Oxidative Modifications

Analysis of protein-associated carbonyls Consistent with the ''free radical theory'' of aging, many tissues show an age-dependent increase of the steady-state concentrations of oxidized proteins. So far most reports on the accumulation of oxidized proteins have analyzed ''protein-associated carbonyls.'' The term ''protein-associated carbonyl'' classifies a carbonyl-containing covalent protein modification, which results either from the direct chemical transformation of an amino acid residue into a carbonyl product or from the covalent attachment of a carbonyl-containing molecule (e.g., 4-hydroxynonenal, 4-HNE) (Berlett and Stadtman, 1997). The most frequently used analytical method for the detection of protein-associated carbonyls is deri-vatization with 2,4-dinitrophenylhydrazine (Scheme 1, as shown in Figure 9.3), followed by UV spectroscopy or Western blot detection with anti-2,4-dinitrophenyl antibodies (Shacter et al., 1994).

The specificity of these antibodies was evaluated through specific reaction with DNP conjugated to proteins (Eshhar et al., 1980; Shacter et al., 1994), and 1 pmol was reported as a lower limit of detection of protein-associated carbonyls (Shacter et al., 1994). A comparable detection limit (0.64 pmol) was recently achieved with a slight modification of the methodology, using biotin-hydrazide labeling of the carbonyls followed by staining with fluorescein isothiocyanate (FITC)-labeled avidin (Yoo and Regnier, 2004). A number of studies have used stationary phase bacterial cells as a model for aging somatic cells of higher eukaryotic organisms in order to delineate the routes leading to the accumulation of




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