what does SDH do
The mitochondrial membrane enzyme succinate dehydrogenase (SDH) oxidizes the compound succinate to fumarate in the krebs cycle of aerobic respiration in cells. SDH releases electrons to the Electron Transport Chain during this reaction.
what normally happens to the SDH released electrons
The electrons released during the activity of SDH are normally passed to the intermediate high-energy compounds flavine adenine dinucleotide (FAD) and iron sulfur clusters located within the enzyme before being transferred to ubiquinone.
what does sodium azide do
In this experiment an inhibitor, called sodium azide blocks the final transfer of electrons to oxygen. The electrons that can no longer be transferred to oxygen are then available to react with an artificial electron acceptor.
what did DCIP do
The rate of activity of a mitochondrial respiratory enzyme, such as SDH, can be determined by colorimetric test using spectrophotometry. The artificial electron acceptor 2,6-dichlorophenol indophenol (DCIP), is used in this experiment to intercept the electrons produced by the activity of SDH. DCIP is reduce to DCIP-H2 with the acceptance of electrons produced by SDH activity and undergoes a change in colour.
why did we need a standard curve of DCIP
A standard curve of absorbance and DCIP concentration is used to measure the concentration of DCIP resulting from SDH activity at specific time intervals. From a graph of DCIP concentration over time, the rate of change in DCIP concentration is equivalent to the rate of SDH activity.
what is the anatomy of the mitochondria
Mitochondria are cellular organelles each having an inner and outer mitochondrial membrane seperated from each other by an intermembrane space. The inner membrane is folded so as to form numerous finger like projections called cristae.
what are the cristae
The cristae project into the fluid interior of the mitochondria, a region known as the matrix. During glycolysis in eukaryotes the 6 carbon sugar glucose is enzymatrically oxidized to two three carbon molecules of pyruvate. During this process a small amount of DTP is produced.
what does the Krebs cycle and electron transport do
However the atomic bonds that hold the pruvate molecule together are still energy rich and eukaryotes are able to access this energy through the reactions of the Krebs cycle and the electron transport chain. In both these series of aerobic processes a succession of enzymatically-catalyzed oxidation reactions releases electrons, which in turn can be used to generate a much greater amount of ATP (36 molecules as opposed to 2 in glycolysis)
where are the enzymes associated with the Krebs cycle located
Most of the enzymes associated with the krebs cycle are within the mitochondrial matrix, but succinate dehydrogenase is located on the inner mitochondrial membrane. This makes it relatively easy to isolate by differential centrifugation in order to assay its rate of activity.
what is succinate
The intermediate compound succinate is produced during the oxidation of pyruvate in the Krebs cycle. SDH catalyses the oxidation of succinate to a lower-energy molecule, fumarate.
what does FAD do
The electrons and protons released by this reaction (Succinate->fumarate) are captured by the electron acceptor FAD resulting in its reduction to FADH2. This molecule transfers protons and electrons to another acceptor molecule, ubiquinone, from which they enter the electron transport chain of aerobic respiration.
what does the ETC do
The ETC creates a H+ gradient across the inner membrane, this is then used by ATP sunthase to generate most of the ATP produced during cellular respiration. All cells use ATP as a readily accessible source of energy for carrying out other metabolic activities and growth.
what happens to any remaining electrons in the ETC
Any remaining electrons at the end of the transport chain are transferred from cytochrome a3 to oxygen. Therefor, oxygen is the terminal electron acceptor in cellular respiration.
what does Sodium azide block
The addition of the poison Sodium azide specifically inactivates the electron transport chain by blocking the final electron transfer.
what is Malonate
Malonate is a structural analog of succinate and competitively inhibits SDH activity by binding to the active site of the enzyme. thus reducing overall SDH activity. It has also been found to increase the production of reactive oxygen species and induce a mitochondrial potential collapse which leads to a release of pro-apoptotic factors.
how is the rate of active usually measured
In enzyme studies, the rate of activity is usually measured as the amount of depletion of the substrate or amount of increase in the product, per unit time.
how can cellular component be isolated
cellular components of a whole organism or tissue can separated from each other by mechanical disruption, enzymatic treatment, etc. After which they can be separated from extracellular components, and further treated to release organelles and intracellular molecules into suspension. From suspension they can be isolated by differential centrifugation.
what does centrifugation do
Organelles have differences in size, shape and density. As a result the effect of gravity on each other is different. By putting the suspension in a variable speed centrifuge and rotating it at a high speed. This creates a centrifugal force that is much greater than earth’s gravity. This causes solutes to precipitate very rapidly.
what is the pellet
The pellet is a concentration of solute at the bottom of the sample. The fluid above is called the supernatant and the pellet can be concentrated by removing as much supernatant as desired.
what is the RCF
The relative centrifugation force (RCF) generated for a given speed of radius of rotation and is calculated by the following equation:
RCF = (1.119 x 10^-5)(rpm)^2(r)
Where: RPMs is the revolutions per minute. r is the distance in cm from the particles form the axis of rotation.
What is RCF at low and high speeds
The centrifugal force created at low speeds is small (around 600G) and only very large or dense particles, such as nuclei, whole cells, and large cellular debris will fall out of suspension.
At high speeds, the force can be as much as 300,000G. And these forces smaller particles will precipitate, and only very small macromolecules will remain in suspension.
what does differential centrifugation do
Differential centrifugation involves stepwise increases in the speed, and therefor force of centrifugation. At each step the more dense particles are separated from less dense particles, and the successive speed of rotation is increased until the target particle is pelleted out.
what can you do with the final pellet
Once the final supernatant is removed, the pellet can be further purified or studied. The separations made are usually not absolute purifications, but relative enrichments of cells or organelles.
how do you inhibit autolysis
Cellular organelles break down by autolysis very quickly following their isolation. To inhibit autolysis, all steps in the isolation must be done on ice or in a refrigerated chamber. Some have built in refrigeration for longer runs.
