Development of Western analysis of oxidised proteins as a biomarker of oxidative damage in liver of fish

Western analysis of non-peptide bond free carbonyls by derivatisation with 2,4-dinitrophenylhydrazine was applied to hepatic proteins of fish as a potential biomarker of contaminant-mediated oxidative damage. Analysis of 1000 and 9000 g supernatants and sonicated hepatocytes of flounder (Platichthys flesus) liver revealed multiple free carbonyl-containing bands (approximate molecular weight 20–60 kD). RNase and protease treatments and nucleic acid precipitation demonstrated that the assay was specific for protein carbonyls in fish liver. Free carbonyl levels increased with in vitro oxidation by a hydroxyl radical-generating system, indicating that specific target proteins should be identified as possible biomarkers of contaminant-mediated oxidative damage.