水稻谷胱甘肽磷脂氢过氧化物酶的热稳定性研究  

The Heat Stability Profiles of the Recombinant Phospholipid Hydroperoxide Glutathione Peroxidase from Oryza sativa

作　　者：王丰[1] 周惠萍[2] 孔宝华[2] 范静华[2] 陈海如[2] 刘进元[1] 

机构地区：[1]清华大学生物科学与技术系分子生物学实验室教育部蛋白质科学重点实验室,北京100084 [2]云南农业大学云南省植物病理重点实验室,昆明650201

出　　处：《中国生物工程杂志》2007年第8期34-39,共6页China Biotechnology

基　　金：国家自然科学基金资助项目(30170080;39770078);国家"973"计划资助项目(2006CB101706);清华-裕元医学科学研究基金资助项目;云南省校合作基金资助项目(98Y9010)

摘　　要：谷胱甘肽磷脂氢过氧化物酶是唯一能够直接还原生物膜上脂类过氧化物的过氧化物酶。利用圆二色光谱(CD)、内源荧光光谱和差示扫描量热仪(DSC)研究了温度对谷胱甘肽磷脂氢过氧化物酶(OsPHGPx)活性及其构象变化的影响。在温度为20～27.5℃期间,随着温度的逐渐升高,OsPHGPx的活性逐渐上升,到27.5℃时达到最大值;在27.5～45℃时,随着温度逐渐升高,其活性迅速下降;当温度超过45℃时,其活性完全丧失。在20～40℃,CD光谱、内源荧光光谱和DSC均没有发生明显变化,暗示OsPHGPx的结构基本保持完整;在40～55℃,CD光谱显示该酶二级结构发生去折叠;内源荧光光谱的变化暗示该酶三级结构发生去折叠。其中在40～45℃,DSC显示该酶可能存在两个去折叠中间体。当温度超过55℃时,整个酶的构象不再发生变化,呈现去折叠状态。Phospholipid hydroperoxide glutathione peroxidase （PHGPx） is the only antioxidant enzyme which could reduce membrane-bound hydroperoxy lipids. The circular dichroism （CD）, fluorescence, and differential scanning calorimetry （DSC） were employed to investigate the effect of temperature on enzymatic activity and the conformational change of a recombinant Oryza sativa PHGPx （OsPHGPx）. PHGPx activity was increased slowly from 10℃ to 27.5℃ and reached the maximum at 27.5℃ ,then descended rapidly from 27.5℃ to 45℃. At temperatures exceeding 45℃, the enzyme activity was completely lost. A classic thermal unfolding of OsPHGPx was found with temperature increasing. First, from 20℃ to 40℃, Far UV CD, intrinsic fluorescence and DSC spectra did not change obviously with increased temperature, indicating the whole structure nearly maintained integrity. Second,from 40℃ to 55℃, an abrupt secondary level change was found in the far UV CD spectra, indicating the secondary structure started unfolding; the change of intrinsic fluorescence spectra revealed that the tertiary structures began partial unfolding and the region near active site was buried into the unfolding structure. The DSC indicated two unfolding intermediates of OsPHGPx appeared at 41℃ and 43℃ during thermal transitions. Third, at over 55 ℃, the whole conformation of the protein kept unchanged.

关 键 词：谷胱甘肽磷脂氢过氧化物酶 热致去折叠 荧光光谱 CD光谱 差示扫描量热法 

分 类 号：Q554.6[生物学—生物化学]