【作者】 郭绍芬；

【导师】 黄晓华； 周青；

【作者基本信息】 江南大学 ， 环境工程， 2008， 博士

【摘要】 近年来,稀土元素在各个领域广为应用。特别是在中国和其他一些国家,稀土微肥被成功用于促进作物生长和提高产品品质,已有30余年的历史。据报道,适量稀土对植物生长、产品品质及抗逆性方面有促进作用,但是当稀土浓度过高时,则起抑制作用,表现为“低促高抑（hormesis effect）”现象。植物体内的保护酶系统,如过氧化氢酶,过氧化物酶（POD）和超氧化物歧化酶对稀土敏感。因此,保护酶如过氧化物酶可作为生物标记物来研究酶与稀土的相互作用。然而,由于植物细胞壁的阻隔作用,稀土能否进入细胞及其对POD在植物细胞的分布、活性和结构的影响机理,以及POD生物活性和结构的关系至今仍不清楚。辣根过氧化物酶（HRP）为POD酶超家族的一员,其主要源自辣根,且HRP分离纯化及其结构研究透彻。本论文中,HRP作为过氧化物酶的典型模型,通过多学科交叉合作,研究了轻稀土离子La³⁺和重稀土离子Tb³⁺对植物体内外的HRP活性与结构的影响。结果主要归纳如下:模拟生理溶液中,轻稀土离子La³⁺对HRP活性的影响随稀土浓度升高呈现“低促高抑（Hormesis effect）”现象。产生这种效应的成因是La³⁺改变了HRP的构象。低浓度的La³⁺使HRP的多肽链构象的有序结构含量增加,无序结构含量减少,使血红素卟啉环中非平面性增加,导致HRP分子血红素活性中心Fe（III）暴露程度的增加,使得HRP分子电子传递更加容易,因而HRP分子的活性增加。高浓度的La³⁺会使HRP分子中的多肽链构象的有序结构含量减少,无序结构含量增加,整个分子更趋近于松散,使血红素卟啉环中平面性增加,导致HRP分子血红素活性中心Fe（III）的暴露程度减弱,使HRP分子电子传递更为困难,因而HRP分子的活性被抑制。La³⁺可以和HRP分子中多肽的酰胺发生相互作用,改变多肽链的构象,从而对活性中心产生微扰。低浓度的La³⁺和HRP相互作用,只是改变了HRP的构象,其中并没有La-HRP配合物生成。高浓度的La³⁺和HRP相互作用,有La0.88-HRP配合物生成,0.44摩尔Ca（II）被La³⁺取代。HRP中Ca（II）的丢失将导致HRP活性的降低。进一步解释了HRP的活性被高浓度的La³⁺所抑制的现象。在La0.88-HRP配合物中,La³⁺和HRP多肽链酰胺的氧原子或氮原子形成共价键,使整个分子电子云密度重新分配,从而导致HRP分子生物活性的变化。辣根体内HRP的活性随La³⁺浓度的升高也呈现出低促高抑现象。低浓度的La³⁺处理辣根,稀土离子不能进入细胞内部,在细胞壁和细胞膜上有少量稀土存在,细胞超微结构完整,叶绿体类囊体的片层结构增加,能促进HRP的酶蛋白的合成和营养元素的吸收利用,如,钙,铁。低浓度La³⁺对辣根生长起到促进作用,此时,辣根体内没有La-HRP生成。高浓度的La³⁺处理辣根,稀土能进入细胞内部,细胞结构受损,HRP大多集中在细胞壁上,造成细胞衰老,对辣根植物生长起到抑制作用。在该条件下,辣根体内生成La-HRP配合物,其中La³⁺和HRP肽链上的氧原子或氮原子结合,La-HRP的分子量为43833 Da,pI 8.76,大约1摩尔的La³⁺键合到1摩尔的HRP上形成1摩尔的La-HRP。HRP和La-HRP的性质对比研究表明, La-HRP的构象和微结构不同于HRP。两者相比,La-HRP血红素基团的平面性增加,活性中心Fe（III）的电子云密度减小,从而使La-HRP的电化学和催化活性被抑制,这可能是La³⁺抑制辣根体内过氧化物酶的机理之一。荧光显微镜的结果表明La-HRP经食物链进入生物体,将被吸附在细胞膜上而影响膜的正常生理功能,对细胞造成伤害。在模拟生理溶液中Tb³⁺和HRP相互作用,主要是抑制HRP催化活性,Tb³⁺的这种抑制作用程度随Tb³⁺浓度的增加而增强。Tb³⁺可以和HRP分子中多肽的酰胺发生相互作用,改变了HRP多肽链的构象,从而对活性中心产生微扰。Tb³⁺使HRP分子中的多肽链构象的有序结构含量减少,无序结构含量增加,整个分子更趋近于松散,使血红素卟啉环中平面性增加,导致HRP分子血红素活性中心Fe（III）暴露程度的减弱,使得HRP分子电子传递更为困难,因而HRP分子的活性受抑制。MALDI-TOF/MS和XPS结果表明Tb³⁺对HRP活性产生抑制作用的实质是有Tb₂-HRP配合物生成,其中平均每摩尔Tb₂-HRP含有约2摩尔的Tb³⁺。在Tb₂-HRP配合物中,Tb³⁺和HRP多肽链的氧原子形成共价键,使整个分子电子云密度重新分配,从而导致HRP分子生物活性的变化。Tb³⁺处理辣根,Tb³⁺对HRP的抑制作用是主要的,对各项生理指标都表现出伤害效应,此时Tb³⁺起到重金属离子的作用。电镜自显影结果显示Tb³⁺大多集中分布在细胞壁上,少量Tb³⁺能进入原生质体,并主要分布在液泡内,叶绿体内和叶绿体膜上。而此时HRP大多集中在细胞壁上,造成细胞衰老,对辣根植物生长起到抑制作用。在该条件下,辣根体内生成Tb4-HRP配合物,Tb4-HRP的分子量为44336 Da, pI 8.80,大约4摩尔的Tb³⁺键合到1摩尔的HRP上形成1摩尔的Tb4-HRP,同时有0.21摩尔的钙被Tb³⁺取代。HRP和Tb4-HRP的性质对比研究表明,Tb4-HRP的形成改变了HRP的构象和微结构,导致血红素基团的平面性增加,活性中心Fe（III）的暴露程度减小,从而使Tb4-HRP的电化学和催化活性被抑制,这可能是Tb³⁺抑制辣根体内过氧化物酶活性的机理之一。荧光显微镜的结果表明Tb4-HRP经食物链进入生物体,将被吸附在细胞膜上而影响膜的正常生理功能,对细胞造成伤害。在稀土农用时,我们要慎重选择稀土离子应该为轻稀土离子如镧,并控制在较低的浓度范围。重稀土离子如铽对植物生长是有害的,应避免施用。更多还原

【Abstract】 In the recent years, the rare earth elements （REEs） have been widely applied in many fields. Especially,the microfertilizers of REEs have been widely and successfully used in agriculture in China and other countries to promote the plant growth and to improve the quality of products for more than 30 years. It was reported that the concentration of REEs is suitable, the REEs can play a positive role in promoting the plant growth, improving the quality of products and increasing the resistance of adversity of plants. When their concentration is high, they would result in a negative effect on plants. The above effect was called the“hormesis effect”. The biological investigation indicated that the protective enzymes, such as catalase, peroxidase （POD）, superoxide dismutase are very sensitive to the REEs in the plants, thus POD served as an indicator to investigate the interaction between enzymes and REEs is meaningful. However, until now, the effect and its mechanism of the REEs on the distribution and the activity of POD in the plant cells as well as the relationship between the biological activity and the structure of POD in the plants with increasing the concentration of the REEs are not known due to