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IntroductionIn Eukaryotic cells the mitochondria is the organelle specializing in energy extraction This extraction happens in a process referred to as cellular respiration In cellular respiration sugars fatty acids and amino acids are broken down into carbon dioxide water and cellular energy called adenosine triphosphate or ATP During cellular respiration most of the ATP is generated through electron transport in the mitochondrial matrix However there is a very important enzyme that is located outside of the mitochondrial matrix This enzyme is succinate dehydrogenase and its coenzyme flavin adenine dinucleotide or FAD These two enzymes together are responsible for the oxidation of succinate to fumarate in the Krebs cycle portion of cellular respiration as seen in Figure 1 Figure 1 Krebs Cycle Diagram httpccnmtlcolumbiaeduprojectsbiologylecture9krebshtmTo determine the rate of reaction of succinate to fumerate the reduction of an artificial electron acceptor is monitored and measured spectrophotometrically To use an artificial electron acceptor the normal path of electrons must be blocked A poison called sodium azide is used to achieve this result Sodium azide inhibits the electron transfer from cytochrome A to the final oxygen in the reaction The electrons are detoured to the artificial electron acceptor which in this case is 26 dichlorophenolindophenol or DCIP The reduction of DCIP can be measured spectrophotometrically because the oxidized form is blue and the reduced form is colorless This reaction is summarized as In this experiment the rate of reaction of succinate to fumarate is measured using mitochondrial fraction isolated from cauliflower cells ProcedureIsolation of mitochondrial fractionTo isolate the mitochondrial fraction from the cauliflower 20 grams of the outer 2-3mm of cauliflower surface was removed with a straight razor The cut cauliflower was added into a chilled mortar with 40mL of ice-cold mannitol grinding medium and 5g of cold sand The mixture was ground with a chilled pestle for approximately 4 minutes until an even mixture was made The mixture was then filtered through four layers of cheesecloth
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