牦牛肉肌原纤维蛋白在不同pH值条件下的分子结构变化

Molecular Structure Changes of Yak Myofibrillar Proteins via Protein Oxidation at Different pH Values

该研究以不同pH值下不同H_(2)O_(2)浓度的羟自由基氧化体系孵育24 h的牦牛肉肌原纤维蛋白为研究对象,通过对羰基、巯基、二级结构、三级结构、十二烷基硫酸钠聚丙烯酰胺凝胶电泳等指标分析,探究不同pH值下氧化对牦牛肉肌原纤维蛋白分子结构的影响。结果表明:不同pH值下随着H_(2)O_(2)浓度的增加,羰基含量从最初的27.2 nmol/mg升至36.44 nmol/mg(pH值5.0)、36.98 nmol/mg(pH值6.0)、33.64 nmol/mg(pH值7.0)、34.53 nmol/mg(pH值8.0),表面疏水性从383.64(pH值5.0)、275.58(pH值6.0)、245.21(pH值7.0)、238.99上升至484.23、410.92、306.69、291.13,总巯基含量从最初的92.1 nmol/mg左右降至80.68 nmol/mg(pH值5.0)、78.72 nmol/mg(pH值6.0)、87.6 nmol/mg(pH值7.0)、86.28 nmol/mg(pH值8.0),溶解度下降了1.8倍左右、内源荧光强度降低,二级结构比例下降,氧化后蛋白分子内部发生交联。随着pH值远离等电点,羰基上升速率降低,巯基、游离氨基下降速率降低,溶解度、内源荧光强度增加,疏水性降低,靠近等电点的肌原纤维蛋白质具有较高的β-折叠结构和较低的α-螺旋结构。试验表明在等电点附近,肌原纤维蛋白的氧化程度更高。

Myofibrillar proteins from yak meat were incubated for 24 h in a hydroxyl radical oxidation system with different H_(2)O_(2) concentrations at various pH values.By analyzing carbonyl,mercapto,secondary structure,tertiary structure,SDS-PAGE separation,and other indicators,the effects of oxidation at different pH values on the molecular structure of yak muscle myofibrillar proteins were investigated.The results show that with the increase of H_(2)O_(2) concentration at different pH values,the carbonyl content increases from the initial level of 27.2 nmol/mg to 36.44 nmol/mg(5.0),36.98 nmol/mg(6.0),33.64 nmol/mg(7.0),and 34.53 nmol/mg(8.0),whereas the surface hydrophobicity increases from 383.64(5.0),275.58(6.0),245.21(7.0),and 238.99 to 484.23,410.92,306.69,and 291.13,respectively.Moreover,the total mercapto content decreases from the initial level of 92.1 nmol/mg to 80.68 nmol/mg(5.0),78.72 nmol/mg(6.0),87.6 nmol/mg(7.0),and 86.28 nmol/mg(8.0).The solubility was reduced by 1.8 times,and the endogenous fluorescence intensity dropped.The proportion of secondary structures decreased.After oxidation,cross-linking was observed within the protein molecules.As the pH value is farther away from the isoelectric point,the increased rate of the carbonyl content was reduced,whereas the decline rate of the sulfhydryl and free amino contents decreased.Meanwhile,the solubility and endogenous fluorescence intensity increased,and the hydrophobicity decreased.Myofibril proteins close to the isoelectric point have more β-pleated sheet structures and fewer α-helix structures.The results demonstrate that myofibrillar proteins have a higher degree of oxidation near the isoelectric point.