不同价态铈离子对HRP构象的影响
摘要
在悬浮培养的东北红豆杉细胞中,Ce~(4+)作为诱导子促进信号分子活性氧迸发从而增加紫杉醇的产量。为了探讨Ce~(4+)作为诱导子影响细胞氧化还原酶类的结构和功能进而调控活性氧迸发的机制,本文以Ce~(4+)和Ce~(3+)作用下辣根过氧化物酶的构象改变为切入点,综合运用多种检测手段在分子水平上进行了较全面的研究,取得了以下结果:
(1) Ce~(4+)较Ce~(3+)更有效的改变了辣根过氧化物酶的酶促反应动力学参数,酶的活力、最大反应速率以及酶与底物的亲和力都随着Ce~(4+)和Ce~(3+)浓度的增加而逐渐降低。
(2)应用紫外分光光度计、荧光分光光度计以及电子顺磁共振仪对Ce~(4+)和Ce~(3+)诱导下的辣根过氧化物酶的三级结构进行了研究,经Ce~(4+)和Ce~(3+)处理后辣根过氧化物酶与其天然状态下的光谱特征不同,且这种改变随Ce~(4+)和Ce~(3+)浓度的增加而更加明显。虽然Ce~(4+)和Ce~(3+)所引起的辣根过氧化物酶的紫外特征吸收峰变化趋势相同,但是Ce~(4+)引起的特征峰位移和强度的改变都强于Ce~(3+);并且Ce~(4+)和Ce~(3+)引起的色氨酸荧光发射峰变化趋势相反,g因子的值也有明显的差别。
(3)通过圆二色和傅立叶红外光谱拟和数据发现,Ce~(4+)和Ce~(3+)使得辣根过氧化物酶的二级结构的相对百分含量发生改变,平衡从α螺旋向β转角和β折叠移动。这种二级结构间的转化对于Ce~(4+)和Ce~(3+)具有浓度依赖性;并且相同条件下Ce~(4+)引起的转化程度远大于Ce~(3+)。
Ce~(4+)和Ce~(3+)对于辣根过氧化物酶的二级结构和三级结构所造成的影响不同可能与它们的离子半径、得失电子能力、电荷排布的不同有关。Ce~(4+)作为诱导子促进活性氧的迸发可能与其对过氧化物酶的二级结构和三级结构的影响有关。
Our previous studies have demonstrated that Ce~(4+) could induce ROS (reactive oxygen species) burst as a signal to promote pacilitaxel biosynthesis in suspension cultured Taxus cuspidate cells. To further understand the mechanism of effects of cerium ion on the ROS burst, we carefully studied the changes of the conformation of HRP treated by cerium ions, and we gained some results below:
1. Ce~(4+) is much more efficient in reduced the value of Vmax, Km, and HRP activity than Ce~(3+). The extent of the reduced value increases accompanied by the increase of the concentration of cerium ions.
2. This thesis used synchronous fluorescence spectrum, uv-visible spectrum and electron paramagnetic resonance (EPR) to detect the change of HRP tertiary structure. The change of florescence spectrum caused by Ce~(4+) and Ce~(3+) is opposite; although the current of uv-visible spectrum changes caused by cerium ions is similar, the changed caused by Ce~(4+) is still much evident than that caused by Ce~(3+); furthermore, g factor (gx and gy) in EPR induced by Ce~(4+) and Ce~(3+) was significantly different.
3. Circular dichroism (CD) and infrared spectrum are used to compute the content of secondary structure. The percentage of transition from helical content and other structure toβstrands andβturns caused by Ce~(4+) is more than Ce~(3+). These results indicate that the different valence of cerium ion induces various changes of HRP conformation, and Ce~(4+) is more effective than Ce~(3+). This suggests that Ce~(4+) affects the burst of ROS through changing the conformation of redoxases.
