Measurement of protein carbonyls in human brain tissue

The carbonyl assay has been developed as a general assay of oxidative protein damage to assess steady-state protein damage in animal tissues and body fluids. The assay is based on the fact that several ROS can attack amino acid residues in proteins (particularly histidine, arginine, lysine, and proline) to produce carbonyl functions that can react with 2,4-dinitrophenylhydrazine (DNPH) to generate chromophoric dinitrophenylhydrazones. This reaction can, therefore, be used to estimate the carbonyl content of proteins in human tissues and body fluids. Western blotting assays based on the use of anti-dinitrophenol antibodies have also been developed to identify oxidatively damaged proteins in tissues and body fluids after their extraction and derivatization with DNPH. The carbonyl assay has become widely used and many laboratories have developed individual protocols for it. Sometimes, the assay procedures used are not precisely specified, and when they are, they may differ from those originally proposed by the group of Stadtman et al. This point is important, because there is considerable variation in the baseline levels of protein carbonyls in certain tissues, depending on how the assay is performed.