A.  Learning Objectives

In this lab, students will:

• use a respiration chamber to assess the rate of cellular respiration of germinating peas.

• design and conduct an experiment to test the role of enzymes in cellular respiration.

• determine the fermentation rate of different sugars by yeast.

B. Textbook Correlation: 

Please review Sections 7.1 and 7.2 of Chapter 7: Cellular Respiration, Fermentation, and Secondary Metabolism when preparing for the lab.

C.  Introduction

Cellular respiration is the method that cells use to get energy from food molecules and release waste products.  The three major stages of respiration are glycolysis, the Krebs cycle, and the electron transport chain. Glycolysis occurs in both aerobic and anaerobic respiration and causes a glucose molecule to be broken down into 2 pyruvate molecules. This process produces a net gain of 2 ATP and 2 NADH molecules and occurs in the cytosol. In between glycolysis and the Krebs cycle, pyruvate is broken down into an acetyl group after entering thee mitochondrial matrix, producing a CO₂ molecule and an NADH molecule in the process. The citric acid, or Krebs, cycle occurs in the mitochondrial matrix. In this stage, each acetyl group is incorporated into an organic molecule, liberating 2 CO₂ molecules. 1 ATP, 3 NADH, and one FADH₂ are are produced per acetyl group as well. Oxidative phosphorylation is the final stage, and involves two components: the electron transport chain and ATP synthase. The energy stored in the NADH and FADH₂ from the previous steps is used to create an electrochemical gradient of H+ that is used to synthesize ATP from ADP and Pi. In eukaryotes, oxidative phosphorylation occurs along the cristae of the mitochondrial membrane. The electron transport chain is composed of a group of proteins complexes and organic molecules that embedded in the inner mitochondrial membrane. They pass electrons from one component to the next in a series of redox reactions. NADH and FADH₂ are energy intermediates that donate their electrons at different points in the electron transport chain. The final electron acceptor in the chain is O₂. The energy released from the movement of the electrons is used to pump H+ across the membrane, creating a H+ electrochemical gradient.  The passive flow of H+ across back into the matrix is an exergonic process, as they pass through ATP synthase. ATP synthase harnesses some of the free energy release from the flow of H+ to synthesize ATP. The metabolic pathways for carbohydrate metabolism are also connected to the pathway for amino acid and fat metabolism. Proteins and fats can enter into glycolysis or the citric acid cycle at different points. Proteins are first broken down into amino acids by enzymes, then either enter at later steps of glycolysis or become attached to CoA after an acetyl group is removed. Fats can also be broken down into glycerol and fatty acids. Cellular metabolism is efficient because the same enzymes can be used to breakdown different starting molecules.

Oxidative Phosphorylation: Includes the Electron Transport Chain and ATP Synthase http://www.doctortee.com/dsu/tiftickjian/bio100/respiration.html	How Proteins, Carbohydrates, and Fats are Catabolized http://biology-forums.com/index.php?action=gallery;sa=view;id=339

