Abstract

Competitive reactions between additives and probes towards peroxyl radicals (ROO•) are usually employed to determine the antioxidant activity (AC) of bioactive peptides. In this work, we investigated the AC of free and peptide Trp and Tyr residues, employing pyranine (PYR) as the probe and AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride) as the ROO• source. Solutions containing PYR and 10 mM AAPH were incubated at 37 °C in the absence and presence of additives. The initial consumption rates (R0) of PYR (5 µM) were affected by the type of peptide, with free Trp showing a higher effect than short peptides (R0 = Gly-Trp > Gly-Trp-Gly > Trp-Gly > free Trp), while the order of R0 of Tyr residues was as follows: free Tyr ~ Tyr-Tyr-Tyr > Gly-Tyr. Experiments carried out at 1 µM PYR, and employing larger peptides showed that the AC of Trp and Tyr cannot be explained by a simple mechanism. While the generation of lag times in the kinetics would not be necessarily associated with PYR repairing, their absence would not exclusively reflect competition for ROO•. These results demonstrate that the AC of Trp and Tyr follows complex mechanisms, implying that particular care should be taken when amino acids and peptides are proposed as antioxidants. © 2025 by the authors.