title: Protein oxidation during long storage: identification of the oxidation sites in dihydrofolate reductase from Escherichia coli through LC-MS and fragment studies.
abstract: An LC-MS study revealed some heterogeneity in terms of molecular mass of a cysteine-free mutant of dihydrofolate reductase (DHFR) after long storage of the highly purified protein as an ammonium sulfate precipitate, but not in the case of a cysteine- and methioneine-free mutant of DHFR. One-third of the cysteine-free DHFR sample stored for a long time, around 18 months, comprised molecular species with molecular masses increased by 16, 32 and 48 Da. A peptide mapping study revealed that at least one of the methionine residues at positions 1, 16 and 20 was oxidatively modified to a methione-sulfoxide residue, while those at positions 42 and 92 were essentially unaffected. Each of the oxidized species of the DHFR exhibiting different degrees or sites of oxidation was further purified to essentially homogeneity in terms of molecular mass from the stored sample, and its enzyme activity was determined. One oxidized DHFR showed higher activity than that of the non-oxidized enzyme, while the other four oxidized DHFRs showed less activity. This agrees with the observation that the enzyme activity of the stored sample, a mixture in terms of oxidation, was apparently the same as that of the non-oxidized enzyme. This suggests that the activity itself is not a proper measure for quality control of proteins.
authors.affiliation: Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Tatsuyuki
authors.initials: T
authors.lastname: Takenawa
authors.forename: Akiko
authors.initials: A
authors.lastname: Yokota
authors.forename: Masanao
authors.initials: M
authors.lastname: Oda
authors.forename: Hisashi
authors.initials: H
authors.lastname: Takahashi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Iwakura
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; D010446:Peptide Fragments; D006861:Hydrogen Peroxide; D013762:Tetrahydrofolate Dehydrogenase