Final presantation mike

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  • The electron transport chain in Mitochondria, which transfers electrons from NADH in a chemical path that ends in the four-electron reduction of O 2 to H 2 O during respiratory ATP synthesis. sometimes the flow of electrons through the electron transport chain a glitch occurs, and occasionally oxygen molecules undergo one- or two-electron reduction reactions to form ROS, particularly H 2 O 2 and [O 2 ] •− sometimes the flow of electrons through the electron transport chain a glitch occurs, and occasionally oxygen molecules undergo one- or two-electron reduction reactions to form a ROS.
  • Because supper oxide can not pass thought membraines
  • The excitation spectra of purified HO-mito-Edt+ showed that there was an excitation peak at 396 nm that is not present for mito-Edt+ (figure 3b). excitation at 396 nm enhanced the fluorescence emission of HO-mito-Edt+ and reduced spectral overlap(figure 3c). Therefore excitation at 396nm is a more selective way to show the oxidation due to superoxide using Mito-HE.
  • The second peak was determined to be the HO-mito-Edt+ peak by comparison with standards and mass spectrometry results(figure 4). The first peak of the HPLC was the oxidation of Mito-He by other reactive oxygen speices or other oxidations Figure 4 shows the ion trap mass spectrometry spectra, the ions at m/z=316.0 and 630.5 were identified as Mito-Etd+. The doubly charged ion at m/z= 324.0 corresponded to the molecular weight of the hypothesized structure of HO-Mito-Etd+, and the singly charged ion at m/z= 646.0 lead to the proposal of the carbonyl structure, O=Mito Etd
  • Now that we are sure the only oxidation that we are detecting is from superoxide we can start looking in mitochondrial cells This is about .1% of the total superoxide production due to electron transfer, and because the mitochondria consumed more oxygen at a concentration larger than 100 uM, antimycin stimulation of superoxide production must be used
  • Using this we can discriminate between superoxide oxidation and other oxidations of Mito-HE
  • Final presantation mike

    1. 1. Reactive oxygen species:detection and reactivity By: Michael Janucik
    2. 2. OutlineReactive oxygen species Definition Formation Function Controlling proteinsDetection of superoxides
    3. 3. Reactive oxygen speciesWhat are they?Why are they important?How do they effect the body?How are they detected? (probe selectivity)
    4. 4. What are reactive oxygen species? Reactive oxygen species are chemically reactive molecules that contain oxygen. Superoxide, hydroxyl radical, hydroxyl ion most reactive oxygen species are highly reactive due to the presence of unpaired valence shell electrons. Reactive oxygen species form as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis.
    5. 5. Reactive oxygen species The electron transport chain in Mitochondria, which transfers electrons from NADH in a chemical path that ends in the four- electron reduction. 1–5% of total oxygen that is consumed in aerobic metabolism produce superoxide anions.
    6. 6. Reactive oxygen species A problem with these molecules is that when they react with non antioxidants they tend to form new free radical species. The life-span of different ROS varies considerably, from less than 1 ns of .OH to even hours of H2O2
    7. 7. Why are ROS important? Reactive oxygens serve to some capacity as signaling molecules in cells. These molecules usually activate transcription factors that produce different proteins in the body
    8. 8. How are ROS controlled? Humans have developed a superoxide scavenging enzyme called superoxide dismutase which catalyzes the neutralization of superoxide Other ROS regulating enzymes are glutathione peroxidases, catalase and peroxiredoxins. M3+-SOD + O2− → M2+-SOD + O2 M2+-SOD + O2− + 2H+ → M3+-SOD + H2O2.
    9. 9. ROS damage The problem is when ROS is produced in excess and can oxidize membranes and DNA leading to aging and other biological damage Superoxide is created from the electron transfer but it is not able to pass through the membrane of the mitochondria.
    10. 10. Reactivity of superoxide? Superoxide is one of the main initiators of creating free radicals and other reactive oxygen species.
    11. 11. How are superoxide anions detected? Hydroethidine (HE) is joining to a hexyl triphenylphosphonium cation (Mito-HE) HE is in the body naturally ad is oxidized to Etd+, this oxidation increases its florescence when using 535-nm excitation and 610- nm emission wavelengths So the fluorescence of Etd+ does not definitely prove that superoxide’s are being produced and used to oxidize HE.
    12. 12. Detection Because Mito-HE can be oxidized by oxidants other than super oxide we need a way to selectively pick out the product that is oxidized by the superoxide, So superoxide was generated by xanthine oxidase and allowed to react with our Mito-HE
    13. 13. Selectivitya) Emission spectra exciting at 510 nmb) excitation spectrum shows a distinct excitation for HO-Mito-Etd+ at 396 nma) Emission spectra using an excitation wavelength of 396 nmMito-Etd+(____) HO-Mito-Etd+(----)
    14. 14. Determining product To make sure our structure was correct ion catch mass spec was used
    15. 15. Superoxide in mitochondria Mito-HE was allowed to accumulate in isolated mitochondrial cells. To test the ability of Mito-HE detect superoxide using a excitation wavelength of 396nm. When oxygen levels in the buffer reached near saturation levels, mitochondria oxidized the Mito-HE at a rate of .23 nmol O2 •-/min*mg of protein and consumed 190 nmol O atm/min mg protein
    16. 16. Superoxide in mitochondria The rate of oxidation in mitochondria was increased to .81 nmol superoxide/ min*mg of protein due to antimycin stimulation of superoxide. The rate of antimycin stimulated superoxide fluorescence was 31% faster at a wavelength of 510 nm, as compared with a wavelength of 396 nm. Because HO-Mito-Etd+ is less fluorescent at a wavelength of 510 nm, the 31% faster rate indicated that antimycin must have increased the formation of Mito-Etd+ in addition to HO- Mito-Etd+.
    17. 17. Selectivity of probe  This table shows fluorescence of other oxidation products with respect to superoxide.
    18. 18. Conclusions Superoxides are an inevitable byproduct of the electron transfer process in the mitochondria. An over production of ROS molecules can cause inter cellular damage Superoxide anions react with a number of biological molecules to produce different radical molecules. The fluorescent probe used here is one way to detect with precision and selectivity superoxide anions.
    19. 19. References 1)Robinson, K.M. et al. Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc. Natl. Acad. Sci. USA 103, 15038–15043 (2006) 2) Murphy, M.P. How mitochondria produce reactive oxygen species. Biochem. J. 417, 1–13 (2009). 3) Ray G, Husain SA. Oxidants, antioxidants and carcinogenesis. Indian J Exp Biol 2002;40:1213–32 Scott K. Powers, Jose Duarte, Andreas N. Kavazis, and Erin E. Talbert Reactive oxygen species are signalling molecules for skeletal muscle adaptation Exp Physiol January 1, 2010 95 (1) 1-9; published ahead of print October 30, 2009, doi:10.1113/expphysiol.2009.050526
    20. 20. Questions

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