Cells commonly metabolize organic molecules in the absence of oxygen. Some organisms live in environments where oxygen is not present. Similarly, when someone exercises under extreme exertion, the rate of their oxygen consumption by muscle cells may exceed the rate of oxygen delivery. Organisms have evolved different strategies to metabolize organic molecules in the absence of oxygen. One approach is to use a substance other than O2 as the final electron acceptor of an electron transport chain. This process is called anaerobic respiration. Another strategy is to produce ATP only through substrate-level phosphorylation. Animals, yeast and many other organisms can use only O2 as the final electron acceptor of their electron transport chains. Under anaerobic conditions, these organisms must find a different way to produce adequate ATP. Glycolysis is one method of making ATP, as it can occur under anaerobic or aerobic conditions. A key problem is that glycolysis requires NAD+. In aerobic respiration, the electron transport chain turns NADH back to NAD+ with the aid of oxygen, thus avoiding any NAD+ shortage and letting glycolysis to take place. In anaerobic respiration, cells have to find another way to turn NADH back to NAD+. Anaerobic muscle cells reduce pyruvate from glycolysis and use it to make lactate. The electrons to reduce pyruvate come from NADH, which is oxidized to NAD+. The decrease of NADH prevents or at least decreases the potential of its harmful effects. At high NADH concentrations, NADH will chaotically donate its electrons to other molecules and promote the formation of free radicals. Also, from the reduction of pyruvate the level of NAD+ increases, which allows glycolysis to continue. Once oxygen is restored, the lactate created during strenuous exercise can be taken up by cells, converted back to pyruvate, and used for energy. Yeast cells cope with anaerobic conditions in a different manner. Yeast cells metabolize sugar under anaerobic conditions during wine making. Pyruvate is broken down to CO2 and acetaldehyde, a two carbon molecule. Then, acetaldehyde is reduced to make ethanol while NADH is oxidized to NAD+. This also decreases NADH and increases NAD+. Fermentation is the breakdown of organic molecules to harness energy without any net oxidation. Examples are the breakdown of glucose to lactate or ethanol. Electrons are removed from the organic molecule (such as glucose) to produce pyruvate and NADH, but the electrons are donated back during production of lactate or ethanol. This means there is no net removal of electrons from an organic molecule. Fermentation produces far less ATP than oxidative phosphorylation. This is because glucose is not oxidized completely to CO2 and water and the NADH made during glycolysis cannot be used to make more ATP. The complete breakdown of glucose in the presence of oxygen yields 34-38 ATP molecules. The anaerobic breakdown of glucose to lactate or ethanol yields only 2 ATP molecules. 

Ethanol fermentation. http://www.bio.davidson.edu/Courses/Molbio/ MolStudents/spring2010/Ghant/Fermentation2.jpg	Lactic acid fermentation. http://schoolworkhelper.net/wp-content/uploads/2010/07/Lactic-Acid-Fermentation.jpg

