Assay of succinate dehydrogenase of after isolation of mitochondria in Cauliflower (Brassica oleracea) using differential centrifugation.
Kelly M. Messick, Rebecca Conner
Department of Biological Sciences, Salisbury University, Salisbury, MD, 21801 U.S.A
Address for correspondence:
Kelly M Messick
Department of Biological Sciences
Salisbury, MD 21801
Running title: Assay of succinate dehydrogenase.
Cell fractionation is a very important procedure in cell biology and can be very useful for studying different organelles. By fractionating, we mean separating or dividing the cell into different component parts. Fractionation results in a series of fractions with only one highly purified organelle or protein present in each one ideally. Fractionation can be done by many different methods including size exclusion chromatography, charge, density, and immunoprecipitation; these are just a few. The two most popular methods are differential centrifugation and density gradient centrifugation because of their flexibility and effectiveness, these methods use gravity to separate organelles based on their size and density(Satori et al 2011). Density gradient centrifugation and differential centrifugation are a lot alike except for the fact that density gradient centrifugation uses a density gradient so the organelles settle at their buoyant densities where as differential centrifugation forms a pellet and supernatant without a density gradient. During differential centrifugation more dense particles pellet before less dense particles so that you are able to separate the original sample into fractions based on density. Fractions are rarely a perfect separation of organelles, normally each fraction will have an organelle that consumes the majority but may be present in other fractions. Because the fractions aren't a perfect separation of the organelles it is necessary to assay for the activity of mitochondria to make sure we fractionated what we thought we were and to make sure we are looking at the purest fraction.
There are certain enzymes that are used to identify certain organelles; in mitochondria those enzymes are succinate dehydrogenase(SDH) and cytochrome oxidase. SDH is one of nine enzymes that catalyzes the steps of the Kreb's cycle and it is also an enzyme only found bound to the inner mitochondrial membrane so that the only fraction it should be present in our fractions containing mitochondria. SDH is responsible for the oxidation of succinate with FAD to produce fumarate. During this reaction FAD accepts the electrons liberated and is reduced to FADH₂. FADH₂ then passes those electrons to the first of several electron acceptors found in the electron transport chain(ETC), ubiquinone (Williams et al 2012). We can use this information in order to assay the activity of SDH by replacing ubiquinone with the artificial electron acceptor, 2,6-dichlorophenolindophenol(DCIP). DCIP changes color from deep blue to colorless when it is reduced and the color change can be measured spectrophotometrically. To make sure the electrons are passed to DCIP instead of ubiquinone the ETC can be effectively bottlenecked by adding the poison sodium azide to the reaction which would block the final transfer of electrons from cytochrome a₃ to oxygen in the ETC therefore bottlenecking it. The rate of the disappearance of the blue color is proportional to the concentration of SDH.
There are two types of enzyme assays; a kinetic assay and a fixed time assay. A kinetic assay measures the consumption of a reactant or formation of a product over time, this time of assay is usually used when some property of the reaction can readily be monitored. In a fixed time assay the consumption of a reactant or formation of a product formed is measured at fixed time intervals, this is generally used when the reaction being measured can only...
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