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




Blood Pressure Health

Blood Pressure Health

Your heart pumps blood throughout your body using a network of tubing called arteries and capillaries which return the blood back to your heart via your veins. Blood pressure is the force of the blood pushing against the walls of your arteries as your heart beats.Learn more...

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