Download AP Laboratory Investigation Lab 13 – Enzyme Activity 2H2O2

AP Laboratory Investigation Lab 13 – Enzyme Activity 2H2O2 Catalase > 2H2O + O2 Problem: How does the reaction rate of an enzyme change when: 1) the enzyme concentration is doubled and the substrate concentration is not changed; 2) the substrate concentration is decreased by one-­‐half and the enzyme concentration is not changed; 3) the enzyme is boiled; 4) the enzyme has been acted upon by a strong acid? Pre-­‐lab questions 1. What do catalysts do? 2. Explain what this equation means: E + S à ES à E + P 3. Identify the enzyme, substrate and products in our experiment 4. Explain how pH might affect an enzyme and the rate of the reaction 5. Explain how temperature might affect an enzyme and the rate of the reaction 6. Explain how concentration might affect an enzyme and the rate of the reaction 7. Explain how activators and inhibitors might affect an enzyme and the rate of the reaction 8. Explain how concentration of the substrate might affect an enzyme and the rate of the reaction 9. Describe catalase & explain why it is so essential that it would be found in all aerobic (oxygen-­‐dependent) organisms. Enzyme Lab INTRODUCTION: 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 (KAT-­‐uh-­‐LAYSS); it speeds up a reaction, which breaks down hydrogen peroxide, a toxic chemical, into 2 harmless substances-­‐-­‐water and oxygen. The reaction is: 2 H2O2 -­‐-­‐-­‐-­‐> 2 H2O + O2 This reaction is important to cells because hydrogen peroxide (H2O2) is produced as a byproduct of many normal cellular reactions. 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. MATERIALS: 1molar HCl solution 1molar NaOH solution 6 Test tubes Measuring Pipette 10-­‐ml Graduated cylinder 40 ml 3% Hydrogen peroxide solution (found in stores) Straight-­‐edged razor blade Scissors and Forceps (tweezers) pH paper (optional) Stirring rod Fresh liver, Apple, and Potato Test tube holders Ice bath Warm water bath Boiling water bath PART A -­‐ Observe Normal Catalase Reaction 1. Place 2 ml of the 3% hydrogen peroxide solution into a clean test tube. 2. Using forceps and scissors cut a small piece of liver and add it to the test tube. Push it into the hydrogen peroxide with a stirring rod. Observe the bubbles. What gas is being released? (consider the equation) ______________________ Throughout this investigation you will estimate the rate of the reaction (how rapidly the solution bubbles) on a scale of 0-­‐5 (0=no reaction, 1=slow, ..... 5= very fast). Assume that the reaction in step 2 proceeded at a rate of "4" Recall that a reaction that absorbs heat is endothermic; a reaction that gives off heat is exothermic. Now, feel the temperature of the test tube with your hand. Has it gotten warmer or colder ___________ Is the reaction endothermic or exothermic? ____________________ 3. Pour off the liquid into a second test tube. Assuming the reaction is complete. What is this liquid composed of? _______ What do you think would happen if you added more liver to this liquid? __________________________________ Test this and record the reaction rate. Reaction Rate ___________ (1 – 5) 4. Add another 2 ml of hydrogen peroxide to the liver remaining in the first test tube. What is the reaction rate? ________ Is catalase reusable? Explain how you know. Part B -­‐ What Tissues Contain Catalase You will now test for the presence of catalase in tissues other than liver. Place 2 ml of hydrogen peroxide in each of 3 clean test tubes and then add each of the three test substances to the tubes. As you add each test substance, record the reaction rate (0-­‐5) for each tube. Substance Rate of Reaction (0-­‐5) Potato Apple Chicken Based on your observations, which tissues contained catalase? _______________________________________________ Do some contain more catalase than others? How can you tell? _____________________________________________ PART C -­‐ What is the Effect of Temperature on Catalase Activity? 