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RESPIRATION AND FERMENTATION: AEROBIC AND ANAEROBIC OXIDATION OF ORGANIC MOLECULES Bio 171 – Week 6 Procedure 1) 2) 3) 4) 5) 6) Label test tubes well, including group name Add solutions listed to small test tubes For 1-6, fill remaining volume with yeast suspension; for 7 fill remaining volume with water. Slide larger test tube over the smaller tubes; hold smaller tube against the bottom of the larger tube and invert. There should be no air trapped at the top of the tube. It may help to practice this with water. Label test tubes well, including group name Incubate tubes at 37˚C for 40 minutes. After 40 minutes, measure the height (in millimeters) of the bubble of accumulated CO2. Record results in table 12.1 Procedure Wear gloves – NaF is corrosive and toxic Shake yeast bottles before using Absolutely NO eating in lab Clean benches immediately when done Rinse test tubes well and put upside-down in rack near sink. Cellular Respiration Cellular Respiration: oxidation of organic molecules into energy in the form of ATP ATP = Adenosine Triphosphate: organic molecule containing high-energy phosphate bonds Cellular Respiration - Summary C6H12O6 + 6O2 Glucose is oxidized [removes electrons], O2 is reduced (oxidation-reduction reaction or REDOX) Remember OIL RIG – Oxidized Is Loss (of electrons) and Reduction Is Gain) Remember that adding/removing a hydrogen is a way of adding/removing an electron 6CO2 + 6H20 + e- + 36-38ATP One glucose yields 36-38 ATP Electrons (as H) moved by coenzymes NAD+ and NADH2 Steps of Cellular Respiration Glycolysis Prep Reactions Krebs Cycle Electron Transport Chain Respiration: Glycolysis Energy Investment Step Two ATP used to split glucose into two 3-carbon molecules Energy-Harvesting Step 1) 3-carbon molecules oxidized by NAD+, resulting in two NADHs 2) Phosphate group added to each. 3) Substrate-level ATP synthesis or substrate-level phosphorylation: enzyme passes high-energy phosphate to ADP, and ATP results (adenosine diphosphate triphosphate (+2 ATP, 3PG (3phosphoglycerate)) 4) 3PG is oxidized by the removal of water (+2H2O, 2 PEP) 5) Substrate-level ATP synthesis again. (+2 more ATP, 2 pyruvate) NET GAIN: 2 ATP, (because we used two in energy investment) + 2 pyruvate. Respiration: Glycolysis Glycolysis: “Sugar-splitting” or “Energy investing” step Occurs in cytoplasm Requires 2ATP Glucose split into 2 Pyruvate Respiration: First Set of Reactions Glycolysis: “Sugar-splitting” or “Energy investing” step Occurs in cytoplasm Requires 2ATP Glucose split into 2 Pyruvate Final Products: 2 NET ATP (4 produced, but 2 were used) 2 NADH 2 Pyruvate If Oxygen is Present… Prep Reactions Citric Acid Cycle Pyruvate oxidation into acetyl-CoA One NADH produced Occurs in matrix of mitochondria Acetyl-CoA oxidized into two CO2 Produces 1 ATP per turn Store energy in electron carries such as NAD+ and FAD+ Electron Transport Chain Electrons from NADH and FADH2 move through a series of proteins called the ETC Potential energy released during these redox reactions creates proton gradient across a membrane; flow of protons across the membrane generates ATP Anaerobes – organisms that live without oxygen Some use nitrate, sulfate or other inorganic compounds as electron acceptors instead of oxygen. Some use glycolysis reduce the pyruvate If No Oxygen is Present… NADH reduces Pyruvate C6H12O6 2CO2 + 2C2H5OH + ATP C6H12O6 2CH3CHOHOCOOH + ATP Occurs in anaerobic organisms (anaerobes) Occurs temporarily in plants and animals Roots in anaerobic soils Animals, some In muscles for rapid bursts of energy microbes Glucose Glycolysis Pyruvate Plants, some microbes NADH NAD+ Lactate CO2 Ethanol Advantages/Disadvantages? Disadvantages: Less ATP produced (2 VS 36/38 in aerobic respiration) Produces toxins (lactic acid or ethanol) Advantages: Can produce ATP without oxygen Byproducts used in many foods (economic value) Back to the lab Pyruvate: product of glycolysis; reduced to ethanol or lactic acid during anaerobic fermentation Magnesium Sulfate (MgSO4): Provides Mg2+ , a cofactor that activates some enzymes of glycolysis Sodium fluoride (NaF) – an inhibitor of some enzymes of glycolysis – inhibits phosphorylation Glucose – a common organic molecule used as an energy source for respiration Lab Results Table 12.1: Experimental Treatments of CO2 Production During Anaerobic Fermentation Tube 3M Na Pyruvate (Activator) 0.1 M MgSO4 (Activator) 0.1 M NaF (Inhibitor) 5.0% Glucose (Activator) Water Fill With CO2 Produced After 40 Min (mm) 1 - - - - 7.5 mL Yeast Suspension 0 2 - - - 2.5 mL 5.0 mL Yeast Suspension 25 3 - 5.0 mL - 2.5 mL - Yeast Suspension 30 4 - - 0.5 mL 2.5 mL 4.5 mL Yeast Suspension 10 5 - - 5.0 mL 2.5 mL - Yeast Suspension 5 6 2.5 mL - 2.5 mL 2.5 mL - Yeast Suspension 40 7 - - - 2.5 mL 2.5 mL Water Predictions Tube 1: Water and yeast suspension. Tube 2: Glucose (activator), Water, Yeast suspension This tube had 10x as much NaF as Tube 5 Tube 6: Pyruvate, NaF (inhibitor), Glucose, Yeast suspension What happened to CO2 production here? Tube 5: NaF (inhibitor), Glucose, Yeast suspension What process does glucose activate? Tube 3: MgSO4, Glucose, and Yeast suspension. Tube 4: NaF (inhibitor), Glucose, Water, Yeast suspension. What purpose does this tube serve? What happened to CO2 production here? What process do you think NaF inhibits? Tube 7: Glucose, water. What is the purpose of this tube?
