title: 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.
authors.affiliation: Center for Pharmaceutical Biotechnology, Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: Renuka
authors.initials: R
authors.lastname: Thirumangalathu
authors.forename: Sampathkumar
authors.initials: S
authors.lastname: Krishnan
authors.forename: Pavel
authors.initials: P
authors.lastname: Bondarenko
authors.forename: Margaret
authors.initials: M
authors.lastname: Speed-Ricci
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
authors.forename: David N
authors.initials: DN
authors.lastname: Brems
mesh_terms: D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000737:chemistry*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins; D013696:Temperature; D013816:Thermodynamics
chemical_list: D053590:Interleukin 1 Receptor Antagonist Protein; D011994:Recombinant Proteins; D008715:Methionine