the barrier effect of the cell wall of the plants. Horseradish peroxidase （HRP） is one of the POD superfamily, it is mainly produced from the horseradish root and has been successfully isolated, purified and characterized. In this thesis, HRP is used as a typical model of POD, the effects of a light rare earth element La³⁺ and a heavy rare earth element Tb³⁺ on the activity and structure of HRP in vivo and in vitro were investigated by the interdisciplinary cooperation. The original results from our experiments can be mainly concluded as follows:The results from the investigation in vitro indicated that La³⁺ could clearly change the activity of HRP in the simulated physiological solution and show the“hormesis effect”with increasing the concentration of La³⁺. It is due to the interaction between La³⁺ and HRP, and changed the conformation of HRP. When the concentration of La³⁺ is low, the content of ordered structure of HRP was increased and the content of random coil was decreased, the non-planarity of the heme group in HRP was increased and then the exposure extent of the active center, Fe（III）, in the heme group was increased. The electron transfer is promoted, thus the activity HRP is enhanced. When the concentration of La³⁺ is high, the content of ordered structure of HRP was decreased and the content of random coil was increased, the planarity of the heme group in HRP was increased and then the exposure extent of the active center, Fe（III）, in the heme group was decreased. The electron transfer is inhibited, and thus the activity of HRP is inhibited. When HRP is in the presence of the low concentration of La³⁺, La³⁺ only altered the conformation of HRP, and the complex of La-HRP is not formed. However, when HRP is in the presence of the high concentration of La³⁺, La³⁺ could interact with O and/or N atoms in the polypeptide chain of HRP, and formed the complex of La0.88-HRP. The formation of the complex of La0.88-HRP disrupted the conformation of HRP and disturbed the active center, leading to the redistribution of electron density in HRP, and thus inhibited the activity of HRP. In horseradish, La³⁺ could significantly change the peroxidase activity and also show the“hormesis effect”with increasing the concentration of La³⁺. When horseradish is treated with the low concentration of La³⁺, La³⁺ is not entered the cell, and mainly distributed on the cell wall and plasma membrane of horseradish cell. Meanwhile, the cell ultrastructure is not destroyed, La³⁺ located on the plasma membrane can promote the uptake of the nutrients elements in cell, such as Ca, Fe. HRP distribution scope is increased, physiological index result also indicated that the low concentration of La³⁺can promote horseradish growth. Under this condition, no La-HRP is formed in horseradish. When horseradish is treated with the high concentration of La³⁺, a large amount of La³⁺ is distributed on cell wall, some of La³⁺ has been entered into the protoplast of horseradish,leading to the distortion of the protoplast in horseradish. Meanwhile,the uptake of the nutrient elements in cell are clearly inhibited by La³⁺. In addition, the cell ultrastructure is destroyed; HRP is mainly distributed on cell wall to accelerated cell senescence; physiological index results also indicated that the horseradish growth is inhibited by La³⁺. Under this condition, a new peroxidase complex containing La³⁺ （La-HRP） was obtained from horseradish treated with the high concentration of La³⁺, in which the molecular weight of the complex of La-HRP is near 43682Da, pI is about 8.76, 1 molar of La³⁺ is bond to 1 molar of HRP. La³⁺ could interact with O and/or N atoms in the polypeptide chain of HRP. Comparing