基于光谱技术分析不同还原糖对Ara h2糖基化反应的影响

Analysis of the Effect of Different Reducing Sugars on Ara h2Glycation Based on Spectral Technology

糖基化反应能诱导食品中蛋白质的结构发生改变;Ara h2是花生中的主要蛋白组分之一,可以作为一种模式蛋白研究花生蛋白糖基化产物的结构变化。不同还原糖对Ara h2糖基化反应的影响目前未见相关报道。以花生蛋白Ara h2为研究对象,通过SDS-PAGE、内源荧光、同步荧光、紫外、圆二色谱、红外等光谱技术研究Ara h2糖基化前后分子量、二级、三级结构以及官能团的变化,分析六种还原糖(核糖、木糖、半乳糖、葡萄糖、果糖、乳糖)对花生蛋白Ara h2糖基化产物结构的影响,阐明经不同还原糖修饰后花生蛋白Ara h2的结构变化。SDS-PAGE电泳表明木糖和核糖修饰的花生蛋白Ara h2电泳条带明显上移,糖基化程度最大;紫外光谱分析表明糖基化反应会改变花生蛋白Ara h2的吸收峰强度。五碳糖修饰的花生蛋白Ara h2具有最强的吸收强度,其中五碳糖中木糖的吸收峰强度最大;内源荧光、同步荧光和三维光谱实验结果表明,糖基化修饰会使花生蛋白Ara h2的荧光强度降低,且五碳糖修饰的Ara h2荧光强度最低。分析认为由于糖基化修饰使花生蛋白Ara h2的结构展开,导致芳香族氨基酸暴露在水环境中,从而引起荧光猝灭;圆二色谱分析表明不同还原糖修饰的Ara h2糖基化产物α-螺旋含量都增加,其中木糖修饰的α-螺旋含量最大(15.6%);红外光谱分析表明木糖和核糖修饰的花生蛋白Ara h2的吸收峰分别从3327.41 cm^(-1)红移至3318.43和3321.09 cm^(-1),1700~1600 cm^(-1)处木糖和核糖修饰的花生蛋白Ara h2吸收峰强度略高于其他还原糖修饰的该蛋白。不同还原糖对Ara h2糖基化反应后的糖基化产物结构的影响不同,还原糖的碳链越短、空间位阻越小,糖基化反应程度越高,对Ara h2的结构影响越大。

Glycation reaction can induce the structural change of protein in food stuff;Ara h2is one of the main proteins in peanuts,and it can be used as a model protein to study the structural change of the glycation products of peanut protein.However,the effects of different reducing sugars on Ara h2glycation have not been reported.Therefore,this article took Ara h2as the research object to study the changes in the molecular weight,the secondary and tertiary structure and the functional groups of Ara h2before and after glycation by SDS-PAGE,endogenous fluorescence,synchronous fluorescence,ultraviolet spectrum,circular dichroism,Fourier transform infrared spectroscopy and other spectroscopic techniques.The effects of six reducing sugars(ribose,xylose,galactose,glucose,fructose and lactose)on Ara h2were analyzed to clarify the structural change of different Ara h2glycation products.The results of SDS-PAGE showed that these electrophoretic bands of Ara h2 modified by xylose and ribose moved up significantly,and their glycation degree was the largest,compared with other reducing sugars.Ultraviolet spectrum analysis showed that glycation reaction would change the absorption peak intensity of Ara h2,and modification with pentoses had the strongest absorption intensity(absorption peak intensity of xylose was the largest).The results of endogenous fluorescence,synchronous fluorescence and three-dimensional spectral scanning showed that glycation reduced the fluorescence intensity of Ara h2and pentose modified Ara h2had the lowest fluorescence intensity.It might be due to the structural unfolding of Ara h2caused by glycation,which exposes aromatic amino acids to the water environment and leads to fluorescence quenching.Circular dichroism analysis showed that the content ofα-helix increased after Ara h2was modified by different reducing sugars,among which modified by xylose showed the highest helix content(15.6%).Fourier transform infrared spectroscopy showed that the absorption peaks of Ara h2(modified by xylose and ribose)shifted from 3327.41to 3318.43and 3321.09cm^(-1),respectively;At 1700~1600cm^(-1),the absorption peak intensity of Ara h2modified by xylose and ribose was slightly higher than that modified by other reducing sugars.Therefore,different reducing sugars have different effects on the structure of Ara h2glycation products;The shorter carbon chain and the less steric hindrance of reducing sugars led to a higher glycation degree and a greater impact on the structure of Ara h2.