The Hydrogen Peroxide Breakdown

Transcription

1 Biology The Hydrogen Peroxide Breakdown Examining Factors That Affect the Reaction Rate of Enzymes MATERIALS AND RESOURCES EACH GROUP aprons 2 beakers, 50 ml calculator, graphing forceps goggles graduated cylinder, 10 ml LabQuest paper towels sensor, gas pressure test tube rack catalase solution filter paper disks hydrogen peroxide, 3% TEACHER 2 beakers, 600 ml blender graduated cylinders, 100 ml hot plate ice and ice chest test tube brushes wood splints filter paper hole punch, single pipettes, thin stem scissors hydrochloric acid, 6 M beef liver matches potato water, distilled ABOUT THIS LESSON This lesson provides students with the opportunity to investigate factors that affect enzymatic reaction rates. The students manipulate both temperature and enzyme concentration to measure how these factors affect the rate at which catalase is able to facilitate the breakdown of hydrogen peroxide. OBJECTIVES Students will: Investigate how temperature and concentration affect enzymatic reaction rates through experimentation Collect data and analyze the data to formulate reasonable conclusions Determine another variable to test, and use the guided procedure to test this variable LEVEL Biology T E A C H E R P A G E S i

2 NEXT GENERATION SCIENCE STANDARDS COMMON CORE STATE STANDARDS (LITERACY) RST PLANNING/CARRYING OUT INVESTIGATIONS ANALYZING AND INTERPRETING DATA Follow precisely a multistep procedure when carrying out experiments, taking measurements, or performing technical tasks, attending to special cases or exceptions defined in the text. (LITERACY) RST CAUSE AND EFFECT STRUCTURE AND FUNCTION SCALE, PROPORTION, AND QUANTITY Translate quantitative or technical information expressed in words in a text into visual form (e.g., a table or chart) and translate information expressed visually or mathematically (e.g., in an equation) into words. ASSESSMENTS LS1: STRUCTURES AND PROCESSES CONNECTIONS TO AP* AP BIOLOGY 4 B.1 Interactions between molecules affect their structure and function. *Advanced Placement and AP are registered trademarks of the College Entrance Examination Board. The College Board was not involved in the production of this product. The following types of formative assessments are embedded in this lesson: Assessment of prior knowledge (Pre-Lab Exercises) Sharing class data The following assessments are located on our website: Chemistry of Life Assessment 2005 Biology Posttest, Free Response Question Biology Posttest, Free Response Question 1 T E A C H E R P A G E S ACKNOWLEDGEMENTS EasyData, Graphical Analysis, LabPro, LabQuest, and Logger Pro 3 used with permission, Vernier Software & Technology. ii

3 TEACHING SUGGESTIONS Students must possess a fundamental understanding of chemical reactions, organic molecules, and protein structure prior to performing this laboratory. For students in need of remediation, the middle grades lesson that covers biomolecules ( Biochemistry ) could be used as a homework assignment. Alternately, the lesson Teaching Strategy for Enzymes is a great introductory activity specifically about enzymes for the entire class. If you need to raise the rigor for students who can be pushed further than the parameters of this lab, you may try the following suggestions: You can lead them through Part I (the effect of enzyme concentration) and make them design their own experiment to test the effect of temperature. You can have them design an experiment using another enzyme, such as amylase in their saliva, which begins the breakdown of starch in the foods that we ingest. If the necessary technology is unavailable, the lesson Enzyme Activity is a wonderful substitution. The following demonstrations can be used to build student understanding of the fundamental concepts of enzyme activity and how it is affected by temperature and ph. To demonstrate the presence of catalase in a variety of tissues, add a few drops of hydrogen peroxide to a slice of potato and a slice of liver. Explain that the bubbles are created as the catalase decomposes hydrogen peroxide into water and oxygen. Using a transfer pipette, add several drops of the liquid from the liver to a 100 ml graduated cylinder containing 50 ml of hydrogen peroxide. This liquid contains blood and interstitial fluid from the liver. The solution will bubble as oxygen gas is released. Demonstrate that oxygen is the gas released by inserting a glowing splint into the oxygen-rich bubbles, which will cause the splint to reignite. To demonstrate that catalase, a protein, is denatured by a significant increase in temperature, heat 5 ml of the blood/interstitial liquid in a microwave for 10 to 30 seconds, then add several drops of this heated fluid to 50 ml of hydrogen peroxide. The students should notice that the liquid appears to be different and produces no response when added to the hydrogen peroxide. Explain that the damage caused during heating destroys the catalase enzyme, rendering it ineffective. To demonstrate that catalase, a protein, is denatured by changes in ph, add 5 ml of the blood/interstitial liquid to 5 ml of 6 M HCl. Students should notice that liquid appears to be different and actually resembles the liquid after it has been cooked. The solution should not bubble. Explain that the lack of bubbles is due to the fact that the acid damages the structure of catalase. T E A C H E R P A G E S iii

