氢气对辣根过氧化物酶活性的影响及其作用机制的研究  

Effect of Hydrogen on Horseradish Peroxidase Activity and Its Mechanism

作　　者：仪杨 张晓康[1] 郭博远 谢飞[1] 张昭[1] 马胜男 赵鹏翔[1] 李英贤[2] 马雪梅[1] YI Yang;ZHANG Xiao-Kang;GUO Bo-Yuan;XIE Fei;ZHANG Zhao;MA Sheng-Nan;ZHAO Peng-Xiang;LI Ying-Xian;MA Xue-Mei(Department of Biotechnology,College of Life Science and Biomedical Engineering,Beijing University of Technology,Beijing 100124,China;China Astronaut Research and Training Center,Beijing 100094,China)

机构地区：[1]北京工业大学生命科学与化学学院生物技术系,北京100124 [2]中国航天员科研训练中心,北京100094

出　　处：《中国生物化学与分子生物学报》2020年第7期811-819,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：载人航天预先研究项目(No.2015SY54A0503);军委后勤保障部开放研究重点项目(No.BHJ17L018)资助。

摘　　要：氢气具有广泛的生物学功能,但氢气发挥生物学效应的靶点仍存在争论。我们前期发现,氢气可以与乙酰胆碱酯酶(AChE)直接作用并提高其活性,提示酶分子可能是氢气的潜在作用靶点。除AChE外,氢气是否还可与其它酶分子相互作用目前尚未见报道。本研究目的是探索氢气对辣根过氧化物酶(HRP)活性的影响。采用荧光光谱和紫外光谱方法检测氢气对HRP活性的影响,并对氢分子作用机制进行了深入探索。研究结果表明,当氢气浓度在饱和状态(800μmol/L)和半饱和状态(400μmol/L)下,HRP酶活性分别提高了18.42%和5.44%;荧光光谱检测表明,氢气使HRP内源性氨基酸荧光强度增强了4.15%,色氨酸到血红素中心的距离由2.85 m缩短至2.69 nm,荧光量子产率由72.8%增加至73.4%;紫外光谱检测表明,氢气可使HRP转化为高价铁的HRP I状态速率降低,同时在间歇性紫外照射后,波长在红移至HRP I状态后存在蓝移现象。以上结果提示,氢气可能改变了HRP中的组氨酸、色氨酸及天冬氨酸的微环境,导致氢键网络重构,从而使反应过程中向血红素中心传递能量的效率增加,His42-Asn70之间氢键增强,酶活增强;同时,氢气可使“H 2 O-H2O2”为桥梁的氢键网络式催化发生变化,降低HRP I的形成速率。本研究不仅进一步证实了酶分子可能是氢分子发挥生物学作用的潜在靶点,同时对氢分子调节HRP酶活性机制的探索,为氢分子作用机制研究提供了更多的线索。Although hydrogen has a wide range of biological functions, the target of hydrogen is still debated. We previously found that hydrogen could directly interact with acetylcholinesterase(AChE) and enhance its activity, suggesting that the enzyme molecules may be the potential targets of hydrogen. Besides AChE, whether hydrogen could interact with other enzymes has not been reported. The purpose of this study is to explore the possibility of hydrogen on horseradish peroxidase(HRP) activity. In this study, fluorescence and ultraviolet spectroscopy were used to determine the effects of hydrogen on HRP activity, and the mechanism underlying the effects of molecular hydrogen was also investigated. The results showed that HRP enzyme activity increased by 18.42% and 5.44%, respectively when the hydrogen concentration reached to saturated(800 μmol/L) and semi-saturated(400 μmol/L) levels;the results from fluorescence spectrum showed a hydrogen-induced increase in fluorescence intensity of endogenous amino acids in HRP by 4.15%, the distance from tryptophan to heme center decreased from 2.85 nm to 2.69 nm, and the fluorescence quantum yield increased from 72.8% to 73.4%;UV spectrum data showed that hydrogen could reduce the conversion rate of HRP to high valent iron state(HRPI). Meanwhile, intermittent UV irradiation could induce blue shift after red shift of the wavelength reached to the HRPI state. The above results suggest that hydrogen might induce alterations in the microenvironment of histidine, tryptophan and aspartate in HRP, and reconstruct of the hydrogen bond network with the "H2O-H2O2" bridge, thus increasing the efficiency of energy transfer to heme center and enhancing the hydrogen bond between His42 and Asn70, which finally result the increase in enzyme activity. This study further confirmed that the enzyme molecules may be potential targets for hydrogen. The exploration of the mechanism underlying the modulatory role of hydrogen on HRP enzyme activity also provided more clues study of the mechanism of hydr

关 键 词：氢气 血红素 辣根过氧化物酶 内源性氨基酸 氢键 

分 类 号：Q5[生物学—生物化学]