Effects Of La(III),Tb(III) On The Activity And Structure Of Horseradish Peroxidase In Vivo And In Vitro

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.