4 TEACHING SUGGESTIONS (CONTINUED) The hydrogen peroxide used in this lab is standard 3% hydrogen peroxide available at local pharmacies and variety stores. Part II of the lab utilizes hydrogen peroxide at 10 C and 35 C. Use a large beaker with ice to cool the hydrogen peroxide down to approximately 10 C and another large beaker with warm water to keep the warm hydrogen peroxide near 35 C. To prepare the catalase solution, place a small amount of the fluid found around the liver (blood and interstitial fluid) into a beaker of distilled water. You want the resulting solution to be a very light pink. The resulting catalase solution will last several hours on ice. However, it is recommended that a fresh batch of catalase be made for afternoon classes if the original solution was prepared early in the day. The reaction chambers are 80 ml test tubes (3 cm 20 cm). Newer pressure probes come with a custom stopper that can be used for this lab. If necessary, stoppers can be made by inserting the glass tubing of an eyedropper through the hole of a singlehole #4 stopper. Be sure to use glycerin to lubricate the glass and paper towels or other hand protection when inserting the glass through the stopper. The glass tube should be inserted so that the pointed end points out of the top of the stopper. In Part III, students are asked to design a plan for collecting data to determine another factor that affects the catalysis rate. There are several factors that affect the catalysis rate besides temperature and enzyme concentration. These include but are not limited to substrate concentration, ph, and the presence of inhibitors (hydroxylamine hydrochloride, a competitive inhibitor, works well). T E A C H E R P A G E S iv

5 PRE-LAB EXERCISES 1. List four characteristics of enzymes. Enzymes are proteins. Enzymes are biological catalysts. Enzymes can be used over and over again. Enzymes react with specific reactants, or substrates. HYPOTHESES As the enzyme concentration increases the enzyme activity will increase, thus producing more oxygen gas. As the temperature of the enzyme increases, the enzyme activity will increase. 2. What is an active site, and what is its relationship to an enzyme? An active site is the actual part of the enzyme involved in binding with the reactants to cause the chemical reaction to take place. 3. What is the energy of activation? The energy of activation is the energy that must be overcome to start a chemical reaction by breaking chemical bonds. 4. How does the enzyme affect the energy of activation? Enzymes lower the energy of activation needed for the chemical reaction to occur. 5. What are some factors that can affect the reaction rate of an enzymatic reaction? Answers may vary. Possible factors include but are not limited to: Temperature Concentration of enzyme Concentration of substrate ph Presence of inhibitors A N S W E R K E Y v

6 DATA AND OBSERVATIONS Table 1. Enzymatic Reaction Part Experiment Pressure (mmhg) Minimum Maximum Total Change 4 disks I Concentration 3 disks disks disk Room II Temperature Cold Warm PART III: INQUIRY EXPERIMENT A. Question The students get to choose a variable that they want to test. Some possibilities include: More varied temperatures, including a temperature high enough to denature the enzyme More varied concentrations Substrate concentration ph Presence of inhibitors B. Hypothesis Answers may vary depending on the variable chosen. The students must state what effect the variable will have on the enzyme catalysis rate. C. Methodology Answers may vary depending on the variable chosen. The students must list the steps that they took to perform their experiment. D. Results Answers may vary depending on the variable chosen. The students must have both data table and graph attached to this part. E. Conclusion Answers may vary depending on the variable chosen and how students chose to communicate their results to the class. The students must state whether or not their data supported their hypothesis. They also must use actual data from their experiment to justify their conclusion, with explanation. The students must present their findings to their peers. Each group s presentation will then undergo peer review where their peers will ask the group questions and offer constructive criticism. A N S W E R K E Y vi

