Problem: What would happen to enzyme activity if enzymes are placed outside their normal conditions? Hypothesis:

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1 Name: Date: Period: Honors Biology: Enzyme Lab Background information What would happen to your cells if they made a poisonous chemical? You might think that they would die. In fact, your cells are always making poisonous chemicals. They do not die because your cells use enzymes to break down these poisonous chemicals into harmless substances. Enzymes are proteins that speed up the rate of reactions that would otherwise happen more slowly. The enzyme is not altered by the reaction. You have hundreds of different enzymes in each of your cells. Each of these enzymes is responsible for one particular reaction that occurs in the cell. In this lab, you will study an enzyme that is found in the cells of many living tissues. The name of the enzyme is catalase; it speeds up a reaction which breaks down hydrogen peroxide (H 2 O 2 ), a toxic chemical, into two harmless substances (1) water and (2) oxygen. If the cells did not break down the hydrogen peroxide, they would be poisoned and die. In this lab, you will study the catalase found in liver cells. You will be using chicken or beef liver. It might seem strange to use dead cells to study the function of enzymes. This is possible because when a cell dies, the enzymes remain intact and active for several weeks, as long as the tissue is kept refrigerated. The reaction is as follows: 2H 2 O > 2H 2 O + O 2 Problem: What would happen to enzyme activity if enzymes are placed outside their normal conditions? Hypothesis: Teacher Prep: Each lab group should have Test tube clamp 5 test tubes Forceps test tube holder Stirring rod 10ml graduated cylinder ml beakers (labeled peroxide & dh2o) Teacher prep tray. Cut up liver Soapy test tube brushes by sinks Acidic peroxide (ph3) Acidic graduated cylinder Basic peroxide (ph10) Basic graduated cylinder Neutral peroxide (ph 7) Neutral graduated cylinder Hot water, Warm water, Ice water baths A - Observe Normal Catalase Reaction 1) Measure and add 2 ml of hydrogen peroxide into test tube #1. 2) Obtain a fresh piece of liver. Push it into test tube #1 with your stirring rod. Observe the bubbles. The bubbles show a chemical reaction is taking place. Throughout this investigation you will estimate the rate of the reaction on a scale of 0-5. (0 = no reaction, 5 = very fast). Assume that the reaction in test tube #1 proceeded at a rate of "4". Fill in the data table. 3) A reaction that absorbs heat is endothermic. A reaction that gives off heat is exothermic. Feel the temperature of test tube #1 with your hand. Discuss with your group if it has gotten warmer or colder? 4) Pour the leftover liquid from test tube #1 into test tube #2 (do not dump the liquid down the drain). Be sure to keep the liver in test tube #1 as you will need it later. Add a fresh piece of liver to test tube #2 and push it down into the used liquid. Record the rate of reaction in your data table. 5) Is catalase reusable? At this point, test tube #1 should have a piece of used liver but no liquid. Add 2ml of fresh peroxide into test tube #1. Record the rate of reaction in your data table.

2 B - What is the Effect of Temperature on Catalase Activity? Test tube #3: Hot Water Bath 1) Put a piece of liver into the bottom of test tube #3 and cover it with a small amount of distilled water. 2) Place this test tube in a boiling hot water bath for 5 minutes. Use the test tube clamp to prevent burns. 3) After 5 minutes, remove the test tube from the hot water bath and allow it to air cool. CAUTION: Use a test tube clamp when handling the hot test tube to prevent burns. 4) After cooling for a couple minutes, pour out the water and add 2 ml of hydrogen peroxide to test tube #3. Record the reaction rate (0-5) in your data table. Test Tube #4: Ice Water Bath 1) Put a piece of chilled liver (obtain from teacher) into the bottom of test tube #4 and cover will a small amount of chilled distilled water (obtain from teacher). 2) Place this test tube into the ice bath for 5 minutes. 3) Drain the water but then add 2ml of chilled hydrogen peroxide (obtain from teacher). Record the reaction rate (0-5) in the data table. Test Tube #5: Warm Water Bath 1) Put a piece of liver into the bottom of test tube #5 and cover it with a small amount of distilled water. 2) Place this test tube into the warm water bath for 5 minutes. 3) After 5 minutes, remove the test tube from the warm water bath 4) Drain the water but then add 2ml of hydrogen peroxide. Record the reaction rate (0-5) in the data table. C - What is the Effect of ph on Catalase Activity? You will need to clean 3 of the used test tubes for this next part. A. Drain the used liquid from three test tubes. B. Throw away any used liver in the trash (not down the drain). Use your forceps if you don t want to touch the liver. C. There are soapy test tube brushes located next to each sink. Use the soapy brushes to clean the inside of your test tubes. Be sure to rinse the tubes well to remove any soap when finished. CAUTION: Be sure to rinse your hands if you get any acids/bases on your skin. 1) Fill three clean test tubes according to the directions below. Treat each tube as follows: Tube #1 Add 2ml of acidic peroxide (ph 3). Tube #2 Add 2ml of basic peroxide (ph 10). Tube #3 Add 2ml of neutral peroxide (ph 7). 2) Put equal quantities of liver into each of the three test tubes and observe the effects of the varying ph. Record your observations in the DATA TABLE. Final Clean Up Rules: If I have to clean up your test tubes, supplies, and/or lab table you will receive a 0% for your grade. 1. Drain any used liquid from the test tubes. 2. Throw away any used liver in the trash (not down the drain). Use your forceps if you don t want to touch the liver. 3. There are soapy brushes located next to each sink. Use the soapy brushes to clean your test tubes, stirring rod, and graduated cylinder. Be sure to rinse well to remove any soap when finished. 4. Return all supplies to your lab table. 5. Wipe down your lab table and then wash your hands.

3 DATA TABLE Normal Liver (test tube #1) Rate of Reaction (0-5) Observations A Liver added to Used Peroxide (test tube #2) Reused Catalase (test tube #1) Boiled Liver (test tube #3) B Ice Bath Liver (test tube #4) Warm Liver (test tube #5) Basic Solution (ph 10) C Acidic Solution (ph 3) Neutral Solution (ph 7) Conclusions Answer the following questions while referring to your data. Be sure to use your best writing skills. In General (Quick Responses): 1. Which test tube is the control group of this experiment? 2. What was the dependent variable in this experiment? 3. In part B, what was the independent variable? 4. In part C, what was the independent variable? Part A 5. From step 2, which gas is being released when the liver and peroxide come in contact?

4 6. When liver and peroxide produced the bubbles, was this reaction endothermic or exothermic? How do you know? 7. From step 4, the left over liquid was put in a new test tube. What was the used liquid and why were few bubbles produced when a fresh piece of liver was added? 8. Based on the directions from step 5, is catalase reusable? Explain how you know this. Part B 9. What is the effect of temperature on catalase? 10. What is the optimum temperature for catalase? Part C 11. What is the effect of ph on catalase? 12. Does there appear to be an optimum ph?

5 General 13. Amylase is an enzyme that can be used to break down carbohydrates, like those found in bread and crackers to individual subunits of sugars. This is why bread begins to taste sweet as we chew it, amylase is found in saliva. Benedict's solution is a chemical that changes color in the presence of sugar. In water, Benedict's is a blue color, but it will turn orange in the presence of glucose. In the space below, explain how would you design an experiment to test the reaction rate of amylase in breaking down starch into sugars?