1. Put a piece of liver into the bottom of a clean test tube and cover it with a small amount of water. Place this test tube in a boiling water bath for 5 minutes. Remove the test tube from the hot water bath, allow it to air cool, then pour out the water. Add 2 ml of hydrogen peroxide. CAUTION: Use a test-­‐tube holder for hot test tubes. What is the reaction rate for the boiled liver and peroxide? __________ 3. Put equal quantities of liver into 2 clean test tubes and 1 ml H2O2 into 2 other test tubes. Put one test tube of liver and one of H2O2 into an ice bath. Place the other set in a warm water bath (not boiling). After 3 minutes, pour each tube of H2O2 into the corresponding tube of liver and observe the reaction What is the reaction rate for the cold liver/peroxide? ______ What is the reaction rate for the warm liver/peroxide? ______ PART D -­‐ What is the Effect of pH on Catalase Activity 1. Add 2 ml hydrogen peroxide to each of 5 clean test tubes. Tube 1-­‐-­‐add 4 drops of acetic acid (vinegar) pH =_______ Tube 3 – add 4 drops of sodium bicarbonate (base) pH =______ Tube 5 – add 3 drops of water (neutral) pH =_____ *(Optional) Using pH paper, determine the pH of your samples. Now add liver to each of the test tubes (try to do it all at about the same time, so you can easily compare) Rate of Reaction for: Acid _____ Neutral ______ DATA ANALYSIS 1. Describe the relationship between catalase and hydrogen peroxide. Indicate which is the enzyme, which is the substrate and what occurs during the reaction. It may be helpful to write the equation. (2) 2. Is catalase reusable? Use your data to support your answer. (1) 3. How does temperature and pH affect the reaction rate of catalase? Propose a way to refine your experiment to find the exact, or OPTIMAL pH and temperature of catalase. (3) 4. In 2-­‐3 sentences, summarize the data and information displayed in this graph. Lab Version #2 Materials: Pan, rubber tube, glass bend for water displacement Flask 100mL graduated cylinder 50mL graduated cylinder Turnip, Potato, or Yeast Extract Water Hydrogen Peroxide Graph Paper Timer Procedure: Part I – Control 1. Place 300mL of water in a blender and add a medium turnip or potato (sliced). Blend for 30 seconds. Strain through cheesecloth or paper towels. Collect the fluid in a beaker and place the beaker on ice. The fluid contains the enzyme catalase. 2. Fill the container ¾ full of water and submerge a 100mL graduated cylinder in the water. 3. In a beaker, combine 10mL of water and 10mL of hydrogen peroxide. 4. In a second beaker, collect 10mL of the enzyme solution. 5. Place the rubber tubing into the submerged graduated cylinder, and keeping the mouth of the graduated cylinder under the water, invert the cylinder (make sure the cylinder is full of water – no air pockets at the top). 6. Pour the hydrogen peroxide and catalase solutions into the flask, stopper, and swirl the flask. 7. Begin timing when the first bubble or gas appears in the cylinder. 8. Collect the data. Slope = Change in Y / Change in X Use this format for each experiment, and be sure to give each table a descriptive title. Time Interval (min) mL O2 Produced Slope 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Now that a baseline has been established through a controlled experiment, each experimental group should choose one variable from the problem to change and compare the graph and the reaction rates to the results of the controlled experiment. Note: Catalase solution may be frozen. If this lab cannot be completed in one class period, freeze half of the catalase solution for the second day lab. Each fresh batch of catalase solution will have a different enzyme concentration. The following page contains the questions for the analysis of these experiments. 1. What is the relationship between slope and the reaction rate? 2. Why did the reaction rate during the latter part of the experiments? 3. What is happening in the flask when the reaction rate is “0”? 4. When is the rate the highest? Explain why. 5. When is the rate the lowest? For what reason is the rate low? 6. Explain the inhibiting effect of sulfuric acid on the function of catalase. Relate this to enzyme structure and chemistry. 7. Predict the effect that lowering the temperature would have on the rate of enzyme activity. Explain your prediction.