Overview
Free radicals are an unwanted bi-product of cellular redox reactions, and in particular of electron transfer within mitochondria. They are being continually produced even under the most favorable of conditions, but rapidly destroyed. Cells have endogenous anti-oxidant proteins such as superoxide dismutase, and chemicals such as glutathione, to rapidly inactivate these radicals. Inside mitochondria, electrons are passed for NADH and succinate through flavins, hemes and non-heme iron centers, before final reaction with molecular oxygen catalyzed by cytochrome c oxidase. A small amount of oxygen inevitably reacts with partially reduced prosthetic groups, particularly the flavins, to generate superoxide, but as discussed above, this superoxide is rapidly detoxified by glutathione as well as a mitochondrially-located superoxide dismutase and other redox proteins such as thioredoxins. When respiratory chain activity is slowed through transient hypoxia, or because of a variety of genetic and environmental causes, the lifetime of partly reduced prosthetic groups increases, and as a result the levels of free radicals increase, sometimes to the point of overwhelming the anti-oxidant defences. This results in oxidative modification of proteins, lipids and DNA, which can accumulate to the point of generating pathology.

In addition to oxidative damage, there is nitrative modification of proteins in many cells as a consequence of the reaction of superoxides with NO a cellular messenger found in many tissues, and this generates the highly reactive peroxinitrite anion. Nitrative modification of proteins generates nitrotyrosine, that contributes significantly to protein alterations as an overall part of oxidative damage.

Assay Details
The OxStress™ assay involves arraying multiple mitochondrial proteins by immunocapture for subsequent detection of carbonyl and/or nitrotyrosine incorporation into each. In conjunction with the MITOTOX expression assay, which determines the levels of each protein, the extent of oxidative modification can be expressed as post-translational modification levels per unit of each protein. This makes comparison between different samples simple and quantitative.

The proteins arrayed for analysis on the OxStress™ plate are generators of free radicals (Complexes I and III), closely positioned enzymes to these (Complex IV), and proteins susceptible to oxidative damage because of their prosthetic groups (pyruvate dehydrogenase, with a lipoate, and AIF with its flavin group). Our studies show that the modification of these are representative of the global oxidative damage as a result of mitochondrially-generated oxidative stress.

Monoclonal antibodies specific to each of the 5 proteins are provided on a 96 well plate to allow analysis of 24 samples in triplicate. After capture, the modification of each protein for introduction of carbonyls can be measured quantitatively, and when this assay is multiplexed with the assay for the expression of each protein by using the detector antibodies for each, relative labeling in separate samples can be compared directly. Finally levels of nitrotyrosine modification can be determined using a highly specific nitrotyrosine monoclonal antibody which we have recently generated.

Figure 1. Percent change in carbonylation of 11 proteins in HepG2 cells after dosing with 40µM chloramphenicol for 24 hours relative to dosing with DMSO.

Figure 2 & 3. Activity of Complex IV (COX) and Pyruvate dehydrogenase (PDH) declines as nitration of these enzymes increases.