(1) Ce~(4+)较Ce~(3+)更有效的改变了辣根过氧化物酶的酶促反应动力学参数,酶的活力、最大反应速率以及酶与底物的亲和力都随着Ce~(4+)和Ce~(3+)浓度的增加而逐渐降低。
(2)应用紫外分光光度计、荧光分光光度计以及电子顺磁共振仪对Ce~(4+)和Ce~(3+)诱导下的辣根过氧化物酶的三级结构进行了研究,经Ce~(4+)和Ce~(3+)处理后辣根过氧化物酶与其天然状态下的光谱特征不同,且这种改变随Ce~(4+)和Ce~(3+)浓度的增加而更加明显。虽然Ce~(4+)和Ce~(3+)所引起的辣根过氧化物酶的紫外特征吸收峰变化趋势相同,但是Ce~(4+)引起的特征峰位移和强度的改变都强于Ce~(3+);并且Ce~(4+)和Ce~(3+)引起的色氨酸荧光发射峰变化趋势相反,g因子的值也有明显的差别。
(3)通过圆二色和傅立叶红外光谱拟和数据发现,Ce~(4+)和Ce~(3+)使得辣根过氧化物酶的二级结构的相对百分含量发生改变,平衡从α螺旋向β转角和β折叠移动。这种二级结构间的转化对于Ce~(4+)和Ce~(3+)具有浓度依赖性;并且相同条件下Ce~(4+)引起的转化程度远大于Ce~(3+)。
Ce~(4+)和Ce~(3+)对于辣根过氧化物酶的二级结构和三级结构所造成的影响不同可能与它们的离子半径、得失电子能力、电荷排布的不同有关。Ce~(4+)作为诱导子促进活性氧的迸发可能与其对过氧化物酶的二级结构和三级结构的影响有关。
Our previous studies have demonstrated that Ce~(4+) could induce ROS (reactive oxygen species) burst as a signal to promote pacilitaxel biosynthesis in suspension cultured Taxus cuspidate cells. To further understand the mechanism of effects of cerium ion on the ROS burst, we carefully studied the changes of the conformation of HRP treated by cerium ions, and we gained some results below:
1. Ce~(4+) is much more efficient in reduced the value of Vmax, Km, and HRP activity than Ce~(3+). The extent of the reduced value increases accompanied by the increase of the concentration of cerium ions.
2. This thesis used synchronous fluorescence spectrum, uv-visible spectrum and electron paramagnetic resonance (EPR) to detect the change of HRP tertiary structure. The change of florescence spectrum caused by Ce~(4+) and Ce~(3+) is opposite; although the current of uv-visible spectrum changes caused by cerium ions is similar, the changed caused by Ce~(4+) is still much evident than that caused by Ce~(3+); furthermore, g factor (gx and gy) in EPR induced by Ce~(4+) and Ce~(3+) was significantly different.
3. Circular dichroism (CD) and infrared spectrum are used to compute the content of secondary structure. The percentage of transition from helical content and other structure toβstrands andβturns caused by Ce~(4+) is more than Ce~(3+). These results indicate that the different valence of cerium ion induces various changes of HRP conformation, and Ce~(4+) is more effective than Ce~(3+). This suggests that Ce~(4+) affects the burst of ROS through changing the conformation of redoxases.
引文
[1]潘登魁,郭春绒。PrCl3对油松种子萌发及幼苗生长的生理生化效应。稀土,1997, 18(2):64~66
[2]马广岳,施国新,王学,等。镧、锌及其交互作用对浮萍活性氧清除系统的影响。农旬生态环境,2004,20(2):42~45
[3]侯彩霞,沈博礼,刘戈,等。镧和铈对小麦幼苗过氧化物酶和淀粉酶活性及同工酶的影响。稀土,1997,18(2):64~66
[4] Dosemedios C G. Ion radius selectivity of skeletal muscle membranes. Nature, 1977, 270(5639): 750~751
[5] Heuck A. Inhibition of La3+, Eu3+, and Tb3+ on F-1,6-Disesterase, Anasoc. Quim. Argent, 1993, 81(2): 147~148
[6] Zhu B, Xue D, Wang K. Lanthanide ions promote the hydrolysis of 2, 3-bisphosphoglycerate. Biometals, 2004, 17: 423~433,
[7] Sun Y, Liu D L, Yu Z Q, et al. An apo-plastic mechanism for short-term effects of rare earth elements at lower concentrations. Plant Cell Environ, 2003, 26: 887~896,
[8] Chen S A, Zhao B, Wang X D, et al. Promotion of the growth of Crocus sativus cells and the production of crocin by rare earth elements. Biotechnology letter, 2004, 26: 27~30
[9] Zhang C, Zhou C, Li G, et al. Effects of manganese with different valences to apoptosis of SH-SY5Y cell. Journal of Hygiene Research, 2003, 32: 421~422,
[10] Bagchi D, Stohs S J, Downs B W, et al. Cytotoxicity and oxidative mechanisms of different forms of chromium. Toxicology, 2002, 180: 5~22
[11] Venisse J S, Barny M A, Paulin J P, et al. Involvement of three pathogenicity factors of Erwinia amylovora in the oxidative stress associated with compatible interaction in pear. FEBS Letters, 2003, 537: 198~202
[12] Gajhede M, Schuller D J, Henriksen A, et al. Crystal structure of horseradish peroxidase C at 2.15 A resolution. Nat. Struct. Biol., 1997,4(12):1032~1038
[13] Al-Delaimy W K, Vankappel A L, Ferrari P, et al. Plasma levels of six carotenoids in nine European countries: report from the European Prospective Investigation into Cancer and Nutrition (EPIC). Public Health Nutr., 2004, 7(6):713~722.
