. It is also formed continually as a by-product of chemical reactions in cells, but is poisonous. It must be immediately removed or broken down by the cells. In living cells, hydrogen peroxide is converted into two harmless substances, oxygen and water. Lab Objectives: You will see the effect of an enzyme called catalase, which speeds up the breakdown of hydrogen peroxide. You can detect the breakdown of peroxide by testing for the production of oxygen. (A glowing splint will reignite in the presence of O 2.) You will have an opportunity to compare catalase with an inorganic catalyst (manganese dioxide, MnO2) that accelerates the same reaction. You will investigate the effect of grinding, temperature, and pH on the enzyme activity of catalase. Pre-Lab Questions: 1. What is a catalyst? 2. What are catalysts called in living things? 3. What type of biomolecule is an enzyme? 4. What is hydrogen peroxide used for? 5. Why does the cell need to eliminate this byproduct? 6. The chemical formula for hydrogen peroxide is: H202 Write a balanced equation showing the break down of H202 into harmless substances. 7. Which enzyme will we will use in this investigation? 8. Write a hypothesis based on one of the following questions: Is the inorganic catalyst, MnO2, more or less effective than the organic compound catalase? What effect does grinding up the liver have on the enzymatic reaction? What effect does temperature have on the enzymatic reaction? What effect does pH have on the enzymatic reaction? Hypothesis: Materials: 3% hydrogen peroxide (H2O2) HCl, 0.1 M mortar and pestle liver NaOH, 0.1 M matches manganese dioxide (MnO2) test tube Bunsen burner or sand test tube holder hot water bath forceps wood splints Procedure NOTE: You must wear safety goggles and appropriate skin coverage (clothing must cover arms to elbow, midriff, legs, and feet) to participate in this investigation! 1. Pour 3% hydrogen peroxide into a test tube to a depth of about one centimeter. 2. Sprinkle a pinch of fine sand into the test tube. 3. Place your thumb over the mouth of the tube, shake gently and observe the reaction. 4. Hold a glowing splint in the tube. Be careful not to touch it to the side of the tube. Record the reactions on your chart in the second and third columns. 5. Rinse your test tube. 6. Manganese dioxide is an inorganic catalyst for the breakdown of hydrogen peroxide. Pour about one cm of peroxide into your test tube and sprinkle in a pinch of manganese dioxide powder. (Try to make your pinches of constant size.) Place your thumb over the mouth of the tube. If necessary, shake gently. Note the reaction and hold a glowing splint in the tube. Record your results on the chart. Rinse your test tube. 7. Liver is an organic material that contains the enzyme catalase. Pour about one cm of peroxide into your test tube. Using forceps, pick up a small piece of fresh liver and drop it into the tube. Place your thumb over the mouth of the tube and shake gently. Observe the reaction and test with a glowing splint. Record your results. Rinse your test tube. 8. Take a small piece of liver and put it in a mortar along with a small amount of sand. Grind the liver with the pestle and use this material to repeat the test of catalytic activity described in Step 3. Be sure to use the same size piece of liver as you did in Step 3 and to get all the ground material into the test tube. Record your results. Rinse your test tube. 9. Place a small piece of liver of the same size you have used before in a test tube. Fill the test tube half full of water and carefully boil the water over a Bunsen burner or place in a hot water bath. Boil the liver for three minutes. Pour off the water and use the boiled liver to repeat the test for catalytic activity as described above. Record your results. Rinse your test tube. 10. Pour about one cm of acid into a test tube. Again, use peroxide and a glowing splint to test for the catalytic activity of the acid. Record your results. Rinse your test tube. 11. Place another small piece of liver in a test tube. Carefully pour in just enough acid solution to cover the liver. (Be sure to clean up any acid you spill and especially to rinse off any you get on your hands.) Let the liver soak in acid for three minutes, pour off the solution and rinse the meat with water. Pour about 1 cm of hydrogen peroxide into the tube then repeat the test for catalytic activity using the liver soaked in acid. Record your results. Rinse your test tube. 12. Pour about one cm of base into a test tube. Test for the catalytic activity of the base as described above. Record your results. Rinse your test tube. 13. Repeat Steps 7 and 8 using base instead of acid. Be careful. Base is more dangerous to your skin than acid. Record the results of the test for catalytic activity on your chart. Rinse your test tube. 14. While you remember your observations, fill in the fourth column of your data chart, estimating the relative activity of each catalyst on a scale of 0-10 (0 = no catalytic activity; 10 = very strong catalytic activity). 15. Carefully clean all the glassware, forceps, and mortars & pestles you have used. Once your group has cleaned up check out with you teacher: ________________(teacher’s initials) Data In the middle column record: Record the amount of bubbles released. Whether or not the glowing splint indicated the presence of oxygen (Did it relight? Once? More?) In the column on the right record an estimate of the different rates of catalytic activity. Use a scale of 0 to 10 (0 = no detectable activity; 10 = extremely high activity). Test Tube Contents Observations Bubbling Activity Scale (1-10) H2O2 + sand H2O2 + MnO2 H2O2 + liver H2O2 + ground liver H2O2 + boiled liver H2O2 + HCl H2O2 + liver + HCl H2O2 + NaOH H2O2 + liver + NaOH Data Analysis and Conclusion Questions: 1. Why was it necessary to test the sand for catalytic activity? 2. Why was it necessary to test the acid for catalytic activity? 3. Why was it necessary to test the base for catalytic activity? 4. How are manganese dioxide and liver similar? 5. How are manganese dioxide and liver different? 6. Which of these two catalysts is more active? How might you explain this result? 7. What effect does grinding the liver have on its catalytic activity? How might you explain this result? 8. What effect does boiling the liver have on its catalytic activity? How might you explain this result? 9. What effect does acid have on the catalytic activity of liver? How might you explain this result? 10. What effect does base have on the catalytic activity of liver? How might you explain this result? 11. Hydrogen peroxide is an unstable compound that breaks down when exposed to light, air, and many other compounds. Given that this is so, why does your body bother to make catalase? 12. Why is it important that your body carefully control the pH of its tissues?
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