Oxidative stress may cause reversible or irreversible changes in proteins. Such changes are meant to modulate protein function (redox regulation) or protect against irreversible damage that causes the inactive proteins to accumulate or become degraded.
Carbonylation, an irreversible oxidative damage, involves the oxidation of side chains of amino acids to aldehydes or ketones. Lysine, arginine, proline, and threonine side-chains can be oxidatively converted to reactive aldehyde or ketone groups causing inactivation, crosslinking or breakdown of proteins.
Protein carbonylation can be detected and quantified at the global level in proteins and protein mixtures using derivatization of carbonyl groups with hydrazines followed by spectrophotometric measurement.
Protein carbonylation occurs providing an integrated assessment of oxidative damage. The most employed method for evaluation of the content of carbonylated proteins is based on 2,4-dinitrophenylhydrazine (DNPH) and was originally developed by Levine et al. This molecule reacts with carbonyl groups leading to the formation of the stable 2,4-dinitrophenylhydrazone. The dinitrophenyl group (DNP) can be detected and quantified spectrophotometrically because it is characterized by a typical absorption spectrum with a maximum at 365–375 nm.