The glycolytic pathway is comprised of 10 enzyme catalyzed steps. The different enzymes involved in glycolysis act as kinases, mutases, dehydrogenases, cleaving enzymes, isomerases or enolases. They split/rearrange the intermediates, are added onto phosphate groups, and move those phosphate groups onto ADP to make ATP. The first step in glycolysis involves the enzyme hexokinase.  In mammals, there are four isozymes of hexokinase: types I, II, III and IV (glucokinase).  These isozymes vary in their catalysis, localisation and regulation, thus contributing to the diverse patterns of glucose metabolism in different tissues.  Type I, II and III hexokinases are able to phosphorylate a variety of hexose sugars, including glucose, fructose and mannose, and as such are involved in a number of metabolic pathways.  It is thought that type I hexokinase might have a catabolic function, producing G6P for energy production in glycolysis, while types II and III may have an anabolic function, providing G6P for glycogen or for lipid synthesis.  Type I hexokinase binds to the mitochondrial membrane, enabling the coordination of the rate of glycolysis with that of the TCA cycle.  Type IV hexokinase (glucokinase) is a liver/pancreatic b-cell enzyme that is specific for a-D-glucose, and whose level is controlled by insulin, and not G6P.  Thanks to the lack of inhibition by G6P, during times of high blood glucose levels the liver can accumulate G6P, converting it to glycogen for usage later.  In pancreatic b cells, type IV hexokinase acts as a glucose sensor to modify insulin secretion.  Mutations in type IV hexokinase have been associated with diabetes mellitus. The enzyme used in step 2 is phosphoglucose isomerase. Phosphoglucose isomerase , or PGI, catalyzes the inter-conversion of G6P and F6P during glycolysis and gluconeogenesis.  The shift of the carbonyl oxygen from the C1 position in G6P to the C2 position in F6P is needed in order to add another phosphate group at the C1 position in a later reaction. The enzyme used in step 3 is phosphofructokinase. PFK is an inducible, extremely regulated, allosteric enzyme that is a key regulator of glycolysis.  PFK is activated by AMP, ADP, Pi, and fructose-2, 6-bisphosphate (F2,6PP), and is inhibited by ATP, citrate, and H+. The enzyme in step 4 is fructose-bisphosphate aldolase. Fructose-bisphosphate aldolase catalyzes the reversible cleavage of F1,6PP to two triose phosphates, both of which continue through glycolysis. Next, in step 5, is triosephosphate isomerase. Triosephosphate isomerase (TIM) catalyses the reversible interconversion of G3P and DHAP.  Only G3P can be used in glycolysis, therefore TIM is essential for energy production, allowing two molecules of G3P to be produced for every glucose molecule, doubling the energy yield. Step 6 uses the enzyme glyceraldehyde 3-phosphate dehydrogenase. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis by reversibly catalyzing the oxidation and phosphorylation of G3P to the energy-rich intermediate 1,3BPG.  NAD+ is a co-substrate for this reaction. Step 7 uses the enzyme phosphoglycerate kinase. Phosphoglycerate kinase (PGK) reversibly catalyzes the formation of ATP to ADP, using one of the high-energy phosphate groups from 1,3BPG.  The reaction forms two ATP molecules per glucose (one per 1,3BPG molecule), which reimburses the spending of 2 ATP in phase I of glycolysis.  The ATP is made by substrate-level phosphorylation, where a phosphate group is transferred from 1,3BPG directly to ADP.  This reaction is vital in most cells for the generation of ATP in aerobes, for fermentation in anaerobes and for carbon fixation in plants. The enzyme used in step 8 phosphoglycerate mutase. Phosphoglycerate mutase (PGAM) catalyzes the transfer of the phospho group from the C3 position to the C2 position, in preparation for the synthesis of ATP.  PGAM enzymes from different sources exhibit different reaction mechanisms. Step 9 uses enolase. Enolase (phosphopyruvate hydratase) is a crucial glycolytic enzyme that catalyzes the reversible dehydration of 2-phosphoglycerate to the high-energy intermediate phosphoenolpyruvate.  Enolase is strongly inhibited by fluoride ions, which forms a fluorophosphate complex with magnesium at the active site. Step 10 uses pyruvate kinase. The enzyme pyruvate kinase (PK) catalyses the final step in glycolysis, the conversion of PEP to pyruvate with the concomitant transfer of the high-energy phosphate group from PEP to ADP, thereby generating ATP.  PK requires both magnesium and potassium for activity.  

Kinase is an enzyme that adds phosphate groups (PO43−) to other molecules. A huge number of kinases exist—the human genome comprises at least 500 kinase-encoding genes. Metabolism of dietary sugars, glycolysis, involves a few steps of phosphorylation by distinct kinases. These phosphate groups are ultimately used to make ATP. In glycolysis, dehydrogenase is the glycolytic enzyme responsible for catalyzing the reaction that changes acetaldehyde to ethanol in the alcoholic fermentation of pyruvate.

Cells control glycolysis by feedback inhibition when it has an appropriate amount of ATP. At high concentrations, ATP binds to an allosteric site in phosphofructokinase, which catalyzes the third step in glycolysis. This is the step thought to be rate limiting. When ATP binds to the site, a conformation change happens and the enzyme is rendered inactive. This prevents the further breakdown of glucose and averts overproduction of glucose.

In these experiments, you will first learn how to measure the respiration rate of germinated peas using a respiration chamber. Then you will be able to design and conduct an experiment to investigate the role of enzymes on pea respiration rate. Although glucose is the preferred carbohydrate for glycolysis and fermentation, you will perform an experiment to measure the fermentation rate of sugars other than glucose by yeast.

D. Respiration

When a seed germinates into a seedling, energy is required.  To meet this energy requirement, seeds store carbohydrates as a source of energy.  These carbohydrates are catabolized and the energy released drives the production of ATP.  

In this experiment you are going to measure the respiration rate of germinating peas by measuring the amount of CO₂ they produce. Refer back to the cellular respiration equation—as glucose is broken down, carbon dioxide is produced. Each group will perform the experiment on germinating peas and non-germinating peas. 

Hypothesis:  Which set of peas will demonstrate the highest rate of cellular respiration?  Explain.