[14]洪伟杰,张朝晖,芦国营.辣根过氧化物酶的结构与作用机制.生命的化学,2005,25(1):33~36
[15] Manescu C, Krause J L, Mvller K B, et al. Suppressing the spreading of continuum wave packets via chirped laser pulse. Phys. Chem., 2004, 108: 8840~8841
[16]赵开弘,周明,李月。藻红蓝蛋白α-亚基的低温紫外-可见吸收光谱和圆二色光谱的研究。武汉大学学报(自然科学版), 1999, 45(2):207~210
[17]王海人,肖忠柏,宋功武,等。荧光素与牛血清蛋白作用的光谱研究与分析应用。分析测试学报,2001, 20(4): 45~47
[18]魏晓芳,丁西明,刘会泌。pH诱导牛血清白蛋白芳香氨基酸残基微环境变化的光谱分析。光谱学与光谱分析,2000, 20(4):556~558
[19]阎隆飞,孙之荣。蛋白质分子结构。北京:清华大学出版社,1999,11~170,
[20]杨频,高飞。生物无机化学原理。北京:科学出版社,2002,22~352
[21] Petchanikow C, Saborio G P, Anderes L, et al. Biochemical and structural studies of the prion protein polymorphism. FEBS letters, 2001, 509:451~456,
[22] Sreerama N, Venyaminov S Y, Woody R W. Estimation of protein secondary structure from CD spectra: Inclusion of denatured proteins with native protein in the analysis. Anal. Biochem., 2000, 287: 243~251
[23]沈星灿,梁宏,何锡文,等。圆二色光谱分析蛋白质构象的方法及研究进展。化学学报,2004, 32(3):388~394
[24]Schleifenbaum A,Stier G,Gasch A,et al. A genetically encoded FRET probe for PKC activity based on pleckstrin. Am. Chem. Soc., 2004, 126: 11786~11787
[25] Seitz A, Surrey T. Processive movement of single kinesins on crowded microtubules visualized using quantum dots. EMBO, 2006, 25(2):267~277.
