大分子拥挤条件下光诱导辣根过氧化物酶还原的光谱研究

doi:10.7517/j.issn.1674-0475.2015.04.321

影像科学与光化学 ›› 2015, Vol. 33 ›› Issue (4): 321-329.DOI: 10.7517/j.issn.1674-0475.2015.04.321

大分子拥挤条件下光诱导辣根过氧化物酶还原的光谱研究

曹洪玉^1,2, 王珍^1,2, 唐乾^1,2, 郑学仿^2,3

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;
3. 大连大学环境与化学工程学院, 辽宁大连 116622

收稿日期:2015-04-29 修回日期:2015-06-16 出版日期:2015-07-16 发布日期:2015-07-16
通讯作者: 郑学仿
基金资助:
国家自然科学基金 (21271036,20871024)和辽宁省教育厅科技(L2013470,L2013471)资助项目

Spectral Study on the Photoreduction of Horseradish Peroxidase under Macromolecular Crowding Conditions

CAO Hongyu^1,2, WANG Zhen^1,2, TANG Qian^1,2, ZHENG Xuefang^2,3

1. School of Life Science and Biotechnology, Dalian University, Dalian 116622, P. R. China;
2. Liaoning Key Laboratory of Bio-organic Chemistry, Dalian University, Dalian 116622, P. R. China;
3. College of Environmental and Chemical Engineering, Dalian University, Dalian 116622, P. R. China

Received:2015-04-29 Revised:2015-06-16 Online:2015-07-16 Published:2015-07-16

摘要/Abstract

摘要：

稀溶液中光诱导辣根过氧化物酶(Horseradish Peroxidase, HRP)能够发生还原反应,但这一研究忽略了生物体细胞内因存在各种分子而高度拥挤的真实环境。本文采用紫外可见、荧光、同步荧光及圆二色光谱法,研究了大分子拥挤环境中光诱导HRP的还原过程及外界环境对其光还原的影响。UV-Vis光谱和同步荧光光谱结果表明,采用403 nm单色光照射时,HRP在拥挤试剂葡聚糖70(Dextran70)中还原程度较好,而HRP在聚蔗糖70(Ficoll70)拥挤环境下不发生光还原反应;采用280 nm单色光照射时,在拥挤试剂Dextran70和Ficoll70中HRP光还原反应程度增加,且HRP的光还原程度在Ficoll70溶液中远高于在Dextran70溶液中;光诱导HRP还原的最适温度在Dextran70和Ficoll70环境中分别为4 ℃和24 ℃;HRP在Dextran70溶液中的最佳光还原浓度是100 g/L,在Ficoll70环境中HRP还原程度随着Ficoll70浓度的增加而增大;CD光谱结果表明拥挤环境中HRP被光还原后,蛋白的二级结构基本没有变化。

关键词: 大分子拥挤环境, 辣根过氧化物酶, 光照还原, 光谱法

Abstract:

The ferric Horseradish Peroxidase(HRP) can be photoreduced to ferrous state by photoexcitation in dilute solution. However, these researches neglected the high crowding conditions in cell environment. We applied UV-Vis absorption, synchronous fluorescence and CD spectra to study the photoreduction process of HRP and its dependence on the external environment under macromolecular crowding conditions. The UV-Vis absorption spectrum data showed that the degree of HRP photoreduction was higher in Dextran70 solution when 403 nm monochromatic light, but HRP was not photoreduced in Ficoll70 solution. The photoreduction process of HRP was facilitated in Dextran70 or Ficoll70 solution when 280 nm monochromatic light. In addition, the photoreduction extent was much larger in Ficoll70 solution than that in Dextran70. The optimum temperatures of photoreduction process were 4 ℃ and 24 ℃ in the presence of Dextran70 and Ficoll70, respectively. The optimum concentration of Dextran70 for the photoreduction was 100 g/L and photoreduction extent showed an increase with the rise of Ficoll70 concentration. The CD spectra results illustrated that the secondary structure of protein remained the same after irradiation under macromolecular crowding solutions.

Key words: macromolecular crowding environment, horseradish peroxidase, photoreduction, spectroscopy

曹洪玉, 王珍, 唐乾, 郑学仿. 大分子拥挤条件下光诱导辣根过氧化物酶还原的光谱研究[J]. 影像科学与光化学, 2015, 33(4): 321-329.

CAO Hongyu, WANG Zhen, TANG Qian, ZHENG Xuefang. Spectral Study on the Photoreduction of Horseradish Peroxidase under Macromolecular Crowding Conditions[J]. Imaging Science and Photochemistry, 2015, 33(4): 321-329.

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大分子拥挤条件下光诱导辣根过氧化物酶还原的光谱研究

Spectral Study on the Photoreduction of Horseradish Peroxidase under Macromolecular Crowding Conditions

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