## 3 Results and Discussion  
Consumption of PYR by ROO\({}^{\bullet}\) followed a zero-order kinetic limit in PYR over a wide concentration range, corroborating its high reactivity towards these radicals [13]. In the presence of 10 mM AAPH, a progressive decrease in the fluorescence intensity of PYR over the incubation time was observed (Figure 1), giving initial consumption rates (R\({}_{0}\)) between 0.3 \(\pm\) 0.04 and 0.5 \(\pm\) 0.06 mM/min for PYR concentrations between 1 and 100 mM. At 5 \(\upmu\)M PYR, a R\({}_{0}\) of 0.32 mM/min was calculated, implying that 2.5 moles of ROO\({}^{\bullet}\) were trapped per mole of PYR (a flux of 0.8 \(\upmu\)M/min of ROO\({}^{\bullet}\) is generated from 10 mM AAPH at 37 \({}^{\circ}\)C [10]). In agreement with previous reports [13; 18], this value would imply a total trap of ROO\({}^{\bullet}\) by PYR, suggesting a minimal yield of RO\({}^{\bullet}\) production (Reaction (6)). As depicted in Panel A of Figure 2, free Trp protected PYR without generating lag times in the kinetics, reflecting only changes in R\({}_{0}\). At 1 mM Trp, a R\({}_{0}\) = 0.12 \(\pm\) 0.01 mM/min was determined, indicating a 2.5-fold-times slower consumption of PYR than control experiments. The absence of lag times suggests that competition between PYR and Trp for ROO\({}^{\bullet}\) is the main process, with a minimal occurrence of Reaction (8). Aiming to obtain new insights regarding the protection of PYR elicited by peptides containing a single Trp residue, we carried out experiments using two dipeptides (Gly-Trp and Trp-Gly), and Gly-Trp-Gly. Due to these peptides having similar reactivities towards ROO\({}^{\bullet}\) (inferred from their direct consumption mediated by ROO\({}^{\bullet}\)[22]), we speculated that simple competitions with PYR for ROO\({}^{\bullet}\) should be reflected in similar values of R\({}_{0}\). In contrast to such hypothesis, as depicted in Figure 2, Panel A, the chemical structure of the peptides affected R\({}_{0}\), with values (expressed as mM/min) following the order as shown:  
Gly-Trp (0.28 \(\pm\) 0.02) > Gly-Trp-Gly (0.19 \(\pm\) 0.02) > Trp-Gly (0.14 \(\pm\) 0.01) \(\sim\) Trp (0.12 \(\pm\) 0.01)  
Figure 1: Kinetics of PYR consumption mediated by AAPH-derived ROO\({}^{\bullet}\). PYR solutions (75 mM phosphate buffer, pH 7.4) were incubated in the presence of 10 mM AAPH at 37 \({}^{\circ}\)C. [PYR] = 1 (black circles), 5 (green diamonds), 7.5 (blue inverted triangles), 10 (magenta triangles), and 100 (orange squares) \(\upmu\)M. Aside from PYR 100 \(\upmu\)M (followed by visible absorbance at 475 nm), PYR consumption was followed by its emission of fluorescence at 510 nm (excitation at 460 nm). 1/1\({}_{0}\) refers to normalized fluorescence or absorbance intensity. This order is not in consonance with the bond dissociation energy (BDE) of the hydrogen-nitrogen bond at position 1 of the indole ring of the Trp residues, which follows the order as shown [22]:  
\[\text{Trp}>\text{Trp-Gly}>\text{Gly-Trp-Gly}>\text{Gly-Trp}\]  
Thus, despite the apparent simplicity of the protection of PYR afforded by Trp, Gly-Trp, Trp-Gly, and Gly-Trp-Gly, the observed behavior should not be interpreted as a simple competitive scheme where the changes in R\({}_{0}\) are only explained by the ability of peptides to neutralize ROO\({}^{\bullet}\). The addition of high concentrations of free Gly (1-3 mM) to solutions containing 5 \(\upmu\)M PYR and 10 mM AAPH did not affect the rate of PYR consumption (Supplementary Figure S1), discarding some effect of Gly in the obtained results. In such a context, it seems reasonable to postulate that intermediates and oxidation products generated during the oxidation of Trp could have some participation in the protection of PYR. The latter could involve reactions involving the oxidation products of Trp, or Trp-derived radicals with PYR or PYR\({}^{\bullet}\). These reactions could be affected by the chemical structure of the peptides, probably modulating the PYR-based AC index.