The germinated peas will demonstrate the highest rate of cellular respiration. A germinating seed will require more energy than a non-germinating seed because it is growing. Thus, it will have a higher rate of cellular respiration as it obtains the energy it needs from the carbohydrates that it has stored.

Procedure:

Part I:  Assembly and Incubation

1. On your bench, there are two bottles labeled germinated and ungerminated.   Fit the rubber stopper with the attached glass tubes into the respiration bottles.

2. Submerge the rubber tubing into the water-filled test tubes at your bench.

3.  Insert the rubber stoppers and take note of the time. 

4.  Incubate for 60 minutes.

Part II:  Measuring the rate of respiration

As stated earlier, we are measuring the rate of respiration as a function of CO₂ gas production.  It is difficult to measure the volume of gas produced because it is colorless and odorless.  To overcome this obstacle, we will bubble CO₂ though water, which produces carbonic acid (H₂CO₃).  We will then use a pH indicator to directly measure the amount of H₂CO₃ in our jars.  In this manner, we will indirectly measure the amount of CO₂ produced in these reactions. 

5.  Replace the water in each test tube with phenol red.  Re-submerge the rubber tubing below the surface of the liquid. 

6.  Remove the rubber stopper from the top of the jar and slowly pour water into the jar.  The carbonic acid will be displaced and move through the rubber tubing into the tube filled with phenol red.  A yellow color is an indication of acidic conditions.  Please obtain video of this step and narrate what is happening in each tube.

E.  Effect of enzymes on the rate of respiration:

The chemical reactions of respiration are controlled by enzymes.  Therefore, the respiration rate in germinating peas can be influenced by environmental factors like temperature. In this exercise you will design and conduct an experiment to test the impact of enzymes on the rate of respiration.  We have provided the materials necessary:  another jar of peas, a beaker of water with boiling chips, a hot plate, and another experimental apparatus.  Design your experiment before attending the lab.

1. Hypothesis: The boiled peas will be unable to carry out cellular respiration.

2. Experimental design:

1. Prepare a boiling hot water bath, then submerge the seeds, keeping them in the boiling water for approximately 15 minutes.

2. Using the germinated seeds, fit the rubber stopper with the attached glass tubes into the respiration bottles.

3. Submerge the rubber tubing into the water-filled test tubes at your bench.

4.  Insert the rubber stoppers.

5.  Incubate for 60 minutes.

6.  Replace the water in each test tube with phenol red.  Re-submerge the rubber tubing below the surface of the liquid. 

7.  Remove the rubber stopper from the top of the jar and slowly pour water into the jar.  The carbonic acid will be displaced and move through the rubber tubing into the tube filled with phenol red.  A yellow color is an indication of acidic conditions.  Please obtain video of this step and narrate what is happening in each tube.

3. Results (Video of bubbling of water)

F.  Fermentation

During fermentation, yeast produce ethanol and CO₂. While glucose is the preferred substrate, other sugars can also be fermented by yeast. In this experiment you will determine yeast’s relative fermentation rates of different sugars. The rate of fermentation can be measured by capturing and measuring the CO₂ as it is produced by the yeast.

Procedure

1.  On your bench, there are four beakers each containing a 15 mL sugar solution: 

            Beaker 1:  Glucose

            Beaker 2:  Glucose

            Beaker 3:  Fructose

            Beaker 4:  Lactose 

2. To beakers 1, 3, and 4 add a 0.5g piece of fresh cake yeast.  Stir with a clean glass rod.  Beaker 2 will not have yeast.

3. Add the entire content of each beaker into separate fermentation tubes called Swan tube due to the curvature of the neck.  Cover the opening of the tube with your thumb and invert each tube so the long tail is filled with solution.

4. Incubate the tubes at 37°C for one hour. 

5. Record the size of the CO₂ bubble in mm every 30 minutes.  Accompany each measurement with a photo of each tube.

Presentation:

You will record two presentations for this lab following the existing format (Introduction, Experimental Design/Execution, Results, Conclusions)

     1.  Cellular Respiration (Sections D and E).

     2.  Fermentation (Section F).