中国农业科学 ›› 2017, Vol. 50 ›› Issue (22): 4382-4388.doi: 10.3864/j.issn.0578-1752.2017.22.014

• 食品科学与工程 • 上一篇    下一篇

磷酸化水平对肌红蛋白稳定性的影响

李蒙,李铮,李欣,杜曼婷,宋璇,张德权   

  1. 中国农业科学院农产品加工研究所/农业部农产品加工重点实验室,北京 100193
  • 收稿日期:2017-05-25 出版日期:2017-11-16 发布日期:2017-11-16
  • 通讯作者: 张德权,Tel:010-62818740;E-mail:dequan_zhang0118@126.com
  • 作者简介:李蒙,Tel:010-62816474;E-mail:Limeng1214@126.com
  • 基金资助:
    国家现代肉羊产业技术体系(CARS-39)、国家农业科技创新工程、公益性行业(农业)科研专项(201303083)

Effect of Phosphorylation Level on Myoglobin Stability

LI Meng, LI Zheng, LI Xin, DU ManTing, SONG Xuan, ZHANG DeQuan   

  1. Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-Products Processing, Ministry of Agriculture, Beijing 100193
  • Received:2017-05-25 Online:2017-11-16 Published:2017-11-16

摘要: 【目的】肌红蛋白是影响肉色最主要的色素物质,主要存在于肌浆中,其绝对含量和3种肌红蛋白(氧合肌红蛋白、脱氧肌红蛋白、高铁肌红蛋白)间的相对含量决定了肉色。已有研究表明蛋白质的磷酸化可能会通过对糖酵解代谢途径以及肌红蛋白的调控进而负向调控肉色的稳定性,本研究旨在探究磷酸化对肌红蛋白稳定性的影响,进而为通过调控磷酸化水平提高肉色稳定性提供理论依据。【方法】用连二亚硫酸钠还原骨骼肌肌红蛋白纯品,再经超滤除去连二亚硫酸钠。随后采用碱性磷酸酶(AP)体外孵育催化肌红蛋白的去磷酸化反应,采用SDS-PAGE凝胶电泳和Pro-Q与Ruby染色的方法测定肌红蛋白磷酸化水平的变化,测定孵育体系pH的变化,紫外分光光度计测定孵育过程中3种肌红蛋白相对含量的变化,圆二色谱测定孵育过程中肌红蛋白的二级结构变化。【结果】磷酸化水平的测定结果表明,碱性磷酸酶处理组(去磷酸化处理)中肌红蛋白的磷酸化水平在孵育6 h时显著低于对照组(P<0.05),表明碱性磷酸酶可以在体外孵育过程中催化肌红蛋白发生去磷酸化反应,降低肌红蛋白的磷酸化水平。3种肌红蛋白相对含量的测定结果表明,从孵育2 h起,碱性磷酸酶处理组中氧合肌红蛋白的相对含量显著高于对照组,高铁肌红蛋白的相对含量显著低于对照组。即与对照组相比,碱性磷酸酶处理组中肌红蛋白的自动氧化速率低,氧化还原稳定性高(P<0.05)。pH的测定结果表明,碱性磷酸酶处理组和对照组孵育体系的pH差异不显著(P>0.05),即添加碱性磷酸酶进行孵育没有改变孵育体系的pH。二级结构的测定结果表明,肌红蛋白的二级结构以α-螺旋为主。从孵育0 min到6 h,碱性磷酸酶处理组中肌红蛋白的α-螺旋和β-折叠的含量基本不变,而对照中肌红蛋白的α-螺旋含量增加,β-折叠的含量减少,表明碱性磷酸酶处理组中肌红蛋白二级结构的稳定性高于对照组。【结论】肌红蛋白发生磷酸化修饰后,可能会通过改变肌红蛋白的二级结构,降低肌红蛋白二级结构的稳定性,增加肌红蛋白的自动氧化速率,进而加速高铁肌红蛋白的积累,不利于肉色稳定性,这可能是蛋白质磷酸化负向调控肉色稳定性的原因之一

关键词: 肉色, 肌红蛋白, 磷酸化, 氧化还原稳定性, 二级结构

Abstract: 【Objective】Myoglobin is the main pigment responsible for meat color, which is mainly in sarcoplasm. Meat color is determined by the absolute content and the dynamics of myoglobin redox form interconversions. Studies have shown that protein phosphorylation may play a negative role in meat color development by regulating glycolysis and the redox stability of myoglobin. The effects of phosphorylation on myoglobin stability was investigated in this study, which will provide theoretical basis for improving meat color stability through regulating protein phosphorylation level.【Method】The pure metmyoglobin from skeletal muscle was used. Metmyoglobin was reduced by sodium dithionite, which was then removed by ultrafiltration. After that, alkaline phosphatase (AP) was added to catalyze the dephosphorylation of myoglobin in vitro. The phosphorylation level of myoglobin was determined by using SDS-PAGE and then stained by Pro-Q & Ruby reagent. pH value was measured by a pH meter. The relative content of three myoglobin forms (oxygenated myoglobin, deoxy myoglobin and iron myoglobin) and the secondary structure of myoglobin during the incubation were measured by ultraviolet spectrophotometer and circular dichroism spectrum, respectively.【Result】According to the results, the phosphorylation level of myoglobin was significantly lower (P<0.05) in AP group than that in control group at 6 h, which indicating that alkaline phosphatase can catalyze the dephosphorylation of myoglobin in vitro, thus decrease the phosphorylation level of myoglobin. The relative content of oxymyoglobin in AP group was significantly higher than that in control group, and the relative content of metmyoglobin was significantly lower than that in control group after 2 h. In brief, the automatic oxidation rate of myoglobin was lower and the redox stability of myoglobin was higher in AP group than that in control group. However, no significant difference (P>0.05) was observed betweenAP group and control group, which means that pH value of the incubation system was not changed by adding alkaline phosphatase. The results showed that the secondary structure of myoglobin was mostly α-helix. From 0 min to 6 h incubation, the contents of α-helix and β-sheet of myoglobin were almost unchanged in AP group, while the α-helix content of myoglobin increased and β-sheet content of myoglobin decreased in control group, indicating that the secondary structural stability of Mb was increased after dephosphorylation.【Conclusion】It was speculated that the secondary structure of myoglobin might be changed after phosphorylation. The secondary structure stability was decreased and automatic oxidation rate of myoglobin was increased after phosphorylation. Thus, leading to the accumulation of metmyoglobin and color deterioration, and this might be one of the reasons by which protein phosphorylation play a negative role in regulating meat color stability.

Key words: meat color, myoglobin, phosphorylation, redox stability, secondary structural