The fact that proteins are the main target of reactive species formed in the cells and extracellular fluids has led to the realization of a great deal of research devoted to revealing the molecular and biological consequences associated with the presence of intermediary protein radicals. This review article describes and comments upon the main chemical pathways involving primary proteic radicals.

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Chemical and structural alterations to lysozyme (LYSO), glucose 6-phosphate dehydrogenase (G6PD), and bovine eye lens proteins (BLP) promoted by peroxyl radicals generated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions were investigated. SDS-PAGE analysis of the AAPH-treated proteins revealed the occurrence of protein aggregation, cross-linking, and fragmentation; BLP, which are naturally organized in globular assemblies, were the most affected proteins. Transmission electron microscopy (TEM) analysis of BLP shows the formation of complex protein aggregates after treatment with AAPH. These structural modifications were accompanied by the formation of protein carbonyl groups and protein hydroperoxides. The yield of carbonyls was lower than that for protein hydroperoxide generation and was unrelated to protein fragmentation. The oxidized proteins were also characterized by significant oxidation of Met, Trp, and Tyr (but not other) residues, and low levels of dityrosine. As the dityrosine yield is too low to account for the observed cross-linking, we propose that aggregation is associated with tryptophan oxidation and Trp-derived cross-links. It is also proposed that Trp oxidation products play a fundamental role in nonrandom fragmentation and carbonyl group formation particularly for LYSO and G6PD. These data point to a complex mechanism of peroxyl-radical mediated modification of proteins with monomeric (LYSO), dimeric (G6PD), and multimeric (BLP) structural organization, which not only results in oxidation of protein side chains but also gives rise to radical-mediated protein cross-links and fragmentation, with Trp species being critical intermediates.

The pyraninoxyl radical is readily formed from the MnO 2 -promoted oxidation of pyranine. The free radical can be formed in high concentrations (mm), and presents a characteristic EPR spectrum that indicates a high spin-density delocalization. It is relatively stable under nitrogen (half-life ca. 50 min) but readily decays in presence of O 2 . In spite of its high stability, the radical readily reacts with antioxidants (phenols and ascorbic acid) with a partial recovery of the parent pyranine. High concentrations of the pyraninoxyl radical (ca. 9 mm) are present when pyranine is exposed to a free radical source (10 mm 2,2'-azobis[2-amidinopropane], 378). The fact that these radicals readily react with antioxidants (ascorbic acid and caffeic acid) supports the proposal that protection by antioxidants of peroxyl radical-promoted pyranine bleaching is mainly due to the occurrence of a repair mechanism.Introduction. -The protection afforded to a target molecule damaged by peroxyl radicals by the addition of a given compound is generally related to its capacity to scavenge the damaging radicals. This simple relationship between reactivity of the additive and afforded protection is the basis of a large number of methods aimed at evaluating the antioxidant capacity of pure compounds and complex mixtures [1 -3]. These methods are based on competitive experiments in which the target molecule (TH) and the tested additive (antioxidant, XH) compete for a ROS (i.e., peroxyl radicals, hydroxyl radicals, superoxide, singlet oxygen, or hypochlorite) or RNS (i.e., peroxynitrite) [4] [5]. When a source of radicals is employed to damage the target molecule, this direct relationship between the additive reactivity and the afforded protection is based on a simplified mechanism comprising Processes 1 and 2: