Name: Class: Date: Objectives * Measure the effects of changes in temperature, ph, and enzyme concentration on reaction rates of an enzyme catalyzed reaction in a controlled experiment. * Explain how environmental factors affect the rate of enzyme-catalyzed reactions. 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 H 2 O 2 ----> 2 H 2 O + O 2 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. Page 1
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. Page 2
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 H 2 O 2 into 2 other test tubes. Put one test tube of liver and one of H 2 O 2 into an ice bath. Place the other set in a warm water bath (not boiling). After 3 minutes, pour each tube of H 2 O 2 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. Page 3
Tube 1--add 4 drops of hydrochloric acid, HCl (acid) ph = Tube 3 add 4 drops of sodium hydroxide, NaOH (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 Base Part E - Design an Experiment Lactaid is a product designed to help people who cannot digest milk sugar (lactose) because they are missing the enzyme lactase. Many people are lactoseintolerant, a condition that is mainly genetic. Lactase breaks down lactose into two subunits: glucose and galactose. To test for the presence of monosaccharides and reducing disaccharide sugars in food, the food sample is dissolved in water, and a small amount of Benedict's reagent is added. The solution should progress in the colors of blue (with no glucose present), green, yellow, orange, red, and then brick red or brown (with high glucose present). Design an experiment where you would determine how quickly lactaid works to break down milk sugar. Be specific in your description, use drawings if necessary. 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) Page 4
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. Page 5
Name: Class Period: Date: Enzyme Lab Quiz 1. What is an enzyme? a. carbohydrate b. protein c. lipid d. nucleic acid 2. What enzyme were you testing in this lab? a. hydrogen peroxide b. liver c. chicken d. catalase e. apple f. potato 3. What was the gas being released when you added the liver to the hydrogen peroxide? a. hydrogen b. hydrogen peroxide c. oxygen d. water e. catalase 4. The test tube should have felt warmer after the reaction. That s because this reaction was a. endothermic. b. exothermic. 5. After the reaction, in part A, step 3, you poured the remaining liquid into a second test tube. Then you added some more liver to this liquid. Nothing should have happened because this liquid is a. water. b. hydrogen peroxide. c. blood. d. oxygen. e. hydrogen. 6. After the reaction, you were instructed to add more hydrogen peroxide to the remaining liver. It should have bubbled up significantly. What does this prove? (part A, step 4) a. This proves that there s a lot of oxygen in liver. b. This proves that hydrogen peroxide is very reactive. c. This proves that the catalase is reusable. d. This proves that it doesn t matter what you put with liver, it will always bubble. 7. After testing the potato, apple, and chicken you should have observed that the chicken made the hydrogen peroxide break down fastest. WHY? a. The chicken was the only raw food. b. The potato & apple oxidized so they didn t allow for the reaction to happen. c. Although most foods contain catalase, meats (chicken) generally have more protein and we know that enzymes are proteins. d. All foods contain equal amounts of catalase. 8. After cooking the liver in boiling water, the reaction didn t seem to work. But warming the liver slightly seemed to make the reaction go faster. WHY? a. If the enzyme gets too hot, it denatures (breaks) & will no longer work. b. Enzymes ONLY work at room temperature. c. Enzymes make reactions go faster when they re exactly 32ºF. 9. Changing the ph of an enzyme can affect its effectiveness. How could we refine our experiment to determine the OPTIMAL ph? a. We could have tested one solution with lemon juice, one solution with bleach, and one solution with vinegar. b. We could have had several solutions ranging from 1-14 and chosen the one that bubbled the most. c. We could have heated the acidic solutions. Page 6