Protein oxidation occurs during multiple human pathologies, and protein radicals can damage other cell components. Such damage may be modulated by agents that scavenge protein radicals. Other radicals may be especially efficacious due to the rapid reaction kinetics of radical-radical reactions.
The potential protective reactions of the nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl radical) against tyrosine- and tryptophan-derived radicals (TyrO•/TrpN•) have been investigated. Pre-treatment of macrophage cells with TEMPO provided protection against Tyr oxidation and photo-oxidation-induced cell death, with nitroxides more effective than hydroxylamines or parent amines. These data are consistent with TEMPO either preventing initial protein oxidation, or repairing damage to Tyr residues.
Pulse radiolysis was employed to determine rate constants, k, for reaction of TEMPO with TyrO• and TrpN• generated on amino acids, peptides and proteins. For TyrO• and TrpN• from N-Ac-Tyr-amide and N-Ac-Trp-amide respectively, k for reaction with TEMPO were ~108 and 7 x 106 M-1s-1 respectively.
For the corresponding radicals formed on the protein pepsin, k for reaction with TEMPO was determined as 1.1 x 108 M-1s-1 for TrpN•, and 4.3 x 107 M-1s-1 for TyrO•, but rapid electron transfer from TyrOH to Trp• was not prevented by TEMPO, as internal electron transfer is very rapid. The corresponding reactions of lysozyme-bound TrpN• were slower (k 1.5 x 107 M-1s-1) allowing TEMPO to inhibit TrpN• to TyrO• conversion. These data indicate that TEMPO has the capacity to inhibit secondary reactions of both TyrO• and TrpN• on proteins, though this is dependent on the protein structure. Studies with TEMPO derivatives demonstrate that substituents barely affect these reactions providing they do not dramatically increase the nitroxide reduction potential.
These results suggest that protein radical scavenging contributes to the beneficial effects of nitroxides such as TEMPO in vivo, and that nitroxides may be tuned for optimal efficacy.