不同种类酸诱导罗非鱼肌球蛋白去折叠行为的研究

Unfolding beheavior of Tilapia myosin induced by different acids

以罗非鱼肉为原料,采用硫酸铵沉淀法提取肌球蛋白,固定蛋白浓度2 mg/m L,分别采用HCl、H2SO4、H3PO4和C6H8O7(Citric acid)调节体系p H值为2.0、3.0,以溶解度、表面疏水性、总巯基和活性巯基含量、色氨酸荧光光谱和圆二色谱为指标,研究不同种类酸诱导肌球蛋白去折叠过程中分子结构的变化。结果表明,肌球蛋白经过酸处理后分子部分展开,表面疏水性和活性巯基含量增加,色氨酸荧光强度下降、α-螺旋含量降低,且p H2.0条件下处理的肌球蛋白去折叠程度大于p H3.0;比较而言,p H2.0条件下,C6H8O7处理的肌球蛋白展开程度最大,α-螺旋含量由41.7%降低至20.5%;p H3.0条件下H2SO4处理的肌球蛋白结构展开程度最大,溶解性下降最明显;而HCl诱导的肌球蛋白去折叠过程中,蛋白分子变性程度最小,α-螺旋含量下降不明显,分子稳定性较好,呈"熔球态"构象。

Myosin was extracted from Tilapia meat using ammonium sulfate precipitation in this study. The acid-induced unfolding was carried out at pH2.0 and pH 3.0 by HC1, H2SO4, H3PO4 and C6H8O7 at protein concentration of 2 mg/mL. Solubility, surface hydrophobicity, total and active sulfhydryl content, intrinsic tryptophan fluorescence spectra and circular dichroism were determined, changes of conformation of tilapia myosin during acid- induced unfolding were studied. Results showed that surface hydrophobicity and reactive SH content of myosin increased by different acid treatments, tryptophan fluorescence intensity and α-helix content decreased, suggesting acid-induced unfolding of myosin. And at pH 2.0, myosin had a greater degree of unfolding than that at pH 3.0. In comparison, theC6H8O7-induced unfolding degree was the highest at pH 2.0 and the α-helix content of myosin decreased from 41.7% to 20.5%. However, at pH 3.0, the H2SO4-induced unfolding degree was the highest, and resulted in the significantly decline of solubility. The denaturation of myosin during HCl-induced unfolding was the minimum, and no obvious change of α-helix content was obtained, indicating that myosin molecules was stable and retained a “molten globule” state.