title: Comparative oxidation studies of methionine residues reflect a structural effect on chemical kinetics in rhG-CSF.
abstract: The effect of protein conformation on the rate of chemical degradation is poorly understood. To address the role of structure on chemical degradation kinetics, comparative oxidation studies of methionine residues in recombinant human granulocyte colony-stimulating factor (rhG-CSF) were performed. The kinetics of oxidation of methionine residues by hydrogen peroxide (H2O2) in rhG-CSF and corresponding chemically synthesized peptides thereof was measured at different temperatures. To assess structural effects, equilibrium denaturation experiments also were conducted on rhG-CSF, yielding the free energy of unfolding as a function of temperature. A comparison of the relative rates of oxidation of methionine residues in short peptides with those of corresponding methionine residues in rhG-CSF yields an understanding of how protein tertiary structure affects oxidation reactions. For the temperature range that was studied, 4-45 degrees C, the oxidation rate constants followed an Arrhenius equation quite well, suggesting the lack of temperature-induced local structural perturbations that affect chemical degradation rates. One of the four methionine residues, Met 122, exhibited an activation energy significantly different from that of the corresponding peptide. Extrapolation of kinetic data predicts non-Arrhenius behavior around the melting temperature. Three phenomenological models based on different mechanisms are discussed, and an application to shelf life prediction of pharmaceuticals is presented.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room E19-502B, Cambridge, Massachusetts 02139, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Pan
authors.forename: Jeff
authors.initials: J
authors.lastname: Abel
authors.forename: Margaret S
authors.initials: MS
authors.lastname: Ricci
authors.forename: David N
authors.initials: DN
authors.lastname: Brems
authors.forename: Daniel I C
authors.initials: DI
authors.lastname: Wang
authors.forename: Bernhardt L
authors.initials: BL
authors.lastname: Trout
mesh_terms: D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D014364:Tryptophan; D008715:Methionine