[26]王守业,徐晓龙,刘清亮,等。荧光光谱在蛋白质分子构象研究中的应用。光谱学与光谱分析,2001,13(4):257~260
[27] Sirinnapa S, Jinda S, Sumio K. Prediction of ripe-stage eating quality of mango fruit from its harvest quality measured nondestructively by near infrared spectroscopy. Postharvest Biology and Technology,2004, 31:137~145,
[28] Jacobsen K, Oga S, Hubbell W L, et al. Determination of the orientation of T4 lysozyme vectorially bound to a planar-supported lipid bilayer using site~directed spin labeling. Biophysics, 2005, 88(6): 4351~4365
[29] Nelson W D, Blakely S E, Nesmelov Y E, et al. Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin. Proc.Natl. Acad. Sci. USA, 2005, 102(11): 4000~4005
[30]王长振,吴可。位置定向自旋标记-电子顺磁共振技术在蛋白质结构研究中的应用。军事医学科学院院刊,2006,30(5):72~475
[31] Kurt W. Studies of structure and function of biological macromolecules. Chemical Nobel Lecture,2002, 8:1~33
[32] Fiaux J,Bertelsen E, Horwich A,et al. NMR analysis of a 900 KGxoEL GxoES complex. Nature, 2002, 4(18):207~211
[33]高荣强,范世福。现代近红外光谱分析技术的原理及应用。分析仪器,2002, 8(3):9~13
[34]周妍娇,李令媛,茹炳根。金属硫蛋白及其结构域突变体的紫外和圆二色光谱学研究。中国生物化学与分子生物学报,2000, 16(4):483
[35] Fu D,Lobson A, Ylierke LJ, etal,Structure of a glycerol conducting and the basis for its selectivity,Science,2000,290(5491):481~486
[36] Doyle D, Cabral J, Pfuetzner R, et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science, 1998, 280(5360):69~77
[37]艾伦·弗斯塔,酶的测定方法,北京:中国轻工业出版社, 1992,190~192
[38]王凡强,王正祥,邵蔚蓝,等。重组大肠杆菌热稳定性过氧化氢酶的纯化及性质研究。微生物学报,2002,42(3):348~353
[39]张树政,孟广震,何忠效.酶学研究技术(上).北京:科学出版社,1987, 338~369
[40]程传煊,生物物理化学,北京:科学技术文献出版社, 1998, 279~281
[41]袁联群,杨永桃,沈鹤柏,等。铈氨基酸配合物的UV和CV方法。上海师范大学学报(自然科学版),2002, 31(4):62~65
[42]张朝平,夏敏.蛋白质-银(I)-铜(II)络合物的生成及其紫外与红外光谱.光谱学与光谱分析,1996, 16(5):43~49
[43]陈惠黎,生物大分子的结构和功能,上海:上海医科大学出版社,1999, 37~40
[44]冯永君,李德顺,孙丽,等.火茹素酪氨酸微区的研究.化学学报,2000, 8(8):1037~1042
[45]刘媛,谢孟峡,康娟.三七总皂甙对牛血清白蛋白溶液构象的影响.化学学报,2003,61(8):1305~1310
[46]陶慰孙,蛋白质分子基础,北京:人民教育出版社,1981, 227~231
[47] Smulevich G, Paoli M, Burke J F, et al, Characterisation of recombinant horseradish peroxidases (HRP-C*) and three site-directed mutants F41V, F41W, and R38K by resonance Raman spectroscopy. Biochemistry, 1994, 33: 7398~7407
[48]陈国珍,黄贤智,许金钩,等.荧光分析法(第二版).北京:科学出版社,1990, 88~92
[49] Chou J, Qu X,Lu T,et al, The effect of solution pH on synchronous fluorescence spectra of cytochrome c solutions. Microchem, 1995, 52:159~165
[50]杜秀莲,李荣昌。用同步荧光光谱法研究铽(III)与脱铁运铁蛋白的作用。科学通报,2001, 46(5):394~396
[51] Chapeaurouge A, Johansson J S, Ferreira S T. Folding intermediates of a model three-helix bundle protein. Biol Chem.,2001, 276(18): 14861~14866
[52] Price N C. Conformational issues in the characterization of proteins. Biotechnol. Appl. Biochem., 2000, 31: 29~40
[53]范小娜,李蕾,佘磊.稀土-蛋白质配合物合成及其傅立叶红外光谱分析,赣南医学院学报, 2001, 21(2):107~110
[54]黄惠华,梁汉华,郭乾初.超声波对大豆胰蛋白酶抑制剂活性及二级结构的影响.食品科学, 2004, 25(3):29~33
[2]马广岳,施国新,王学,等。镧、锌及其交互作用对浮萍活性氧清除系统的影响。农旬生态环境,2004,20(2):42~45
[3]侯彩霞,沈博礼,刘戈,等。镧和铈对小麦幼苗过氧化物酶和淀粉酶活性及同工酶的影响。稀土,1997,18(2):64~66
