In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization.
The effect of oxidation of the methionine residues of Escherichia coli-derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine-containing peptides, using RP-HPLC of Asp-N endoproteinase digests. The initial oxidation rates for Met159, Met-1, Met27, Met36, and Met48 were 0.11 min-1, 0.098 min-1, 0.033 min-1, 0.0063 min-1, and 0.00035 min-1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295-nm Trp peak in the near-UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two- and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.
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Amgen Inc., Amgen Center, Thousand Oaks, California 91320, USA. ahsu@amgen.com
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D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D017434:Protein Structure, Tertiary; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D019089:Stem Cell Factor / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007457:Iodine Radioisotopes; D016877:Oxidants; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D008715:Methionine; D006861:Hydrogen Peroxide; D019009:Proto-Oncogene Proteins c-kit; D014357:Trypsin; D013936:Thymidine