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Abstract

In this study, hydrogen peroxide was used to study the oxidation of rhRlx under various conditions. Oxidation of rhRlx occurred at both of the two methionines on the B chain, Met B(4) and Met B(25), as expected from the three-dimensional structure of the molecule, which shows that these two residues are located on the surface of the molecule and exposed to solvent. The reaction produced three different oxidized forms of rhRlx containing either Met B(4) sulfoxide, Met B(25) sulfoxide, or both residues oxidized. The corresponding sulfone was not formed under these conditions. The oxidation at the two methionines proceeded independently from each other but Met B(25) was oxidized at a significantly faster rate than Met B(4). The fact that the rate of oxidation at Met B(25) was identical to the rate of oxidation of free methionine and that of two model peptides mimicking the residues around Met B(4) and Met B(25) suggests that the lower reactivity at Met B(4) was due to steric hindrance, and at least in this case, neighboring groups do not influence the oxidation kinetics of methionine residues. The reaction was independent of pH, ionic strength, and buffer concentration in the range studied. The enthalpy of activation for the reaction was approximately 10-14 kcal mol-1, with an entropy of activation of the order of -30 cal K-1 mol-1. These data are consistent with previously published mechanisms for organic sulfide oxidation by alkyl hydroperoxides.

Identification of Potential Sites for Tryptophan Oxidation in Recombinant Antibodies Using tert-Butylhydroperoxide and Quantitative LC-MS

“…The oxidative degradation of pharmaceutical proteins has been reviewed by several authors [1], [2], [3], [4]. Amino acid residues that are particularly susceptible to oxidation include cysteine, methionine, tryptophan, histidine, and tyrosine, in that order.…”

Identification and characterization of oxidation and deamidation sites in monoclonal rat/mouse hybrid antibodies

“…Methionine oxidation results in the formation of methionine sulfoxide and, under extreme conditions, sulfones. Peroxides have been widely used for studying the kinetics and mechanisms of methionine oxidation in proteins [8][9][10]. Peroxides react with metal ions to form free radicals that can initiate oxidation of proteins [11,12].…”

Antioxidants for Prevention of Methionine Oxidation in Recombinant Monoclonal Antibody HER2

“…8 In fact, peroxides such as hydrogen peroxide have been widely used for studying the kinetics and mechanisms of methionine oxidation in proteins. [9][10][11] Peroxides react with metal ions to form free radicals that can initiate oxidation of proteins. 12,13 Methionine can also be photooxidized by a free radical pathway, 14 or via singlet oxygen intermediate formation.…”