羟自由基氧化对鲢鱼肌原纤维蛋白结构的影响

Effect of Hydroxyl Radical Oxidation on the Structure of Silver Carp Myofibrillar Protein

以鲢鱼肌原纤维蛋白为研究对象,用芬顿体系(H2O2的浓度为0.1、1、5、10和50 mmol/L)产生不同浓度的羟自由基对蛋白进行模拟氧化,研究羟自由基氧化对肌原纤维蛋白氧化和结构的影响。结果表明:随着H2O2浓度的升高(H2O2浓度为0~10 mmol/L),羰基含量和二聚酪氨酸含量显著提高(P<0.05),总巯基和活性巯基含量在H2O2浓度大于1 mmol/L时显著下降(P<0.05)。氧化使内源荧光下降,使表面疏水性升高,粒径分布向粒度大的方向移动。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)表明,氧化后的蛋白质发生了不同程度的交联或聚集,交联方式主要是二硫键。氧化还改变了蛋白的二级结构,主要表现为H2O2浓度0~1 mmol/L时α-螺旋向β-折叠转化,H2O2浓度1~50 mmol/L时β-折叠向无规卷曲转化。氨基酸分析表明,氧化过程中几乎所有氨基酸都会参与反应,但是对羟自由基的敏感程度为:半胱氨酸(Cys)>丙氨酸(Ala)>赖氨酸(Lys)>酪氨酸(Tyr)。综上,羟自由基氧化可使肌原纤维蛋白的结构发生显著改变。

In this paper,myofibrillar protein from sliver carp were artificial oxidized in a hydroxyl radical-generating systems( the concentration of H2O2 were 0.1,1,5,10 and 50 mmol/L,respectively),and the effects of oxidization and the structural characteristics of myofibrillar protein were investigated.The results showed that with the increasing of H2O2 concentration at the range of 0~10 mmol/L,the carbonyl contents and di-tyrosine contents increased significantly( P < 0.05),and the total thiol group levels and active thiol group levels decreased significantly( P < 0.05) when concentration of H2O2 was higher than1 mmol/L. Endogenous fluorescence decreased significantly,surface hydrophobicity increased significantly and particle size distribution moved in the direction of large particle size after oxidization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis( SDS-PAGE) result showed that hydroxyl radical oxidation could induce the different degrees of cross-linking or aggregation of protein molecules,and the cross-linking mode was mainly disulfide bonds. In addition,the effect on the secondary structure of the protein was that the α-helix was transformed into β-sheet when the concentration of H2O2 was0~1 mmol/L. The β-sheet was transformed into random coil when the concentration of H2O2 was 1 ~ 50 mmol/L. Amino acid analysis indicated that almost all amino acids were involved in the oxidation process,but the sensitivity of amino acids to hydroxyl radicals was cysteine > alanine > lysine > tyrosine.In conclusion,hydroxyl radical oxidation has changed the structure of myofibrillar proteins.