Experiment 3

Experiment 3 - BC368 Biochemistry of the Cell II Experiment...

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1 BC368 – Biochemistry of the Cell II Experiment 3 Dehydrogenase Enzymes of Cauliflower Mitochondria Introduction Contained within the mitochondria is the biochemical machinery for cellular respiration, the aerobic process by which sugars, fatty acids, and amino acids are broken down to carbon dioxide and water and their chemical energy captured as adenosine triphosphate (ATP). A key series of reactions in this aerobic process is the citric acid (Krebs) cycle, a complex pathway involving many enzymes and metabolic intermediates. In this project, you will perform an in vitro analysis of the Krebs cycle with the mitochondrial fraction of whole cell lysates of cauliflower. Cauliflower tissue is ideal because of its availability, because its use eliminates the need for animal sacrifices, and because it lacks chloroplasts, simplifying the isolation of mitochondria. You will first characterize the kinetic activity of one of these enzymes, succinate dehydrogenase, which catalyzes the oxidation of succinate to fumarate. You will next check the isolated mitochondria for the presence of several other dehydrogenases of the citric acid cycle. Finally, you will expand your kinetic investigations in an experiment of your own design. Measurement of Dehydrogenase Activity In this project, you will be measuring dehydrogenase activity in isolated plant mitochondria by monitoring the reduction of an artificial electron acceptor. To use an artificial electron acceptor, the normal path of electrons in the electron transport chain must be blocked. This is accomplished by adding either potassium cyanide or sodium azide to the reaction mixture. These poisons inhibit the transfer of electrons from cytochrome a 3 to the final acceptor, oxygen, so that electrons cannot be passed along by the preceding cytochromes and coenzyme Q. Instead, they are accepted by the artificial electron acceptor 2,6-dichlorophenolindolphenol (DCPIP) . The reduction of DCPIP by E-FADH 2 or E- NADH can be measured spectrophotometrically since the oxidized form of the dye is blue and the reduced form is colorless. For example: E-FADH 2 + DCPIP oxidized E-FAD + DCPIP reduced (blue) (colorless) Thus, the change in absorbance of DCPIP, measured at 600 nm, can be used to monitor the dehydrogenase reactions. Initially, you will assay the isolated mitochondria for the presence of several dehydrogenases. You will provide the mitochondrial preparations with several substrates of specific dehydrogenases and NAD + . The activity of the dehydrogenase will be detected by simple observation of the color change of DCPIP. You will then perform a more quantitative analysis of dehydrogenase activities using the Ocean Optics spectrophotometers. Enzyme Kinetics
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Experiment 3 - BC368 Biochemistry of the Cell II Experiment...

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