[4] Dosemedios C G. Ion radius selectivity of skeletal muscle membranes. Nature, 1977, 270(5639): 750~751
[5] Heuck A. Inhibition of La3+, Eu3+, and Tb3+ on F-1,6-Disesterase, Anasoc. Quim. Argent, 1993, 81(2): 147~148
[6] Zhu B, Xue D, Wang K. Lanthanide ions promote the hydrolysis of 2, 3-bisphosphoglycerate. Biometals, 2004, 17: 423~433,
[7] Sun Y, Liu D L, Yu Z Q, et al. An apo-plastic mechanism for short-term effects of rare earth elements at lower concentrations. Plant Cell Environ, 2003, 26: 887~896,
[8] Chen S A, Zhao B, Wang X D, et al. Promotion of the growth of Crocus sativus cells and the production of crocin by rare earth elements. Biotechnology letter, 2004, 26: 27~30
[9] Zhang C, Zhou C, Li G, et al. Effects of manganese with different valences to apoptosis of SH-SY5Y cell. Journal of Hygiene Research, 2003, 32: 421~422,
[10] Bagchi D, Stohs S J, Downs B W, et al. Cytotoxicity and oxidative mechanisms of different forms of chromium. Toxicology, 2002, 180: 5~22
[11] Venisse J S, Barny M A, Paulin J P, et al. Involvement of three pathogenicity factors of Erwinia amylovora in the oxidative stress associated with compatible interaction in pear. FEBS Letters, 2003, 537: 198~202
[12] Gajhede M, Schuller D J, Henriksen A, et al. Crystal structure of horseradish peroxidase C at 2.15 A resolution. Nat. Struct. Biol., 1997,4(12):1032~1038
[13] Al-Delaimy W K, Vankappel A L, Ferrari P, et al. Plasma levels of six carotenoids in nine European countries: report from the European Prospective Investigation into Cancer and Nutrition (EPIC). Public Health Nutr., 2004, 7(6):713~722.
[14]洪伟杰,张朝晖,芦国营.辣根过氧化物酶的结构与作用机制.生命的化学,2005,25(1):33~36
[15] Manescu C, Krause J L, Mvller K B, et al. Suppressing the spreading of continuum wave packets via chirped laser pulse. Phys. Chem., 2004, 108: 8840~8841
[16]赵开弘,周明,李月。藻红蓝蛋白α-亚基的低温紫外-可见吸收光谱和圆二色光谱的研究。武汉大学学报(自然科学版), 1999, 45(2):207~210
[17]王海人,肖忠柏,宋功武,等。荧光素与牛血清蛋白作用的光谱研究与分析应用。分析测试学报,2001, 20(4): 45~47
[18]魏晓芳,丁西明,刘会泌。pH诱导牛血清白蛋白芳香氨基酸残基微环境变化的光谱分析。光谱学与光谱分析,2000, 20(4):556~558
[19]阎隆飞,孙之荣。蛋白质分子结构。北京:清华大学出版社,1999,11~170,
[20]杨频,高飞。生物无机化学原理。北京:科学出版社,2002,22~352
[21] Petchanikow C, Saborio G P, Anderes L, et al. Biochemical and structural studies of the prion protein polymorphism. FEBS letters, 2001, 509:451~456,
[22] Sreerama N, Venyaminov S Y, Woody R W. Estimation of protein secondary structure from CD spectra: Inclusion of denatured proteins with native protein in the analysis. Anal. Biochem., 2000, 287: 243~251
[23]沈星灿,梁宏,何锡文,等。圆二色光谱分析蛋白质构象的方法及研究进展。化学学报,2004, 32(3):388~394
[24]Schleifenbaum A,Stier G,Gasch A,et al. A genetically encoded FRET probe for PKC activity based on pleckstrin. Am. Chem. Soc., 2004, 126: 11786~11787
[25] Seitz A, Surrey T. Processive movement of single kinesins on crowded microtubules visualized using quantum dots. EMBO, 2006, 25(2):267~277.
