Methionine residue acts as a prooxidant in the •OH-induced oxidation of enkephalins.

Enkephalins are bioactive pentapeptides (Tyr-Gly-Gly-Phe-Leu (Leu-enk) and Tyr-Gly-Gly-Phe-Met (Met-enk)) produced while an organism is under mental and/or physical stress. In the course of their biological action they are exposed to reactive oxygen and nitrogen species. We have reinvestigated the reactions of (•)OH radicals toward these peptides in order to elucidate the oxidation mechanisms and the final products. Nanosecond pulse radiolysis was used to obtain the spectra of the reaction intermediates and their kinetics. Additional insight into details of the oxidation mechanism was gained by identification of main final products by means of UV-vis spectrophotometry, HPLC coupled with fluorescence spectroscopy, and mass spectrometry. The key processes are different in both peptides. In Leu-enk, the first step is an (•)OH radical addition to the aromatic rings of Tyr and Phe residues that leads to hydroxylated residues, dihydroxyphenylalanine (DOPA) from Tyr and tyrosine isomers from Phe, respectively. In Met-enk, these processes are less important, an additional target being the sulfur atom of the methionine residue. Depending on pH either an OH-adduct (hydroxysulfuranyl radical) or a sulfur radical cation undergo intramolecular electron transfer with Tyr residue resulting in a repair of Met and oxidation of Tyr to tyrosyl radicals and a final formation of dityrosine. At low pH, the OH-adducts to Tyr residue are precursors of tyrosyl radicals and dityrosine. Thus, the final products coming from oxidation of the Tyr residue depend strongly on the neighboring residues and the pH.

Superoxide radical anions protect enkephalin from oxidation if the amine group is blocked.

The pentapeptide methionine-enkephalin (Met-enk) is a natural opiate that inhibits signals of pain. The N-terminal tyrosyl residue is important in the recognition of the peptide by its receptor. In oxidative stress, this residue can be oxidized by reactive oxygen species. The one-electron oxidation of Met-enk and of tert-butoxycarbonyl-methionine-enkephalin (Boc-Met-enk) was studied by gamma- and pulse radiolysis in the absence and in the presence of superoxide radical anions (O(2)(.-)) and oxygen, using azidyl radicals as oxidants. Without oxygen, both peptides behaved similarly. The tyrosyl radical resulting from the oxidation of tyrosyl residue produced the dimer linked by dityrosines. Methionine was also oxidized to its sulfoxide; however, this reaction is of minor importance. When O(2)(.-) was present, it added to tyrosyl radical giving a hydroperoxide. For Met-enk, this adduct cyclized via an intramolecular Michael addition of the amine on the aromatic ring. Conversely, for Boc-Met-enk, the adduct eliminated oxygen which led to 97% regeneration of the nonmodified peptide. Blocking the terminal amine group had thus a key role in protection of the tyrosyl residue. This finding might be exploited in the search for new pain inhibitors.