3.5.2. Fukui Function

To predict which atom would be most susceptible to a nucleophilic or electrophilic attack, peptide FSEY was selected to investigate the tendency by Fukui functions. Fukui function is a local reactivity parameter, which is widely used for molecular reactivity analysis, indicating the tendency of a molecule to lose or gain an electron thus predicting which atom in the molecule would be more prone to a nucleophilic or electrophilic attack. When a molecule prefers to accept an electron, the Fukui function is f+, it is the index of nucleophilic attack. While when a molecule has a tendency to lose an electron, the Fukui function is f− and is also termed as the index of electrophilic attack [22]. In our study, the individual atomic charges were calculated by natural population analysis (NPA) with B3LYP/6-311G (d, p) basis set. For all atomic sites of peptide FSEY, their Fukui functions (f−, f+, f0) were presented in Figure 3b,c, respectively.

Blue, red, and green colors in Figure 3b represent nucleophilic, electrophilic, and radical attacks, respectively. It is found that nucleophilic, electrophilic, and radical attacks of peptide FSEY are located at N1, C13, C15, C51, C56, C57, C59, C61, C63, C65, and O66 atoms, especially at the atoms of Tyr residue. These results further highlighted a fact that Tyr residue should act as a biological activity site, as shown in Figure 3c. Moreover, the molecular reactivity site of peptide FSEY, as indicated by Fukui functions, totally corresponded to that of its HOMO.

## *3.6. Effects of Tyrosine Residue's Location on the Antioxidant Activity of Peptides*

To clarify how the position of tyrosine residue governs the antioxidant activity of a peptide against free radicals, three peptides FSEY, FYSE, and YFSE were selected to examine their inhibitions towards the oxidation of linoleic acid (see Figure 4a). The three peptides have the same composition of amino acids but have different locations for tyrosine residue. After incubated with linoleic acid at 40 ◦C for 48 h, the peptide FSEY showed the strongest ability in stopping the oxidation of the fat acid compared to others. The peptide YFSE had the lowest antioxidant activity. It is concluded that if tyrosine residue is located at the C-terminal, the Tyr-containing peptide should have a stronger activity against linoleic acid radical, as shown in Figure 4a.

**Figure 4.** Antioxidant activities of peptides. (**a**) stands for activities of FSEY, FYSE, and YFSE on the oxidation of linoleic acid; (**b**) represents the effects of the antioxidants on oil oxidation, GSH and TBHQ as positive controls.