[26]王守业,徐晓龙,刘清亮,等。荧光光谱在蛋白质分子构象研究中的应用。光谱学与光谱分析,2001,13(4):257~260
[27] Sirinnapa S, Jinda S, Sumio K. Prediction of ripe-stage eating quality of mango fruit from its harvest quality measured nondestructively by near infrared spectroscopy. Postharvest Biology and Technology,2004, 31:137~145,
[28] Jacobsen K, Oga S, Hubbell W L, et al. Determination of the orientation of T4 lysozyme vectorially bound to a planar-supported lipid bilayer using site~directed spin labeling. Biophysics, 2005, 88(6): 4351~4365
[29] Nelson W D, Blakely S E, Nesmelov Y E, et al. Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin. Proc.Natl. Acad. Sci. USA, 2005, 102(11): 4000~4005
[30]王长振,吴可。位置定向自旋标记-电子顺磁共振技术在蛋白质结构研究中的应用。军事医学科学院院刊,2006,30(5):72~475
[31] Kurt W. Studies of structure and function of biological macromolecules. Chemical Nobel Lecture,2002, 8:1~33
[32] Fiaux J,Bertelsen E, Horwich A,et al. NMR analysis of a 900 KGxoEL GxoES complex. Nature, 2002, 4(18):207~211
[33]高荣强,范世福。现代近红外光谱分析技术的原理及应用。分析仪器,2002, 8(3):9~13
[34]周妍娇,李令媛,茹炳根。金属硫蛋白及其结构域突变体的紫外和圆二色光谱学研究。中国生物化学与分子生物学报,2000, 16(4):483
[35] Fu D,Lobson A, Ylierke LJ, etal,Structure of a glycerol conducting and the basis for its selectivity,Science,2000,290(5491):481~486
[36] Doyle D, Cabral J, Pfuetzner R, et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science, 1998, 280(5360):69~77
[37]艾伦·弗斯塔,酶的测定方法,北京:中国轻工业出版社, 1992,190~192
[38]王凡强,王正祥,邵蔚蓝,等。重组大肠杆菌热稳定性过氧化氢酶的纯化及性质研究。微生物学报,2002,42(3):348~353
[39]张树政,孟广震,何忠效.酶学研究技术(上).北京:科学出版社,1987, 338~369
[40]程传煊,生物物理化学,北京:科学技术文献出版社, 1998, 279~281
[41]袁联群,杨永桃,沈鹤柏,等。铈氨基酸配合物的UV和CV方法。上海师范大学学报(自然科学版),2002, 31(4):62~65
[42]张朝平,夏敏.蛋白质-银(I)-铜(II)络合物的生成及其紫外与红外光谱.光谱学与光谱分析,1996, 16(5):43~49
[43]陈惠黎,生物大分子的结构和功能,上海:上海医科大学出版社,1999, 37~40
[44]冯永君,李德顺,孙丽,等.火茹素酪氨酸微区的研究.化学学报,2000, 8(8):1037~1042
[45]刘媛,谢孟峡,康娟.三七总皂甙对牛血清白蛋白溶液构象的影响.化学学报,2003,61(8):1305~1310
[46]陶慰孙,蛋白质分子基础,北京:人民教育出版社,1981, 227~231
[47] Smulevich G, Paoli M, Burke J F, et al, Characterisation of recombinant horseradish peroxidases (HRP-C*) and three site-directed mutants F41V, F41W, and R38K by resonance Raman spectroscopy. Biochemistry, 1994, 33: 7398~7407
[48]陈国珍,黄贤智,许金钩,等.荧光分析法(第二版).北京:科学出版社,1990, 88~92
[49] Chou J, Qu X,Lu T,et al, The effect of solution pH on synchronous fluorescence spectra of cytochrome c solutions. Microchem, 1995, 52:159~165
[50]杜秀莲,李荣昌。用同步荧光光谱法研究铽(III)与脱铁运铁蛋白的作用。科学通报,2001, 46(5):394~396
[51] Chapeaurouge A, Johansson J S, Ferreira S T. Folding intermediates of a model three-helix bundle protein. Biol Chem.,2001, 276(18): 14861~14866
[52] Price N C. Conformational issues in the characterization of proteins. Biotechnol. Appl. Biochem., 2000, 31: 29~40
[53]范小娜,李蕾,佘磊.稀土-蛋白质配合物合成及其傅立叶红外光谱分析,赣南医学院学报, 2001, 21(2):107~110
[54]黄惠华,梁汉华,郭乾初.超声波对大豆胰蛋白酶抑制剂活性及二级结构的影响.食品科学, 2004, 25(3):29~33