The nitroxide TEMPO is an efficient scavenger of protein radicals: Cellular and kinetic studies

Abstract

Protein oxidation occurs during multiple human pathologies, and protein radicals are known to induce damage to other cell components. Such damage may be modulated by agents that scavenge protein radicals. In this study, the potential protective reactions of the nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl radical) against Tyr- and Trp-derived radicals (TyrO/TrpN) have been investigated. Pretreatment of macrophage cells with TEMPO provided protection against photo-oxidation-induced loss of cell viability and Tyr oxidation, with the nitroxide more effective than the hydroxylamine or parent amine. Pulse radiolysis was employed to determine rate constants, k, for the reaction of TEMPO with TyrO and TrpN generated on N-Ac-Tyr-amide and N-Ac-Trp-amide, with values of k∼108 and 7×106M−1s−1, respectively, determined. Analogous studies with lysozyme, chymotrypsin, and pepsin yielded k for TEMPO reacting with TrpN ranging from 1.5×107 (lysozyme) to 1.1×108 (pepsin)M−1s−1. Pepsin-derived TyrO reacted with TEMPO with k∼4×107M−1s−1; analogous reactions for lysozyme and chymotrypsin TyrO were much slower. These data indicate that TEMPO can inhibit secondary reactions of both TyrO and TrpN, though this is protein dependent. Such protein radical scavenging may contribute to the positive biological effects of nitroxides.