Glycolysis: Acetyl-CoA synthesis (Intermediate Step) and Krebs Cycle Electron Transport Chain (ETC):

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1 Name: Bio AP Lab: Cell Respiration (Modified from Carolina Cell Respiration & AP Biology Investigative Labs) BACKGROUND: You are probably familiar with photosynthesis, the process that plants use to harness energy from the sun. But how do plants acquire energy when they germinate underground, out of the reach of sunlight? They metabolize sugar much like humans do, through cellular respiration. All cells need energy, Energy is contained in the molecular structure of organic compounds such as carbohydrates, proteins, and fats. Carbohydrates are the primary source of cellular energy. When the bonds of a carbohydrate molecule are broken (in a series of small steps, with the help of specific enzymes), energy is released from the bonds, The energy, stored in a molecule of adenosine triphosphate (ATP), can then be used by the cell. ATP is the chief energy source of cells. It stores energy in the structure of its three-phosphate tail. The removal of a phosphate from the ATP molecules releases energy that powers almost all metabolic processes. When phosphate is removed, adenosine triphosphate becomes adenosine diphosphate (ADP). Through cell respiration, fermentation, and other metabolic processes, there is a constant cycling between ATP and ADP. In this lab, we will focus on aerobic cellular respiration. The series of reactions that occurs during aerobic respiration is grouped into steps celled glycolysis, Acetyl CoA synthesis (intermediate step), Krebs cycle, and the electron transport chain (ETC). The breakdown of the sugar molecule is summarized by the following equation: C 6 H 12 O 6 + 6O 2 6CO 2 + 6H 2 O + 36 ATP Glycolysis: Glycolysis is a series of reaction that splits a 6-carbon glucose molecule into two 3-carbon molecules called pyruvate. The process occurs in the cytoplasm of the cell. As glucose is broken down, four ATP s and two NADH molecules are produced. Because two ATP molecules are required to start the reaction, there is a net gain of two ATP. If oxygen is present, cells may undergo aerobic respiration to produce more energy by further breaking down the derivatives of glucose. All higher organisms and many microorganisms have the necessary enzymes to perform aerobic cellular respiration. Acetyl-CoA synthesis (Intermediate Step) and Krebs Cycle: If oxygen is present, the pyruvate made during glycolysis enters the mitochondria of the cell, where it is initially converted to acetyl-coa. As this happens, one CO 2 molecule and one NADH molecule are produced. The acetyl CoA is then broken down in a series of reactions referred to as the Krebs cycle (citric acid cycle). For each acetyl-coa broken down, two CO 2 molecules, one ATP molecule, 3 NADH molecules and one FADH2 molecule are produced. Electron Transport Chain (ETC): The NADH and FADH 2, produced during glycolysis, the intermediate step and the Krebs cycle, contain energy and are used to generate more ATP in the ETC. As the electrons from NADH and FADH 2 are passed between the protons of the ETC, energy is released and used to activate proton pumps, which shuttle the H+ ions into 1

