大分子拥挤条件下光诱导细胞色素C还原的光谱研究  

Spectral Study on the Photoreduction of Cytochrome c under Macromolecular Crowding

作　　者：刘艳伟[1] 曹洪玉[1,2] 唐乾[1,2] 郑学仿[1,2] 

机构地区：[1]大连大学生命科学与技术学院,大连116622 [2]辽宁省生物有机化学重点实验室大连大学,大连116622

出　　处：《化学学报》2014年第2期246-252,共7页Acta Chimica Sinica

基　　金：国家自然科学基金(No.21271036;20871024);辽宁省教育厅科学技术研究项目(No.L2013470;2013471)资助~~

摘　　要：稀溶液中高铁细胞色素C(ferric cytochrome c,Fe(III)-Cyt c)能被光照还原成亚铁细胞色素C(ferrous cytochrome c,Fe(II)-Cyt c),但这一研究忽略了生物体细胞的真实环境是高度拥挤的,因此采用紫外可见、同步荧光及圆二色谱光谱法研究了大分子拥挤环境中光诱导Cyt c的还原过程及其对外界环境的依赖情况.UV-Vis光谱和同步荧光光谱结果表明,拥挤试剂葡聚糖70(Dextran70)和聚蔗糖70(Ficoll70)的加入利于Cyt c光还原的进行,且Ficoll70存在时Cyt c光还原程度远高于Dextran70存在时;Dextran70和Ficoll70环境中光诱导Cyt c还原的最适温度分别为37℃和25℃,定波长280 nm光照射利于Cyt c还原;Cyt c的光还原程度随着Dextran70浓度增加而增大,Ficoll70环境中Cyt c的最适光还原浓度为100 g?L-1;拥挤环境下Met,Trp,Tyr和Phe的存在对光诱导Cyt c的还原过程依Phe>Tyr>Trp>Met顺序有催化促进作用;CD光谱结果表明光照射稀溶液中Cyt c蛋白还原后,其α-螺旋含量降低,β-折叠含量升高,而光照射拥挤环境中Cyt c还原后蛋白的二级结构基本没变化.结果表明拥挤试剂具有稳定蛋白质二级结构的作用,且影响光还原Cyt c的电子传递过程及效率.The ferric Cyt c can be photoreduced to the ferrous state by photoexcitation in dilute solution. However, these researches neglected the high crowded conditions in cell environment. In this work, we applied UV-Vis absorption, synchronous fluorescence and CD spectra to study the photoreduction process of Cyt c and its dependence on the external environment under macromolecular crowded conditions. The UV-Vis absorption spectrum data showed that with addition of Dextran70 or Ficoll70 the hydrophobic microenvironment of heme in Cyt c was changed and the photoreduction process of Cyt c was facilitated. In addition, the photoreduction extent was much larger in Ficoll70 solution than that in Dextran70. Under macromolecular crowded conditions, we used four wavelengths of light to irradiate Cyt c samples and found that the 280 nm light was favored for the reduction. The optimum temperatures of photoreduction process were 37℃ and 25 ℃ in the presence of Dextran70 and Ficoll70, respectively. The concentration of macromolecular crowding reagents had massive influence on the photoreduction process. Photoreduction extent showed an increase with rising the concentration of Dextran70 and the optimum concentration of Ficoll70 for the photoreduction was 100 g.L-1. The photoreduction process was promoted by adding external amino acids and the relative rates of photoreduction with amino acids followed the order Phe〉Tyr〉Trp）-Met. Synchronous fluorescence spectra data suggested that Dextran70 and Ficoll70 were able to emit fluorescence which was absorbed by porphyrin. The CD spectra results of Cyt c after irradiation illustrated that the secondary structure of protein varied with a-helix decreased, and β-sheet increased in dilute solution, but remained unchanged after irradiation under macromolecular crowding solutions. Based on the data, macromolecular crowded conditions could maintain a stable structure of Cyt c and promote its photoinduced electronic transfer and reduction process.

关 键 词：大分子拥挤环境 光照还原 细胞色素C 光谱法 蛋白二级结构 

分 类 号：Q559.9[生物学—生物化学]