Alpinetin (Alp),a main active component ofAlpinia katsumadai,was used to study the mechanism underlying its interaction with hemoglobin (BHG) by fluorescence polarization,synchronous fluorescence,3D fluorescence and absorption spectroscopy combined with molecular modeling technique under physiological conditions.The data from the overlapping spectra confirmed that the interaction between Alp and BHG could be matched with the non-radioactive energy transfer theory.Various binding constants (5.880 × 104,4.337 × 104 and 4.935 × 104 L/mol for quenching mechanism;3.239 × 103,5.225 × 103 and 7.692 × 103 L/mol for sensitization effect) and thermodynamic parameters (△ S:182.073 J/(mol.K),△H:34.320 kJ/mol,△ G:-20.119 (299 K),-21.940 (309 K) and-23.760 kJ/mol (319 K)) for Alp-BHG systems were hereby obtained under different temperatures (299,309 and 319 K) based on different action mechanisms.Molecular docking was performed to reveal the binding of Alp moiety to the hydrophobic cavity of BHG and the acting force was mainly a hydrophobic interaction.The low anisotropy values suggested that there was shorter relaxation time and Alp molecules were observed in a motionally unrestricted environment introduced by BHG.The synchronous fluorescence and absorption spectra showed that the addition of Alp impacted the microenvironment around BHG in aqueous solution.The 2D and 3D fluorescence spectroscopy of Alp-BHG system indicated that Alp strongly quenched the intrinsic fluorescence of BHG causing a conformational change of the protein.The results indicated that the interaction between Alp and BHG was strong,implying that Alp may be stored and transferred by BHG in some degree.In addition,the effects of common ions on the constants of alpinetin-BHG complex were also discussed,indicating an increasing trend for binding constants in the present of different metal ions.%利用多种荧光光谱法、紫外光谱法并结合分子模拟等方法,表征模拟生理条件下草豆蔻中的主要活性组分山姜素(alpinetin,Alp)影响牛血红蛋白(bovine hemoglobin,BHG)的结构信息,探讨Alp与BHG的作用机制.重叠光谱数据证实,Alp与BHG的相互作用符合F(o)rster非辐射能量转移,据此依据不同的作用机理,获得3种温度(299、309、319 K)条件下的键合常数(猝灭机理为5.880×104、4.337×104 L/mol及4.935×104 L/mol;增敏机理为3.239×103、5.225×103 L/mol及7.692×103 L/mol)及热力学常数(△S:182.073 J/(mol·K),△H:34.320 kJ/mol,△G:-20.119 (299 K)、-21.940 kJ/mol (309 K)及-23.760kJ/mol (319K));分子模拟表明,Alp键合位点于BHG分子的疏水腔内,并与BHG的键合模式主要是疏水作用;荧光偏振显示,Alp与BHG结合后生成的配合物弛豫时间较短,结合的较松;同步荧光及紫外光谱证实,Alp的存在影响了BHG的微环境;二维及三维荧光光谱表明,Alp可以猝灭BHG的内源荧光,使其构象发生变化.结论表明,BHG对Alp有较强的结合能力,提示BHG对Alp在一定程度上可起到贮存和转运的作用.另外,考察了几种共存金属离子对Alp与BHG相互作用的影响,表明加入共存离子后的键合常数均有较大增加.
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