Detection and localization of oxidized proteins in muscle cells by fluorescence microscopy

In meat, no detailed studies on the intracellular distribution of oxidized proteins during oxidative stress have been performed, to our knowledge. Therefore, we used fluorescence microscopy to detect and locate protein carbonyls, oxidation products of basic amino acids, generated in bovine M. Rectus abdominis during either exposition to a chemical free radical generating system, or refrigerated storage, or cooking. The technique consisted of an immunohistochemical detection of carbonyls by reaction with the specific probe DNPH (2,4-dinitrophenylhydrazine) followed by the sequential addition of a first antibody against DNPH-carbonylated proteins and a CY3-labeled secondary antibody. The fluorescence of the CY3 probe increased regularly with level of free radical generating system and storage time. Moreover, an important heterogeneity of carbonyl distribution was observed, with a higher oxidation level at the periphery than inside the muscle cells. Cooking induced fluorescence increase only at the periphery of cells. Specific coloration of collagen by Sirius red showed that collagen was not involved in fluorescence. We can deduce that accumulation of oxidized proteins observed in the cell periphery was linked to membrane protein oxidation and not to connective tissue oxidation. Biochemical assays were performed in parallel on membrane and myofibrillar proteins to provide complementary quantitative data on level of oxidized proteins.