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
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DOI：
10.1016/j.jpba.2015.04.030
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发表时间：
2015-12-10
影响因子：
3.4
通讯作者：
Masaki, Haruhiko
中科院分区：
文献类型：
--
作者：
Miyamoto, Tetsuya;Takahashi, Nobuyuki;Masaki, Haruhiko
蛋清蛋白是蛋清中的一种主要蛋白质，在有壳鸡蛋的储存过程中，它被转化为一种更耐热的分子形式，称为s -卵清蛋白。我们之前的x射线晶体学研究表明，s -卵白蛋白含有三个D-Ser残基（S164， S236和S320），这可能是其热稳定性的原因。在这里，我们使用氨基酸α -质子氘标记和液相色谱-串联质谱（LC-MS/MS）相结合的技术证实了这些D-Ser残基在卵清蛋白中的存在。取蛋清卵清蛋白和重组卵清蛋白，分别在pH 9.5和37℃条件下孵育约12天，然后在DCl/D2O蒸汽中水解，与4-氟-7-硝基-2,1,3-苯并恶二唑（NBD-F）衍生，采用LC-MS/MS进行分析。在7天的时间内，观察到天然卵清蛋白中D-Ser含量的时间依赖性增加，达到约8%。这对应于每个分子三个丝氨酸残基的值，并且与我们之前基于晶体学分析的预测一致。重组卵清蛋白得到了几乎相同的结果。然后，我们使用该技术研究了在轻度碱性条件下其他蛋白质中是否会出现d -氨基酸残基，并在某些蛋白质中检测到少量但数量显著的D-Ala和/或D-Ser残基，这些残基以时间依赖性的方式增加。(C) 2015 Elsevier B.V.版权所有
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 alpha-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 degrees 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. (C) 2015 Elsevier B.V. All rights reserved.