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Journal of Biochemistry Advance Access published online on January 16, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp003
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Protein Oxidation During Long Storage: Identification of the Oxidation Sites in Dihydrofolate Reductase from Escherichia coli through LC-MS and Fragment Studies

Tatsuyuki Takenawa*, Akiko Yokota*, Masanao Oda, Hisashi Takahashi and Masahiro Iwakura#

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

#To whom correspondence should be addressed: Masahiro Iwakura: Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan Tel: +81-29-861-6179 Fax: +81-29-856-4055 E-mail: masa-iwakura{at}aist.go.jp

Received November 14, 2008; Accepted January 7, 2008


  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, namely 18 months, comprised molecular species with molecular masses increased by 16-, 32-, and 48-daltons. 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.

Key Words: dihydrofolate reductase, fragment studies, liquid chromatography-mass spectrometry (LC-MS), protein aging, protein oxidation

* The first two authors contributed equally to this work.


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