investigation of HRP and La-HRP complex indicated that the conformation and microstructure of the complex of La-HRP is different from HRP. The formation of the complex of La-HRP, redistributed the electron density of HRP, and decreased the electron density of Fe（III） in heme group and then inhibited electron transfer in HRP, thus the activity of HRP is inhibited. Therefore, the formation of the complex of La-HRP might be an inhibition mechanism of La³⁺ on peroxidase activity in horseradish. The fluorescence result indicated that La-HRP entered living organisms through food chain, it would be adsorbed on the cell membrane and disturb the cell membrane physiological function, and do harm to the cell.The investigation results from in vitro indicated that the inhibition effect of Tb³⁺ on the activity of HRP is dominant, and the inhibition effect is increased with increasing of the concentration of Tb³⁺. Tb³⁺ can interact with amide groups of HRP and change the conformation in HRP, and then disturb the active center. The content of ordered structure of HRP was decreased and the content of random coil was increased, the planarity of the heme group in HRP was increased and then the decrease in the exposure extent of the active center, Fe（III）, in the heme group. The electron transfer is inhibited, thus the activity of HRP is decreased. MALDI-TOF/MS and XPS results indicated that the inhibition mechanism of Tb³⁺ on the activity of HRP is the formation of the complex of Tb₂-HRP. In which, 2 molar of Tb³⁺ bound to 1 molar of HRP, Tb³⁺ could interact with O atoms in the polypeptide chain of HRP, leading to the redistribution of electron density of HRP, and the inhibition of the activity of HRP.In horseradish, Tb³⁺ also significantly changes the activity of HRP, the inhibition effect of Tb³⁺ on the activity of HRP in horseradish is dominant. Tb³⁺ acts as a heavy metal element and brings negative effect on horseradish growth. Most of Tb³⁺ is distributed on cell wall, and some of Tb³⁺can enter the protoplast and mainly locate in the vacuole, chloroplast and chloroplast membrane. Meanwhile, the cell ultrastructure is destroyed; HRP is mainly distributed on cell wall, accelerated cell senescence. After the horseradish treated with Tb³⁺, the morphological changes of protoplast are observed, protoplast is collapsed. Under this condition, Tb³⁺ can enter into the horseradish cell and interact with HRP, forming the complex of Tb4-HRP. The molecular weight of the complex of Tb4-HRP is near 44336Da, pI is about 8.80, and 0.21 molar of Ca（II） in 1 molar of HRP is substituted by Tb³⁺, and about 4 molar of Tb³⁺ is bound to 1 molar of HRP. The formation of the complex of Tb4-HRP in horseradish might be the dominant reason for the inhibition of the activity of HRP in horseradish. The formation of Tb4-HRP in horseradish, leading to the increase in the planarity of the heme group in HRP and then the decrease in the exposure extent of the active center, Fe（III）, in the heme group, inhibit the electron transfer of HRP, and thus decrease the activity of HRP. The change in the structure of HRP in horseradish treated with Tb³⁺ may be one of the possible inhibition mechanisms of Tb³⁺ on the activity of HRP in horseradish. The fluorescence result indicated that Tb4-HRP entered living organisms through food chain, it would be adsorbed on the cell membrane and disturb the cell membrane physiological function, and do harm to the cell.When the rare earths were used in agriculture, we should be cautious to select the light rare earths, i.e. La³⁺, and limited the light rare earths with a low concentration, the heavy rare earths would do harm to plants, i.e. Tb³⁺, it should be avoid application.更多还原