[37] Carbonyl assays for determination of oxidatively modified proteins

Enzymes and structural proteins may be attacked whenever free radicals are generated. As a consequence, oxidative modification of proteins may occur in a variety of physiologic and pathologic processes. Although the distinction is sometimes arbitrary, these modifications may be primary or secondary. Primary modifications occur in metal-catalyzed oxidation, radiation-mediated oxidation, and oxidation by ozone or oxides of nitrogen. Secondary modifications occur when proteins are modified by molecules generated by oxidation of other molecules. One important example is the covalent modification of proteins by hydroxynonenal produced by oxidation of lipids. Carbonyl groups (aldehydes and ketones) may be introduced into proteins by any of these reactions, and the appearance of such carbonyl groups is taken as presumptive evidence of oxidative modification. Assay of carbonyl groups in proteins provides a convenient technique for detecting and quantifying oxidative modification of proteins. This chapter presents new methods for determination of carbonyl content, which are based on the reaction of carbonyl groups with 2,4-dinitrophenylhydrazine to form a 2,4-dinitrophenylhydrazone. The assays provide substantial improvements in both sensitivity and specificity.