Oxidation of Methionine Residues in Recombinant Human Interleukin-1 Receptor Antagonist:  Implications of Conformational Stability on Protein Oxidation Kinetics

Abstract

Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.

Stability of Protein Pharmaceuticals: An Update

“…Some evidence has also been presented that the rate of Met oxidation is linked to or correlated with conformational stability (207)(208)(209). Moreover, near the melting temperature of the protein, one can observe non-Arrhenius kinetics due to large-scale structural changes (208).…”

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

“…[7][8][9] In contrast to other chemical degradation pathways, the activation energy of methionine oxidation is rather low, which explains the focus on its role as one of the main potential causes of protein degradation during refrigerated storage. 10,11 When investigating the potential effect of chemical modifications on PK properties in vitro, analysis of the FcRn/IgG interaction is of vital importance. FcRn is a heterodimeric receptor consisting of two polypeptides, a 48-52 kDa class I major histocompatibility complex-like protein (a-FcRn) and a 14 kDa b2microglobulin (b2 min).…”

Effect of Arginine on Pre-nucleus Stage of Interferon Beta-1b Aggregation

“…Protein conformational stability may help to limit protein oxidation (30). However, once oxidation occurs, more destabilization of therapeutic protein structure happens which results in more conformational changes and decreases in stability (30,31).…”

“…However, once oxidation occurs, more destabilization of therapeutic protein structure happens which results in more conformational changes and decreases in stability (30,31). The tertiary structure of oxidized proteins is thermodynamically unstable, and therefore, oxidized proteins tend to expose hydrophobic amino acids to the outside environment which may lead to monomer association.…”