植物多酚-牛血清白蛋白相互作用及对蛋白质结构的影响

Polyphenol-bovine serum albumininteraction and its influence on protein structure

多酚与蛋白质的相互作用取决于蛋白质和多酚的结构与类型,为研究不同植物多酚与牛血清白蛋白(bovine serum albumin,BSA)之间的相互作用,探明不同植物多酚对BSA结构的影响,进而筛选出稳定的植物多酚-BSA复合物。该研究采用原花青素(proanthocyanidins,PC)、儿茶素(catechin,C)、表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)、茶多酚(tea polyphenol,TP),通过紫外光谱法、荧光光谱法、红外光谱法和分子对接研究植物多酚与BSA相互作用及其对蛋白质结构的影响。结果表明:4种植物多酚-BSA复合物的紫外最大波长红移由大到小为EGCG(365 nm)、PC(364 nm)、C(364 nm)、TP(363 nm);红外光谱发现,酰胺Ⅰ带的吸收峰(1657.44 cm^(−1))发生蓝移由大到小为PC(1656.15 cm^(−1))、TP(1632.07 cm^(−1))、EGCG(1631.49 cm^(−1))、C(1631.44 cm^(−1)),烟酰胺II带峰形变宽;BSA二级结构由β-转角、无规卷曲转变为β-折叠、ɑ-螺旋,β-折叠含量增加C-BSA(40.20%)、PC-BSA(39.50%)、EGCG-BSA(39.32%)、TP-BSA(34.04%),表明C更能促进BSA的二级结构稳定;荧光光谱表明4种多酚对BSA的猝灭类型均为静态猝灭,且结合位点约为1,表明存在一个结合位点;分子对接结果表明该结合位点位于Sub-domain IIA的疏水腔中,分子间相互作用力主要是氢键、疏水作用力,结合能由小到大为C(−3.72 kJ/mol)、EGCG(−1.77 kJl/mol)、PC(−1.02 kJ/mol)、TP(−0.38 kJ/mol);4种多酚中C和EGCG与BSA相互作用程度较大,C与BSA的结合能最小,形成的复合物最稳定。通过多酚与蛋白相互作用研究,更好地发挥多酚和蛋白两种组分的功能作用,可为开发功能性多酚-蛋白质复合物产品提供参考。

The interaction of polyphenols with proteins depends on the structure and type of protein and polyphenols.This study aims to determine the interaction between different plant polyphenols and bovine serum albumin(BSA),as well as their influence on protein structure,proanthocyanidins(proanthocyanidins,PC),catechin(catechin,C),epigallocatechin gallate(Epigallocatechin gallate,EGCG),and tea polyphenols(TEA polyphenol,TP).The BSA was combined to form a polyphenols-BSA.The interaction of polyphenols with BSA and their effects on protein structure were analyzed by UV spectroscopy,fluorescence spectroscopy,infrared spectroscopy,and molecular docking.The results showed that the maximum absorption peak of BSA at 286 nm all shared an upward trend in a redshift with the increase of PC,C,EGCG and TP added.There was the UV maximum wavelength redshift EGCG(292 nm)>PC(288 nm),C(288 nm),TP(288 nm).The redshifted absorption peak was attributed to the EGCG as a monomer,more accessible to the hydrophobic amino acid residues of protein molecules.The fluorescence intensity of BSA decreased gradually,as the concentration of the four polyphenols increased.The polyphenols were interacted with BSA to make more tyrosine or tryptophan exposure,eventually resulting in the lower fluorescence intensity.There was the fluorescence spectroscopy maximum wavelength redshift EGCG>PC,C>TP(365 nm>364 nm,364 nm>363 nm).Fluorescence spectrum indicated the static quenching of all four types of polyphenols to BSA.The rate constant of protein quenching of polyphenols was 1013 at temperatures of 290 and 300 K.The Ksv of EGCG-BSA decreased most outstandingly,when the temperature increased.The catechol structure in the EGCG molecule was broken or deformed at high temperature.The binding constant KA with temperature was the same as the quenching constant Ksv.The higher the temperature was,the smaller the binding constant KA was.All level 103 demonstrated the weak interaction force between the four polyphenols and BSA.The binding site n was about 1,indicating the presence of a binding site.The fluorescence intensity of BSA gradually decreased with the concentration of the four polyphenols increased.Therefore,both polyphenols were interacted with BSA to make more tyrosine or tryptophan exposure,eventually resulting in lower fluorescence intensity.The infrared spectroscopy indicated that four polyphenols altered the secondary structure of BSA,and then promoted the protein structure stabilization.Amide I band absorption peak(1657.44 cm^(−1))occurred the blue-shifted PC>TP>EGCG>C(1656.15 cm^(−1)>1632.07 cm^(−1)>1631.49 cm^(−1)>1631.44 cm^(−1)).Nicotinamide II band peak shape was widening.The hydrogen bond of the BSA increased after the interaction.After the addition of TP,PC,and C,the BSA secondary structure was changed fromβ-turn,random coil toβ-sheet,ɑ-helix,and the relative content ofβ-sheet,ɑ-helix increases,indicating the enhanced compactness of BSA conformation.Specifically,β-sheet content increased in the order of C-BSA(40.20%)>PC-BSA(39.50%)>EGCG-BSA(39.32%)>TP-BSA(34.04%),indicated that the C was better promoted the secondary structure stability of BSA.Molecular docking results indicated that the binding site was located in the hydrophobic cavity of Sub-domain IIA.Intermolecular interaction forces were mainly hydrogen bonding,and hydrophobic forces,in the order of the Binding energy C(−3.72 kJ/mol)<EGCG(−1.77 kJ/mol)<PC(−1.02 kJ/mol)<TP(−0.38 kJ/mol).The molecular docking verified that the four polyphenols were bound stably to BSA.The addition of polyphenols made the structure of BSA more compact and stable.The different polyphenols affected the BSA structure,among which C-BSA and EGCG-BSA structures were the most stable.Different plant polyphenols-BSA interactions were clarified to analyze their effects on BSA structure for the better structural stability of the BSA.The interaction of polyphenols and proteins can be expected to regulate the characteristics of proteins during processing,storage and transport.The finding can also provide some theoretical reference for the preparation and development of new polyphenol-protein complex products.