光谱法研究Zn2+对枸杞超氧化物歧化酶JZ活力和结构的影响

针对提取的天然抗氧性物质超氧化物歧化酶量少,稳定性差等问题,实验以枸杞为原料,采用硫酸铵盐析分级分离方法提取了枸杞超氧化物歧化酶(superoxide disutase of lycium,SOD-LY),紫外光谱法确定了其类型;考察了不同种类的锌盐ZnSO4,ZnCl2,Zn(CH3COO)2和Zn(NO3)2对SOD-LY活力的影响,选择出能显著提高其活力的锌盐.采用傅里叶变换红外光谱、紫外光谱、荧光光谱法研究了最佳锌盐同SOD-LY分子的作用方式.结果表明,提取的SOD-LY 属于Cu/Zn-SOD类型.不同浓度锌盐的存在均可影响SOD-LY的活性.其中,Zn(NO3)2对SOD-LY活力影响最大;红外光谱表明,Zn(NO3)2使SOD-LY二级结构中β-片层、β-转角和β-反平行结构减少,α-螺旋和无规则卷曲结构增多.荧光光谱分析得出,Zn2+与SOD-LY之间的相互作用力主要为静电引力,有利于稳定SOD-LY的活性中心,使得SOD-LY活力增强.Zn2+与SOD-LY结合常数(KΛ)为1.666×103 L·mol-1,结合位点数(n)为1.238,结合距离(r0)为3.62 nm,该研究为提高SOD的活力、稳定性及扩展其应用提供理论帮助.

The Activity and Structure of Superoxide Dismutase of Lycium in the Existence of Zn2+ with Spectroscopy

As an important natural antioxidant,superoxide dismutase has been widely applied in fields such as diseases treatment as well as food,cosmetics and agriculture industries.In view of the poor stability of extraction quantity of SOD ,the extraction of superoxide disutase from Lycium (SOD-LY) was carried out by salting-out separation with different levels of ammonium sulphate and reprecipitation with acetone in sequence.Its type was determined with ultraviolet spectrometry.The influence of different types of zinc salts such as ZnSO4,ZnCl2,Zn(CH3COO)2,Zn(NO3)2 on activity of SOD-LY was investigated.The zinc salt enhancing its activity effectively was selected.The mode of interaction between the zinc salt selected and SOD-LY was studied with Fourier transform infrared spectroscopy,ultraviolet spectrum and fluorescence spectrometry.The analysis of results of ultraviolet spectrometry had shown that the SOD-LY extracted in the experiments consisted the type of Cu/zinc-SOD.The activity of SOD-LY can be influenced by different concentration of zinc salts such as zinc nitrate,zinc acetate,zinc chloride,sulfuric acid zinc and ethylene glycol.Among the investigated zinc salts,the activity of SOD-LY can be enhanced through using Zn(NO3)2 greatly.The analysis of infrared spectroscopy demonstrated that β-sheet layer,β-corner and β-antiparallel all decrease,however,α-helix and unordered curling increases for the secondary structure of SOD-LY in the presence of Zn(NO3)2,This may be due to the presence of weakly interacting complex between them.Analysis of fluorescence spectrometry indicated that the fluorescence intensity of SOD-LY decreased due to the addition of Zn(NO3)2 whose binding constant was 1.666×103 L·mol-1 while the binding site number was 1.238 according to the double logarithmic regression curve analysis.The distance is 3.62 nm by overlapping coverage area of the UV absorption spectra of SOD-LY and the fluorescence emission spectra of Zn(NO3)2-SOD-LY.The main action existed between Zn2+ and SOD-LY is electrostatic forces (ΔH<0,ΔS>0).The study has a great theoretical significance for increasing the activity and stability of SOD,including the extension of its application.