7 CONCLUSION QUESTIONS 1. Identify the relationship between pressure and time in these experiments. As time passes more oxygen is released, therefore increasing the pressure in the test tube. 2. Analyzing your data, determine the relationship between temperature and enzyme activity. Justify your answer with evidence from your data. Answers may vary. As temperature increases, so does enzyme activity. In the cold temperature, the pressure only increased 129 mmhg. However, the enzyme activity was greater at room temperature (an increase of 555 mmhg) and increased even more in at warm temperature (an increase of 623 mmhg). 3. Analyzing your data, determine the relationship between enzyme concentration and enzyme activity. Justify your answer with evidence from your data. Answers may vary. With an increasing number of disks (enzyme concentration), the enzyme activity also increased. The trial with only one disk showed a pressure change of 205 mmhg. However, as the number of disks increased (2 disks, an increase of 327 mmhg; 3 disks, an increase of 463 mmhg; 4 disks, an increase of 524 mmhg) the enzyme activity increased. 4. In a cell, free energy is the portion of the cell s energy that is available to do work. Use the graph from Figure 3 to answer the following questions. a. In terms of free energy, interpret the effect that the enzyme has on the breakdown of the product. Enzymes lower the amount of free energy that reactants must invest for the reaction to proceed. b. Enzymes do not affect the amount of free energy released in this reaction. Justify this claim using evidence from the graph. Whether the reaction involves an enzyme or not, the amount of free energy lost from reactants to products remains the same. The only difference lies in the amount of activation energy required. 5. Based on your experience in this lab, construct an explanation for the difference in catalysis rates between the two lines in Figure 4. Answers may vary depending on the variable chosen. For instance, the student could interpret the graph as the results of hydrogen peroxide breakdown by two different temperatures of catalase. In this example, Line A would show the higher temperature and Line B would indicate the lower temperature. This is due to the fact that as temperature rises the energy increases, thus providing for more collisions between substrate and enzyme and causing a greater reaction rate. 6. After analysis of your experimental design and your results from Part III, describe two possible sources of error that might have occurred. For each source of error, explain how it m ight affect the results. Answers may vary. One possible source of error would be a leaky seal between the pressure probe and the test tube. This causes the total amount of pressure (i.e., the total amount of oxygen produced) to be less than the actual amount. Another source of error would be accidentally picking up two disks instead of one. This would cause twice the amount of catalase to be absorbed, and would cause the pressure to be greater than the actual amount intended. A N S W E R K E Y vii

8 Biology The Hydrogen Peroxide Breakdown Examining Factors That Affect the Reaction Rate of Enzymes MATERIALS aprons 2 beakers, 50 ml calculator, graphing forceps goggles graduated cylinder, 10 ml LabQuest paper towels sensor, gas pressure test tube rack catalase solution filter paper disks hydrogen peroxide, 3% Life is made possible by the thousands of chemical reactions that occur in an organism. Most of these chemical reactions would proceed rather slowly without the aid of enzymes. Enzymes are protein catalysts that speed up chemical reactions in a cell. Catalysts are not changed by the reactions they control, and are not used up during the reaction. Therefore, enzymes can be used over and over again. Enzymes are large complex proteins made by the cell that allow chemical reactions to take place at the temperature of the cell. These catalysts are needed in only very small amounts because a single enzyme molecule can complete the same reaction thousands of times a minute. Each enzyme is very specific and can only catalyze a certain reaction. The specific reaction catalyzed by an enzyme depends on the molecular structure and shape of a small area of the enzyme s surface called the active site. The active site can attract and hold only specific target molecules. The target molecule that the enzyme attracts and acts upon is called the substrate. The substrate and the active site of the molecule must fit together very closely (Figure 1). Sometimes the enzyme changes its shape slightly to bring about the necessary fit. This is called the Induced-Fit Hypothesis. Figure 1. Synthesis reaction Substrates + Product Active Site Enzyme Enzyme-Substrate Complex Enzyme 1

9 A chemical reaction requires both the breaking of bonds and the formation of bonds. The initial energy that must be absorbed to break the bonds of the reactant molecules is called the energy of activation (Figure 2). Figure 2. Energy of activation Energy of Activation Free Energy Reactants A+B Products C+D Progress of Reaction Enzymes work by lowering the energy of activation. Consider the role of an enzymatic catalyst in the breakdown of the common household disinfectant hydrogen peroxide, H 2 O 2, which naturally decomposes to form water, H 2 O, and oxygen gas, O 2 (Equation 1). 2H 2 O 2 2H 2 O + O 2 (Eq. 1) Although this is a catabolic reaction (one that breaks down more complex molecules into smaller ones), the rate at which it occurs is slow. Light and temperature increase the rate at which hydrogen peroxide decomposes. As a result, hydrogen peroxide sold at the drug store is packaged in light-blocking brown bottles, and has instructions for storage in a cool dark place. Hydrogen peroxide bottles are marked with an expiration date because even with cool and dark storage, the breakdown of hydrogen peroxide molecules will still occur to some degree. Hydrogen peroxide is toxic to cells, making it useful for treating open wounds as it kills invading bacterial cells. Interestingly, hydrogen peroxide is a byproduct of some biochemical pathways found in many cells, yet the accumulation of hydrogen peroxide can kill a cell. Therefore, cells cannot wait for hydrogen peroxide to naturally decompose because that takes too much time. 2

