Protein Carbonyl (C=O) Colorimetric Assay Kit
Protein carbonylation can be detected and quantified at the global level in proteins and protein mixtures. This can be performed by using derivatization of carbonyl groups with hidrazines followed by spectrophotometric measurement. So the most employed method for evaluation of the content of carbonylated proteins bases on 2,4-dinitrophenylhydrazine (DNPH). This molecule reacts with carbonyl groups leading to the formation of the stable 2,4-dinitrophenylhydrazone (Figure 1). As a result, the user can spectrophotometrically detect and quantify the dinitrophenyl group (DNP). Particularly, it shows a typical absorption spectrum with a maximum at 365–375 nm.
Figure 1. DNPH reaction with carbonyl species. Modified from Uchiyama et al.
Oxidative stress may cause reversible or irreversible changes in proteins. Then, such changes modulate protein function (redox regulation) or protect against irreversible damage that causes the inactive proteins to accumulate or become degraded.
Protein carbonylation is a type of protein oxidation that reactive oxygen species can promote. It usually refers to a process that forms reactive ketones or aldehydes that can react with 2,4-dinitrophenylhydrazine (DNPH) to form hydrazones. Specifically, direct oxidation of side chains of lysine, arginine, proline, and threonine residues, among other amino acids, in the “primary protein carbonylation” reaction produces DNPH detectable protein products . DNPH derivatizable protein products can also be formed in the “secondary protein carbonylation” reaction via the addition of aldehydes, such as those generated from lipid peroxidation processes.