title: Oxidized recombinant human growth hormone that maintains conformational integrity.
abstract: Chemical degradations often induce changes in protein conformation and thus influence protein activity and protein stability in solutions. One difficulty in studying of chemical degradations on protein aqueous properties is to obtain sufficient amount of chemically degraded protein which is well characterized. Chemical degradation protocols that are often used may induce also conformation changes and aggregation of the protein. In this article we studied the effect of methionine oxidation on the conformation of recombinant human growth hormone (r-hGH). In literature it is reported that oxidation of methionine residues induces conformation changes on r-hGH. In our study, oxidation of r-hGH was performed by incubation with hydrogen peroxide under mild conditions. Mass spectrometry and chromatographic analysis revealed that oxidation with hydrogen peroxide resulted in more than 90% of oxidized r-hGH. By extensive spectroscopic characterizations no detectable change in conformation and aggregation of r-hGH after oxidation was found. In conclusion, mild oxidation conditions led to selective oxidation of the two more accessible methionine residues of r-hGH (Met(14) and Met(125)) and did not results in any conformation change of the protein. These findings prove that oxidation of human growth hormone does not influence protein conformation and demonstrate the importance of employing mild conditions during production of oxidized protein.
authors.affiliation: Department of Pharmaceutics and Biopharmaceutics, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CH-1211 Geneva 4, Switzerland.
authors.forename: Filippo
authors.initials: F
authors.lastname: Mulinacci
authors.forename: Steven E J
authors.initials: SE
authors.lastname: Bell
authors.forename: Martinus A H
authors.initials: MA
authors.lastname: Capelle
authors.forename: Robert
authors.initials: R
authors.lastname: Gurny
authors.forename: Tudor
authors.initials: T
authors.lastname: Arvinte
mesh_terms: D000465:Algorithms; D002626:Chemistry, Pharmaceutical; D002942:Circular Dichroism; D019382:Human Growth Hormone / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013059:Spectrum Analysis, Raman; D014465:Ultrasonics / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D019382:Human Growth Hormone; D008715:Methionine; D006861:Hydrogen Peroxide