10 Most tissues produce the enzyme catalase to increase the rate of hydrogen peroxide s decomposition. Catalase lowers the energy of activation needed for the decomposition and as a result, more molecules can be decomposed in a shorter amount of time. Figure 3. Energy of activation with enzyme Energy of Activation without Enzyme Free Energy Reactants A+B Energy of Activation with Enzyme Products C+D Progress of Reaction There are many factors that can affect how fast an enzymatic reaction occurs. These include but are not limited to temperature, ph, enzyme concentration, and substrate concentration. This activity will explore the role of catalase in the decomposition of hydrogen peroxide. Because the reaction produces the gaseous product O 2, the rate of this reaction can be monitored using a gas pressure probe. The more the reaction occurs, the more oxygen gas will be produced and the greater the resulting gas pressure inside the reaction vessel. PURPOSE The purpose of this laboratory exercise is to determine hydrogen peroxide s decomposition rate when either the concentration of catalase or the temperature is varied. You will then choose another variable and use the techniques learned in this lesson to design and carry out an experiment to test it. 3

11 SAFETY ALERT!» Goggles should be worn during this lab.» Take care not to spill any liquids on the data collection equipment or electrical outlets. PROCEDURE Note: You will complete either Part I or Part II, and then share your results with another group BEFORE YOU BEGIN 1. Answer the Pre-Lab Exercises on your student answer page. 2. Formulate two hypotheses: The first hypothesis should predict the effect of enzyme concentration on the reaction rate, and the second hypothesis should predict the effect of temperature on the enzyme reaction rate. Record these on your student answer page. 3. Connect the gas pressure sensor to your data collection device. There should be a piece of plastic tubing from the pressure probe. Some versions of pressure probes have valves for opening and closing the sensor. If the pressure probe has such a valve, ensure that it is open according to the manufacturer s instructions. All secondary openings should be closed. 4. Turn on your device. It should automatically recognize the pressure sensor. Make sure that the pressure probe is reading in mmhg. The probe should be reading between 730 mmhg and 790 mmhg. 5. Pour a small amount of the catalase solution into the 50 ml beaker. Keep this catalase beaker on ice. 4

12 PROCEDURE (CONTINUED) CAUTION! Pressure is building in the test tube. During the course of this experiment, keep your thumb on the stopper to prevent it from popping off as the pressure builds. PART I: EFFECT OF CONCENTRATION Complete Step 1 through Step 6 four times with different numbers of disks to test the effect of enzyme concentration on the reaction rate. Each time, reduce the number of disks using three disks, then two disks, then one disk, respectively. Be sure to wash out the test tube thoroughly between trials, making sure all the catalase is removed. 1. Place the test tube into a test tube rack. Measure 10 ml of hydrogen peroxide with a graduated cylinder and add it to the test tube. 2. Obtain some filter paper disks and separate any disks that may be stuck together. Using the forceps, dip one disk into the catalase solution. Drain the disk against the wall of the beaker several times to remove the excess solution. Use forceps to transfer the disk to the test tube and place it on the interior wall of the test tube somewhere above the hydrogen peroxide solution. The moist disk should stick to the wall of the test tube. Using the forceps, carefully push the disk one half to one third of the way down the wall of the test tube, being careful not to yet allow the disk to contact the hydrogen peroxide. Repeat this procedure until you have four disks stuck to the inside wall of the test tube. 3. Place the stopper connected to the pressure probe on the test tube. Insert the stopper securely to ensure a tight seal. Make sure the valve is closed. 4. Set your device to take a pressure reading every 3 seconds for 5 minutes. Start collecting data. 5. Immediately after activating your data collection device, tip the test tube slightly on its side so the hydrogen peroxide solution touches the disks and the reaction begins. Return the test tube to an upright position. 6. When the experiment is complete, a graph should appear on your data collection device. This constitutes your first run. The graph should display the minimum or initial pressure and the maximum or final pressure. Record this information in the data table on your student answer page. 7. Clean up your area and return your equipment to its original condition. Turn off your data collection device. 8. Construct a graphical representation of your data. Share your data with other lab groups as instructed by your teacher. 5

