物理化学学报 >> 2013, Vol. 29 >> Issue (08): 1785-1792.doi: 10.3866/PKU.WHXB201305271

生物物理化学 上一篇    下一篇

酸诱导拥挤条件下肌红蛋白及突变体去折叠过程

张玉姣1, 唐乾1, 曹洪玉1, 郑学仿1,2   

  1. 1 大连大学生命科学与技术学院, 辽宁 大连 116622;
    2 大连大学辽宁省生物有机化学重点实验室, 辽宁 大连 116622
  • 收稿日期:2013-03-18 修回日期:2013-05-27 发布日期:2013-07-09
  • 通讯作者: 郑学仿 E-mail:dlxfzheng@126.com
  • 基金资助:

    国家自然科学基金(21271036, 20871024)资助项目

Acid-Induced Unfolding Process of Myoglobin and Its Mutant under Macromolecular Crowding Conditions

ZHANG Yu-Jiao1, TANG Qian1, CAO Hong-Yu1, ZHENG Xue-Fang1,2   

  1. 1 School of Life Science and Biotechnology, Dalian University, Dalian 116622, Liaoning Province, P. R. China;
    2 Liaoning Key Lab of Bio-organic Chemistry Dalian University, Dalian 116622, Liaoning Province, P. R. China
  • Received:2013-03-18 Revised:2013-05-27 Published:2013-07-09
  • Contact: ZHENG Xue-Fang E-mail:dlxfzheng@126.com
  • Supported by:

    The project was supported by the National Natural Science Foundation of China (21271036, 20871024).

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摘要：

采用紫外-可见吸收光谱、同步荧光光谱和圆二色(CD)光谱法研究拥挤试剂葡聚糖70 (Dextran70)和聚蔗糖70 (Ficoll70)存在条件下, 酸诱导野生型肌红蛋白Mb(WT)及其突变体Mb(D60K)的去折叠过程. 结果显示: 在Dextran70 和Ficoll70 两种拥挤环境下, Mb(WT)的变性中点pH值由4.25 分别降低到3.78 与3.76, 拥挤试剂加入后增强了Mb(WT)的耐酸能力; 肌红蛋白60位天冬氨酸(Asp)突变为赖氨酸(Lys)后, 变性中点pH值由4.25 降低到4.19, 耐酸性比野生型肌红蛋白有所增强, Mb(D60K)在Dextran70 和Ficoll70 两种拥挤环境下变性中点pH值由4.19 分别降至3.74 和3.12. 以上实验说明肌红蛋白表面氨基酸突变和拥挤试剂的添加起到了稳定血红素微环境、芳香族氨基酸及二级结构和保护蛋白天然状态的作用.

关键词: 拥挤试剂, 酸变性, 肌红蛋白, 突变体, 光谱法

Abstract:

In this paper, we studied the unfolding process of myoglobin (Mb) and its mutant Mb(D60K) induced by acid under macromolecular crowding conditions, using ultraviolet-visible absorption, synchronous fluorescence, and circular dichroism (CD) spectroscopies. The spectroscopic data showed that, with the addition of Dextran70 or Ficoll70, the denaturation midpoint of Mb(WT) decreased from 4.25 to 3.78 or 3.76, respectively. In addition, the acid tolerance of Mb(WT) improved under macromolecular crowding conditions. The denaturation midpoint of Mb(D60K) was 4.19, which was a slight decrease from that of Mb (4.25). Upon addition of Dextran70 or Ficoll70, the denaturation midpoint of Mb(D60K) decreased from 4.19 to 3.74 or 3.12, respectively. The spectroscopic results illustrated that the amino acid mutant and crowding agents could stabilize the microenvironment surrounding the heme and aromatic amino acid as well as the second structure of Mb, protecting its native state.

Key words: Crowding agent, Acid denaturation, Myoglobin, Mutant, Spectrometry