This document discusses reactive oxygen species (ROS), specifically superoxide. It defines ROS as chemically reactive molecules containing oxygen that are produced naturally during cellular metabolism. Superoxide is formed as a byproduct of mitochondrial electron transport and can damage cells when overproduced. The document describes how hydroethidine fluorescence is used to selectively detect superoxide levels in mitochondria, finding increased fluorescence with antimycin stimulation. It concludes that precise superoxide detection aids understanding of its role in signaling and damage.

1. Reactive oxygen species:
detection and reactivity
By: Michael Janucik

2. Outline
Reactive oxygen species
Definition
Formation
Function
Controlling proteins
Detection of superoxide's

3. Reactive oxygen species
What are they?
Why are they important?
How do they effect the body?
How are they detected? (probe selectivity)

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. 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. 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. Why are ROS important?
 Reactive oxygen's serve to some capacity as signaling
molecules in cells.
 These molecules usually activate transcription factors that
produce different proteins in the body

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. 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. Reactivity of superoxide?
 Superoxide is one of the main initiators of creating free
radicals and other reactive oxygen species.

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. 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. Selectivity
a) Emission spectra exciting at 510 nm
b) excitation spectrum shows a distinct excitation for HO-Mito-Etd+ at 396 nm
a) Emission spectra using an excitation wavelength of 396 nm
Mito-Etd+(____) HO-Mito-Etd+(----)

14. Determining product
 To make sure our structure was correct ion catch mass
spec was used

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. 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. Selectivity of probe
 This table shows
fluorescence of
other oxidation
products with
respect to
superoxide.

18. Conclusions
 Superoxide's 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. 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. Questions