13 PROCEDURE (CONTINUED) CAUTION! Pressure is building in the test tube. During the course of this experiment, keep your thumb on the stopper to prevent it from popping off as the pressure builds. PART II: EFFECT OF TEMPERATURE Complete Step 1 through Step 6 three times at different temperatures of reaction. Use room-temperature hydrogen peroxide for your first trial, 10 C hydrogen peroxide for your second trial, and 35 C hydrogen peroxide for your third trial. Be sure to wash out the test tube thoroughly between trials, making sure all the catalase is removed. 1. Place the test tube into a test tube rack. Measure 10 ml of hydrogen peroxide with a graduated cylinder and add it to the test tube. 2. Obtain some filter paper disks and separate any disks that may be stuck together. Using the forceps, dip one disk into the catalase solution. Drain the disk against the wall of the beaker several times to remove the excess solution. Use forceps to transfer the disk to the test tube and place it on the interior wall of the test tube somewhere above the hydrogen peroxide solution. The moist disk should stick to the wall of the test tube. Using the forceps, carefully push the disk one half to one third of the way down the wall of the test tube, being careful not to yet allow the disk to contact the hydrogen peroxide. 3. Place the stopper connected to the pressure probe on the test tube. Insert the stopper securely to ensure a tight seal. Make sure the valve is closed. 4. Set your device to take a pressure reading every 3 seconds for 5 minutes. Start collecting data. 5. Immediately after activating your data collection device, tip the test tube slightly on its side so the hydrogen peroxide solution touches the disk and the reaction begins. Return the test tube to an upright position. 6. When the experiment is complete, a graph should appear on your data collection device. This constitutes your first run. The graph should display the minimum or initial pressure and the maximum or final pressure. Record this information in the data table on your student answer page. 7. Clean up your area and return your equipment to its original condition. Turn off your data collection device. 8. Construct a graphical representation of your data. Share your data with other lab groups as instructed by your teacher. 6

14 PROCEDURE (CONTINUED) PART III: NOW IT S YOUR TURN 1. Choose another variable to test. Make sure that your group chooses a variable that is testable. Record the variable to be tested in the appropriate section of your student answer page. 2. Formulate and record your hypothesis. 3. Use techniques learned in this lesson to design an experiment to test the hypothesis. Write the detailed plan on your student answer page. 4. Create a representation for your experimental data, and then collect your data. Record your results on your student answer page. 5. Evaluate your data to support or reject your hypothesis. Record this information. 6. Communicate the results of your experiment to the class. This could be in the form of a poster, graphic slides, a movie, and so on. Remember to include the following parts of your process in your presentation: Question, Hypothesis, Methodology, Results, and Conclusion. 7

15 PRE-LAB EXERCISES 1. List four characteristics of enzymes. 2. What is an active site, and what is its relationship to an enzyme? 3. What is the energy of activation? 4. How does the enzyme affect the energy of activation? 5. What are some factors that can affect the reaction rate of an enzymatic reaction? 8

16 HYPOTHESES DATA AND OBSERVATIONS Table 1. Enzymatic Reaction Part Experiment Pressure (mmhg) Minimum Maximum Total Change 4 disks I Concentration 3 disks 2 disks 1 disk Room II Temperature Cold Warm 9

17 DATA AND OBSERVATIONS (CONTINUED) GRAPH For this graph, you will need to determine the following: The independent variable: The dependent variable: Use this value to label the horizontal x-axis. Use this value to label the vertical y-axis. Graph: (title) 10

18 DATA AND OBSERVATIONS (CONTINUED) PART III: INQUIRY EXPERIMENT A. Question B. Hypothesis C. Methodology D. Results E. Conclusion 11

19 CONCLUSION QUESTIONS 1. Identify the relationship between pressure and time in these experiments. 2. Analyzing your data, determine the relationship between temperature and enzyme activity. Justify your answer with evidence from your data. 3. Analyzing your data, determine the relationship between enzyme concentration and enzyme activity. Justify your answer with evidence from your data. 4. In a cell, free energy is the portion of the cell s energy that is available to do work. Use the graph from Figure 3 to answer the following questions. a. In terms of free energy, interpret the effect that the enzyme has on the breakdown of the product. b. Enzymes do not affect the amount of free energy released in this reaction. Justify this claim using evidence from the graph. 12

20 CONCLUSION QUESTIONS (CONTINUED) 5. Based on your experience in this lab, construct an explanation for the difference in catalysis rates between the two lines in Figure 4. Figure 4. Catalysis rates A Reaction Rate B Oxygen Concentration 6. After analysis of your experimental design and your results from Part III, describe two possible sources of error that might have occurred. For each source of error, explain how it m ight affect the results. 13