Immunohistochemical detection of protein oxidation

The oxidative modification of proteins by reactive oxygen species (ROS) and other reactive compounds is associated with a number of disease and pathophysiological processes as well as aging (1). Under physiological conditions, almost all oxidative modifications of proteins are resulting in an increase of carbonylated proteins. The three major pathways leading to carbonyl group formation (protein oxidation) are shown in Fig. 1. Carbonyl groups are introduced into proteins as a result of: 1) metal catalyzed oxidation of amino acid residues; 2) lipid peroxidation (the Michael addition of protein amino, sulfhydryl, and imidazole groups to the double bond of α,β unsaturated aldehydes, which are produced during the oxidation of polyunsaturated fatty acids); and 3) protein glycation and glycoxidation reactions. The carbonyl content of proteins is therefore an index of the amount of oxidative protein damage attributable to either direct attack of free radicals or the modification of proteins by oxidation products of carbohydrates or polyunsaturated fatty acids (PUFAs)

Cytofluorescence techniques for the visualization of distinct pools of protein thiols at the single cell level

4-Hydroxynonenal (4-hydroxy-2,3-trans-nonenal; 4-HNE) is the best known and thoroughly studied aldehydic product originating in biological samples during the process of lipid peroxidation (Fig. 1) (1). The latter is an autocatalytic, self-propagating sequence of free radical reactions, ultimately resulting in the fragmentation of the carbon atom chains of unsaturated fatty acids esterified in phospholipids of cellular membranes, which can be set into motion in conditions of severe oxidative stress within the cell (2). Many of the lipid fragments thus originated are aldehydes and other carbonyl products, provided with variable reactivity towards cellular macro molecules. 4-HNE was originally identified in vitro as a specific, dialyzable, cytotoxic product of peroxidation of microsomal phospholipids (3), but subsequent studies have consistently detected it in a number of experimental conditions, in which it has been shown to exert a variety of biological actions (4), as well as in important human diseases such as atherosclerosis, neurodegeneration, and cancer (5–7) Like other α,β-unsaturated aldehydes, 4-HNE is capable of binding covalently to side chains of cysteine, histidine, lysine, and other amino acids in proteins (8), thus originating new epitopes that can be detected by suitable antibodies Fig. 2). Here a convenient procedure is described using polyclonal antibodies (PAbs) and fluorescent revelation