基于多光谱技术分析绿原酸与肌原纤维蛋白相互作用的初步研究

Preliminary Study of the Interaction between Chlorogenic Acid and Myofibrillar Protein Based on Multispectral Techniques

本研究以高白鲑肌原纤维蛋白(Myofibrillar Protein,MP)为研究对象,探究不同浓度(6、30、150、300μmol/g)绿原酸(Chlorogenic Acid,CA)与MP相互作用的结构变化及结合性质。在25℃下孵育形成不同CA-MP复合物,通过测定复合物的理化性质(总巯基,表面疏水性),二级,三级结构变化(傅里叶红外光谱、同步荧光、三维荧光),结合性质(紫外光谱,荧光猝灭及热力学参数)分析CA-MP复合物相互作用情况。结果表明,在300μmol/g CA存在下,蛋白总巯基含量和表面疏水性分别骤降25.65%,40.26%(P<0.05);蛋白质二级结构随之展开,α-螺旋含量下降至11.73%,造成蛋白结构稳定性降低;同步荧光与三维荧光光谱表明CA对蛋白荧光具有猝灭作用,且色氨酸比酪氨酸的猝灭效果高7.90%。紫外光谱和荧光猝灭证实CA-MP形成复合物且二者的结合为静态猝灭,热力学参数进一步验证二者相互作用的主导力为疏水作用力。本研究旨在为多酚与高白鲑MP相互作用提供有价值的信息,为后续对不同结构多酚与高白鲑肌球蛋白的相互作用机制提供研究基础。

In this study,the binding properties and structural changes associated with the interaction of Coregonus peled myofibrillar protein(MP)and different concentrations(6,30,150,and 300μmol/g)of chlorogenic acid(CA)were investigated.The CA-MP complexes were formed at 25°C,and the physical and chemical properties(total sulfhydryl groups,surface hydrophobicity),changes in secondary and tertiary structure(measured by Fourier-infrared spectroscopy,synchrotron fluorescence,and 3D fluorescence),and binding properties(measured by UV spectra,fluorescence quenching,and thermodynamic parameters)of the CA-MP complexes were measured.The results showed that at concentrations of 300μmol/g CA,the total sulfhydryl content and surface hydrophobicity of MP decreased abruptly by 25.65%and 40.26%,respectively(P<0.05).Unfolding of the MP secondary structure and reductions in theα-helix content to 11.73%were observed,adversely affecting the structural stability of the protein.The synchronous and three-dimensional fluorescence spectra of MP showed that CA led to quenching of the protein fluorescence,with tryptophan showing 7.90%higher quenching compared with tyrosine.The UV spectra and fluorescence quenching data confirmed the formation of the CA-MP complexes,with complexes formation inducing static quenching,while the thermodynamic parameters further confirmed that hydrophobic forces dominated the interaction.The findings of the study provide valuable information on the interaction between polyphenols and Coregonus peled MP,as well as a reference for further investigations into the mechanisms underlying interactions between Coregonus peled myosin and polyphenols of different structures.