蛋白质氧化对核桃蛋白质结构的影响

为了深入明晰氧化对核桃蛋白质结构性的影响,本文以核桃分离蛋白为研究对象,采用不同浓度的2,2'-盐酸脒基丙烷(AAPH)热降解形成烷过氧自由基(ROO·)代表脂质过氧化反应过程中产生的脂质自由基,对核桃分离蛋白进行氧化修饰得到不同氧化程度的核桃氧化蛋白。对不同氧化程度蛋白的羰基、巯基和总巯基,表面疏水性(H₀)、内源荧光、粒径分布、相对分子质量和二级结构进行研究和分析。结果表明,随着AAPH浓度的增加,核桃氧化蛋白羰基含量显著增加(p<0.05),巯基和H₀显著降低(p<0.05);圆二色谱结果表明,随着AAPH浓度的增加,核桃蛋白α-螺旋结构和β-折叠含量下降,而无规卷曲结构含量增加,说明氧化破坏了蛋白质的二级结构,使有序结构变为无序结构。最大内源荧光值降低,且出现蓝移现象,说明烷过氧自由基氧化导致分子聚集,使得色氨酸残基包埋;粒径分布结果表明,核桃氧化蛋白可溶性聚集体可被共价交联和非共价聚集转变成不可溶性部分;体积排阻色谱结果表明,核桃氧化蛋白相对分子质量随着氧化浓度的增大聚集程度逐渐增大。研究结果表明,核桃蛋白在烷过氧自由基(ROO·)氧化体系中产生了显著氧化作用并导致其结构发生一定的改变,为进一步阐明核桃蛋白氧化机理提供理论依据。

Effect of oxidative modification of protein on structure of walnut protein

In order to further clarify the effect of oxidation on the protein structure of walnut,walnut protein isolate was studied in this paper,peroxyl radical derived from thermal decomposition of different concentration of 2,2'-azobis(2-amidinopropane)dihydrochloride(AAPH)was selected as representative of lipid peroxidation-derived free radicals. And walnut proteins of oxidative modification by peroxyl radicals were prepared. Effects of peroxyl radical were characterized by protein carbonyl content,free sulphydryl and total sulphydryl content,circular dichroism spectroscopy,intrinsic fluorescence,surface hydrophobicity,particle size distribution and size exclusion chromatogram. The results indicated that,with the increasing of concentration of AAPH,walnut protein carbonyl content increased significantly,free sulfhydryl and total sulfhydryl contents,surface hydrophobicity decreased significantly. The analysis of circular dichroism spectroscopy indicated that exposure of walnut protein to AAPH led to loss of a-helix structure and β-fold structure,and the structure of random coil increased,which indicated that protein secondary structure was damaged by oxidation and converted to disordered structure form ordered structure. The blue shift of wavelength of maximum emission was accompanied by decreased of intrinsic fluorescence intensity,which indicated that peroxyl radical caused molecular aggregation and tryptophan residue was embed. The results of the particle size distribution showed that the soluble aggregate by covalent and non-covalent aggregation could be transformed into an insoluble part. And the results of size exclusion chromatogram showed that the relative molecular mass of walnut protein increased with the concentration of oxidation concentration. Research results in this study indicated that the walnut protein made a significant oxidation in the oxidation system of peroxyl radical and lead to a change in its structure,which would provide theoretical basis for further clarifying walnut protein oxidation mechanism.