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
Reactive oxygen species:detection and reactivity By: Michael Janucik
OutlineReactive oxygen species Definition Formation Function Controlling proteinsDetection of superoxides
Reactive oxygen speciesWhat are they?Why are they important?How do they effect the body?How are they detected? (probe selectivity)
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.
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.
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
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
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.
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.
Reactivity of superoxide? Superoxide is one of the main initiators of creating free radicals and other reactive oxygen species.
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.
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
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+(----)
Determining product To make sure our structure was correct ion catch mass spec was used
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
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+.
Selectivity of probe This table shows fluorescence of other oxidation products with respect to superoxide.
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.
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