Selective Site-Specific Fenton Oxidation of Methionine in Model Peptides: Evidence for a Metal-Bound Oxidant

• Published in: Journal of pharmaceutical sciences 2004-05, Vol.93 (5), p.1122-1130
• Main Authors: Dufield, Dawn R., Wilson, George S., Glass, Richard S., Schöneich, Christian
• Format: Article
• Language: English
• Subjects: Binding Sites - physiology; Biological and medical sciences; Fenton reaction; General pharmacology; Hydrogen Peroxide - analysis; Hydrogen Peroxide - metabolism; Iron - analysis; Iron - metabolism; Medical sciences; Met; Met sulfoxide; Methionine - analysis; Methionine - metabolism; Oxidants - metabolism; Oxidation-Reduction; Peptides - analysis; Peptides - chemistry; Peptides - metabolism; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; reactive oxygen species
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1002/jps.20013

The metal-catalyzed oxidation (MCO) of proteins represents an important pathway for protein degradation. Although many mechanistic details of MCO are currently unknown, such mechanistic information would greatly benefit formulation scientists in the rational design and analysis of protein formulations. Here, we describe the Fenton oxidation (by Fe2+/H2O2) of several Met-, Tyr-, and His containing model peptides, including one derivative containing a conformationally restricted norbornyl Met analogue (Nor), Nor-Gly-His-Met-NH2. Our results will provide evidence for a metal-bound reactive oxygen species selectively oxidizing Met to Met sulfoxide, indicating a Met-specific oxidant and arguing against the involvement of freely diffusible hydroxyl radicals. The Fenton oxidation of Nor-Gly-His-Met-NH2 yields a 2:1 preference for sulfoxide formation at the C-terminal Met versus the N-terminal Nor residue, respectively, while incubation of the peptide with H2O2 alone results in a 1:1 ratio. These results are rationalized by the better access of the thioether side chain of the flexible C-terminal Met residue to the peptide-bound iron compared with the conformationally restricted Nor residue. It is commonly believed that Fenton oxidation reactions involve hydroxyl radicals, and that Met oxidation in proteins is predominantly controlled by the surface-accessibility of the respective Met residues. However, occasionally protein oxidation in formulations shows selectivities, which are not consistent with these paradigms. Our results demonstrate additional features of the Fenton reaction such as the formation of a metal-bound oxidant specific for Met (and not Tyr or His), which may assist formulation scientists in the rationalization of unexpected oxidation selectivities. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1122–1130, 2004