The aim of this experiment was to determine the enzymatic activity of succinate dehydrogenase (SDH) in various solutions, specifically those containing and not containing a reagent. The assigned reagent was malonate, which was previously hypothesized to be an inhibitor of SDH.
Bovine liver was manipulated primarily through centrifugation, and mitochondrial fractions were then created to serve as the enzyme-rich solutions the reagent would act upon. To determine the protein concentration of the mitochondrial fraction, the Bradford assay was implemented; the result will later be used in the calculation of the specific activity of SDH.
Ultimately, the SDH assay provided the data to either support or negate the hypothesis about malonate’s effect on SDH activity. This procedure involved testing tubes containing: potassium phosphate buffer, azide (to block the electron transport chain), buffered sucrose, mitochondrial extract, DCPIP (to accept the electrons from FADH2), succinate (the substrate for SDH), and malonate. These tubes were tested against tubes lacking the reagent malonate, a tube lacking SDH (boiled mitochondrial extract), a tube lacking …show more content…
SDH plays a crucial role in the Krebs cycle and the electron transport chain, both components of cellular respiration occurring within the mitochondria. Basically, SDH, which consists of four subunits – A, B, C, and D, breaks down sugar and transfers electrons from FADH2 to the electron transport chain. The process begins with the Krebs cycle, in which succinate in its reduced form is oxidized to fumarate. FAD is then reduced to FADH2 as it accepts the extra hydrogen molecules from the oxidation of succinate to fumarate. Both of these reactions are catalyzed by SDH. The electrons from FADH2 are then passed to ubiquinone, then down the electron transport chain (Rustin, et al.,
Bovine liver was manipulated primarily through centrifugation, and mitochondrial fractions were then created to serve as the enzyme-rich solutions the reagent would act upon. To determine the protein concentration of the mitochondrial fraction, the Bradford assay was implemented; the result will later be used in the calculation of the specific activity of SDH.
Ultimately, the SDH assay provided the data to either support or negate the hypothesis about malonate’s effect on SDH activity. This procedure involved testing tubes containing: potassium phosphate buffer, azide (to block the electron transport chain), buffered sucrose, mitochondrial extract, DCPIP (to accept the electrons from FADH2), succinate (the substrate for SDH), and malonate. These tubes were tested against tubes lacking the reagent malonate, a tube lacking SDH (boiled mitochondrial extract), a tube lacking …show more content…
SDH plays a crucial role in the Krebs cycle and the electron transport chain, both components of cellular respiration occurring within the mitochondria. Basically, SDH, which consists of four subunits – A, B, C, and D, breaks down sugar and transfers electrons from FADH2 to the electron transport chain. The process begins with the Krebs cycle, in which succinate in its reduced form is oxidized to fumarate. FAD is then reduced to FADH2 as it accepts the extra hydrogen molecules from the oxidation of succinate to fumarate. Both of these reactions are catalyzed by SDH. The electrons from FADH2 are then passed to ubiquinone, then down the electron transport chain (Rustin, et al.,