生物学杂志

生物学杂志 ›› 2020, Vol. 37 ›› Issue (6): 51-.doi: 10.3969/j.issn.2095-1736.2020.06.051

• 研究报告 • 上一篇    下一篇

拟南芥铜锌超氧化物歧化酶解聚的中间态性质

  

  1. 1. 中国科学院 合肥物质科学研究院， 合肥 230031； 2. 中国科学院大学， 北京 100049；3. 安徽省农业科学院 农业工程研究所， 合肥 230001
  • 出版日期:2020-12-18 发布日期:2020-12-21
  • 通讯作者: 吴丽芳，博士，教授，主要从事植物分子生物学研究，E-mail：lfwu@ipp.ac.cn
  • 作者简介:刘祚军，博士，副研究员，主要从事生化与分子生物学研究，E-mail：liuzj1029@163.com
  • 基金资助:
    合肥物质科学技术中心方向项目培育基金（2012FXCX006）；安徽省农科院学科建设项目（17A1329）

Characterization on the intermediate state of dimer depolymerization of Arabidopsis thaliana superoxide dismutase 1#br#

  1. 1. Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China;     2. University of Chinese Academy of Sciences, Beijing 100049; 3. Agricultural Engineering Research Institute,Anhui Academy of Agricultural Sciences, Hefei 230001, China
  • Online:2020-12-18 Published:2020-12-21

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摘要： 拟南芥铜锌超氧化物歧化酶（AtSOD1）是理想的研究植物SOD酶结构与功能关系的模式蛋白，对于理解植物抗逆性有着重要的意义。目前对该酶稳定性、活性等基本性质了解得不够充分，影响了相关研究的开展。研究在大肠杆菌中异源表达出具有生物活性的AtSOD1蛋白，通过电子顺磁共振波谱（EPR）检测，活性中心电子结构准确。对AtSOD1的稳定性进行研究，其中，AtSOD1的尿素解折叠实验的圆二色谱（CD）结果发现，在225 nm附近出现了一个α-螺旋和β-折叠比例变化的拐点，且当尿素浓度为4~5 mol/L时，天然态AtSOD1解折叠曲线出现了一个平台期。在利用差示扫描量热（DSC）检测天然态AtSOD1的物理稳定性时发现，该酶在单体重构为二聚体过程中，也存在两个Tm值，分别为102.2 ℃和100.5 ℃。根据上述结果，推测认为，AtSOD1从二聚体解聚为单体过程中存在一个中间态，与该酶结构稳定性有关。

关键词: 铜锌超氧化物歧化酶, 二聚体, 差式扫描量热法

Abstract: Arabidopsis thaliana superoxide dismutase 1(AtSOD1) has been considered as a critical protein conferring cellular protection against the excessive accumulation of toxic reactive oxygen species, and an ideal model enzyme to understand the relationship between the structure and function of the protein as well. However, the structure stability and catalytic property of AtSOD1 remain poorly understood. In this research, active AtSOD1 with the correct coordinated geometry proved by electron paramagnetic resonance (EPR) was heterogeneous expressed in Escherichia Coli (Rosseta), and then the thermal stability of the enzyme was further analyzed. Urea denaturation of apo and holo AtSOD1 was monitored by circular dichroism (CD), and the results indicated that there was a curve point between α-helix and β-fold in 0-8 mol/L urea while performance at 225 nm wave length. In addition, the curvilinear plateau had been found in holo AtSOD1 when added urea concentration increased from 4 to 5 mol/L. Interestingly, the trace of differential scanning calorimetry (DSC) heating scan of the holo enzyme indicated that the exothermic transition temperature (Tm) value had been observed at 102.2 ℃ and 100.5 ℃ in the enzyme reconstruction from monomer to dimer. According to the CD and DSC results, it was speculated that there is an intermediate state in the dimer unfolding process of AtSOD1 which reflects a type of special structure concerned structure stability.

Key words: copper-zinc superoxide dismutase, protein dimer, differential scanning calorimetry

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