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  • Transition of serine residues to the D-form during the conversion of ovalbumin into heat stable S-ovalbumin.

Transition of serine residues to the D-form during the conversion of ovalbumin into heat stable S-ovalbumin.

Journal of pharmaceutical and biomedical analysis (2015-05-20)
Tetsuya Miyamoto, Nobuyuki Takahashi, Masae Sekine, Tetsuhiro Ogawa, Makoto Hidaka, Hiroshi Homma, Haruhiko Masaki
要旨

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.

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製品内容

Sigma-Aldrich
重水, 99.9 atom % D
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重水, 99.9 atom % D, contains 0.05 wt. % 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt
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塩化重水素 溶液, 35 wt. % in D2O, ≥99 atom % D
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重水, 99.9 atom % D, contains 0.75 wt. % 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt
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リン酸カリウム, 三塩基酸, reagent grade, ≥98%
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アルブミン ニワトリ卵白由来, lyophilized powder
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重水, filtered, 99.8 atom % D
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重水, 70 atom % D
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重水, 99.9 atom % D, contains 1 % (w/w) 3-(trimethylsilyl)-1-propanesulfonic acid, sodium salt (DSS)
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塩化重水素 溶液, 1.0 M in diethyl ether, 97.5 atom % D
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重水, 60 atom % D