Quantitation of oxidative damage to tissue proteins

Abstract

Active oxygen species are thought to be involved in many physiological and pathological processes and are known to oxidatively modify DNA, lipids and proteins. One such modification is the addition of carbonyl groups to amino acid residues in proteins. The number of carbonyl groups on proteins can be quantitated spectrophotometrically using 2,4-dinitrophenylhydrazine (DNPH). The DNPH assay described in the literature was found to be unreliable in samples containing high amounts of chromophore (e.g. hemoglobin, myoglobin, retinoids). By using an HCl–acetone wash, hemes from the chromophores could be extracted, enabling the determination of carbonyl content to be made even in highly colored tissue extracts. Residual DNPH, which was also found to interfere with the assay, was removed by additional washes with trichloroacetic acid and ethanol–ethylacetate. These improvements are known to remove lipids, do not lengthen the time required to do the assay, permit quantification of carbonyl content in 1–4 mg protein from a variety of tissue types and provide a sensitive and reliable method for assessing oxidative damage to tissue proteins.