Reactive oxygen species: detection and

reactivity

By: Michael Janucik

OutlineReactive oxygen species

DefinitionFormationFunctionControlling proteins

Detection of superoxide's

Reactive oxygen species

What 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 speciesThe 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 oxygen's 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 damageThe 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

Selectivity

a) Emission spectra exciting at 510 nm b) 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 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.

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

Questions