2 the Intermembrane space of the mitochondria. As the H+ ions accumulate, the form a gradient across the membrane. The gradient creates a type of pressure that causes the H+ ions to rush through a channel in the membrane called ATP synthase. ATP synthase functions somewhat like a windmill; the rush of H+ ions spins a component of ATP synthase, which drives the production of ATP. From the aerobic respiration of each glucose molecule, approximately 36 ATP molecules are made. Oxygen: Oxygen comes into play only during the final step of the ETC. Remember that electrons are passed from protein to protein. When the electrons get to the end of the chain, they do not simply fall off. The electrons, along with the H+ ions are passed to oxygen, resulting in the formation of water. For this reason, oxygen is called the final (terminal) electron acceptor. As oxygen picks up electrons at the end of the ETC, it enables more electrons to be passed through the chain. If oxygen is not available to pick up electrons, no more electrons can enter the system from NADH and FADH 2. In the absence of oxygen, the ETC AND KREBS CYCLE process CEASE TO FUNCTION. LEARNING OBJECTIVES: To learn how a respirometer system can be used to measure respiration rates in plant seeds or small invertebrates, such as insects. To connect and apply concepts, including the relationship between structure and function (mitochondria); strategies for capture, storage, and use of free energy; diffusion of gases across cell membranes; and the physical laws pertaining to the properties and behavior of gases. To design and conduct an experiment to explore the effect of certain factors, including environmental variables, on the rate of respiration. GENERAL SAFETY: You must wear safely goggles or glasses, aprons, and gloves during this investigation because KOH (or the alternative NaOH in Drano) is caustic, Follow your teacher s directions if you use a hot glue gun. Do not work in the laboratory without your teacher s supervision. THE INVESTIGATION: During this investigation you will use respirometers to measure the rate of respiration of germinating and dormant pea seeds. The respirometer is composed of a vial that contains the peas and a volume of air; the mouth of the vial is sealed with a rubber stopper with a pipet inserted into the hole. During the experiment, the respirometer is submerged in water. If the peas respire, they will use oxygen and release carbon dioxide. Because 1 mole of carbon dioxide is released for each mole of oxygen consumed, there is NO change in the volume of gas in the respirometer, (Avogadro s Law: At constant temperature and pressure, 1 mole of any gas has the same volume as 1 mole of any other gas.) However in this procedure the carbon dioxide produced is removed by potassium hydroxide (KOH). KOH reacts with CO 2 to form the SOLID potassium carbonate (K 2 CO 3 ) through the following reaction: CO 2 + 2KOH K 2 CO 3 + H 2 O 2

3 Thus as O 2 is consumed, the overall gas volume in the respirometer decreases. The change in volume can be used to determine the rate of cellular respiration. As the volume of gas decreases, water moves into the submerged pipet. You will use this decrease of volume, as read from the scale printed on the pipet, as a measure of the rate of respiration. Because respirometers are sensitive to changes in gas volume, they are also sensitive to changes in temperature and air pressure; thus you need to use a control respirometer. As you are working through the procedures, think about this question: What factors can affect the rate of cellular respiration? In Designing and Conducting Your Investigation, you will design and conduct an experiment(s) to investigate at least one of your responses to this question or some other question you have. Your exploration will likely generate even more questions about cellular respiration. INTRODUCTION: After reading through the procedures, answer the question below before you begin the experiment. 1. Why is it necessary to correct the readings of the respirometers containing seeds with the readings taken from the respirometers containing only glass beads? Your answer should refer to the concepts derived from the Ideal Gas Law PV=nRT Where P=pressure of the gas V=volume of the gas N= number of moles of the gas R= the gas constant (its value is fixed) T= temperature of the gas (in Kelvin) MATERIALS: 3

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6 ANALYSIS OF RESULTS: 1. Use your data table to construct your graph. Your goal is to determine the respiration rate for both the germinating and the dormant seeds. Which variable should be on the x-axis and which should be on the y-axis? 2. From your graph determine the rate of respiration for the germinating and dormant seeds. Remember : Rate = yy xx 3. Imagine that you are given 25 germinating pea seeds that have been placed in boiling water for five minutes. You place these seeds in a respirometer and collect data. Predict the rate of oxygen consumption (cellular respiration) for these seeds and explain your reasons. 4. What difficulties would there be if you used a living green plant in this investigation instead of germinating seeds? 5. Imagine that you are asked to measure the rate of respiration for a 25g reptile ( cold-blooded ) and a 25g mammal ( warm-blooded ) at 10 o C. Predict how the results would compare and justify your prediction. 6. Imagine that you are asked to repeat the reptile/mammal comparison of oxygen consumption but at a temperature of 22 o C. Predict how these results would differ from the measurements made at 10 o C, and explain your prediction in terms of the metabolism of the animals. 6