Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa, 761-0795, Japan.
Department of Applied Bioresource Science, The United Graduate School of Agricultural Sciences, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566 Japan.
Poult Sci. 2018 May 1;97(5):1730-1737. doi: 10.3382/ps/pey024.
Chicken egg white protein ovalbumin (OVA) undergoes a conversion to a more thermostable form by alkali treatment, which is assumed to be involved in the physiological functions of OVA. Ovalbumin-related protein X (OVAX), a chicken egg white protein, has 77% sequence similarity to OVA and binds to heparin. In this study, structure characteristics and heparin-binding affinity of alkali-treated OVAX were investigated. Cation-exchange chromatography using SP Sepharose resin showed that alkali treatment (pH 10, 55°C) of OVAX induces the occurrence of a distinct OVAX form with a less positive-charge (acidic OVAX). Circular dichroism and tryptophan-fluorescence analyses showed that the newly-formed acidic OVAX form has an 8% lower α-helical content than its native counterpart, while there is no significant difference in steric environments around tryptophan residues between the 2 forms. The OVAX structure built by homology-modeling showed that OVAX possess a basic cluster domain with α-helix equivalent to 7% of total secondary structures, which does not contain any tryptophan residues. These results suggest that, during alkali treatment, OVAX undergoes mainly a conformational change of the α-helical basic cluster domain and thereby forms acidic OVAX. Acidic OVAX induced by alkali treatment exhibited weaker interactions with Heparin Sepharose resin than native OVAX did. Our results suggest that OVAX basic cluster domain is likely a specific binding site of heparin. Consequently, it is suggested that alkali treatment causes the collapse of the OVAX heparin binding site, which might participate in regulating the functions of heparin.
鸡卵清白蛋白卵清蛋白（OVA）通过碱处理转化为更热稳定的形式，这被认为涉及 OVA 的生理功能。卵清白蛋白相关蛋白 X（OVAX）是一种鸡卵清白蛋白，与 OVA 有 77%的序列相似性，并与肝素结合。在这项研究中，研究了碱处理 OVAX 的结构特征和肝素结合亲和力。使用 SP Sepharose 树脂的阳离子交换层析表明，OVAX 的碱处理（pH 10，55°C）诱导出现一种明显的 OVAX 形式，带正电荷较少（酸性 OVAX）。圆二色性和色氨酸荧光分析表明，新形成的酸性 OVAX 形式的α-螺旋含量比其天然对应物低 8%，而两种形式的色氨酸残基周围的空间环境没有显著差异。同源建模构建的 OVAX 结构表明，OVAX 具有一个碱性簇结构域，α-螺旋相当于总二级结构的 7%，其中不包含任何色氨酸残基。这些结果表明，在碱处理过程中，OVAX 主要经历α-螺旋碱性簇结构域的构象变化，从而形成酸性 OVAX。碱处理诱导的酸性 OVAX 与肝素琼脂糖树脂的相互作用比天然 OVAX 弱。我们的结果表明，OVAX 碱性簇结构域可能是肝素的特定结合位点。因此，建议碱处理导致 OVAX 肝素结合位点的崩溃，这可能参与调节肝素的功能。
