title: Transition of serine residues to the D-form during the conversion of ovalbumin into heat stable S-ovalbumin.
abstract: Ovalbumin, a major protein in chicken egg white, is converted into a more thermostable molecular form, known as S-ovalbumin, during the storage of shell eggs. Our previous X-ray crystallographic study indicated that S-ovalbumin contains three D-Ser residues (S164, S236, and S320), which may account for its thermostability. Here, we confirmed the presence of these D-Ser residues in ovalbumin using a technique combining deuterium labeling of α-protons of amino acids and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ovalbumin from chicken egg white and recombinant ovalbumin were incubated for approximately 12 days at pH 9.5 and 37°C. They were then hydrolyzed in DCl/D2O vapor, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and analyzed by LC-MS/MS. A time-dependent increase in the D-Ser contents in native ovalbumin was observed over a period of 7 days, reaching approximately 8%. This corresponds to a value of three serine residues per molecule, and is consistent with the prediction based on our previous crystallographic analysis. Nearly identical results were obtained with recombinant ovalbumin. We then used this technique to investigate whether D-amino acid residues could arise within other proteins under mild alkaline conditions and detected small but significant amounts of D-Ala and/or D-Ser residues that increased in a time-dependent manner in some proteins.
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; Department of Pharmaceutical and Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Miyamoto
authors.affiliation: Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takahashi
authors.affiliation: Department of Pharmaceutical and Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Masae
authors.initials: M
authors.lastname: Sekine
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Tetsuhiro
authors.initials: T
authors.lastname: Ogawa
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hidaka
authors.affiliation: Department of Pharmaceutical and Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address: hommah@pharm.kitasato-u.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Homma
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: amasaki@mail.ecc.u-tokyo.ac.jp.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Masaki
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D006358:Hot Temperature; D010047:Ovalbumin / Q000737:chemistry* / Q000378:metabolism; D055550:Protein Stability; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: C104026:S-ovalbumin; D012694:Serine; D010047:Ovalbumin
keywords: Isomerization; LC–MS/MS; Ovalbumin; d-Amino acid; d-Serine