Abstract
Publisher Summary This chapter focuses on the detection of carbonyl groups as markers of oxidative protein modification. Protein carbonyls become elevated under various conditions of oxidative stress and are generated readily under experimental conditions in vitro, such as through exposure to iron/ascorbate, ionizing radiation, singlet oxygen, and ozone). Their chemical stability makes them suitable targets for laboratory measurement. Early techniques for the detection of protein carbonyls were based either on incorporation of tritium through reduction with tritiated borohydride or on incorporation of a stable dinitrophenyl (DNP) adduct through reaction of the carbonyls with 2,4-dinitrophenylhydrazine (DNPH). Because DNP absorbs light at 370 nm, the carbonyl groups can be measured spectrophotometrically. While reliable and quantitative, these two techniques suffer from three significant technical drawbacks. They require the use of a substantial amount of purified protein (∼1-2 mg). The derivitizing reagents have to be removed prior to analysis in order to reduce background levels sufficiently to allow detection of protein-bound label (either H 3 or DNP). They are unable to differentiate oxidized from nonoxidized proteins in cell or tissue extracts.
Published Version
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