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

作　　者：曹洪玉[1,2] 王珍[1,2] 唐乾[1,2] 郑学仿[2,3] 

机构地区：[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

出　　处：《影像科学与光化学》2015年第4期321-329,共9页Imaging Science and Photochemistry

基　　金：国家自然科学基金(21271036;20871024);辽宁省教育厅科技(L2013470;L2013471)资助项目

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403nm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280nm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100g/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。The ferric Horseradish Peroxidase（HRP） can be dilute solution. However, these researches neglected the applied UV-Vis absorption, synchronous fluorescence and photoreduced to ferrous state by photoexcitation in high crowding conditions in cell environment. We 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 photoreduetion process of HRP was facilitated in Dextran70 or Ficoll70 solution when 280 nm monochromatic light. In addition, the photoreduction extent was much larger in FicollT0 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.

关 键 词：大分子拥挤环境 辣根过氧化物酶 光照还原 光谱法 

分 类 号：O657.3[理学—分析化学]