沙丘芦苇（Phragmites communis Trin）特有内含物清除羟基自由基机制的研究
摘要
羟基自由基(OH)是一种活泼的活性氧自由基,由于在高温高光强的环境条件下,植物体容易引发产生大量的高反应活性的-OH。一旦形成-OH,由于其极高的氧化活性,可以与生物体内的大分子发生高效、快速的反应,造成细胞内生物膜脂及其他生物大分子的氧化伤害。
PAAC作为沙丘芦苇的特有内含物,分布于叶绿体中,前人研究发现,PAAC在高温高光强的胁迫下主要是通过高效、直接清除-OH来减轻膜脂和蛋白的氧化伤害从而适应这种逆境的,为进一步研究PAAC直接清除-OH的可能机制,本实验经过优化,利用紫外光解初始浓度为10mM的H_2O_2溶液提供外源-OH,在水溶液中,对PAAC在紫外光解过氧化氢体系中反应形成的各产物通过紫外光谱分析、TLC分析来确定PAAC清除-OH后自身结构是否发生变化,然后通过比色法测定其羟基和氨基含量的变化,结合红外光谱分析来探究其与-OH可能的作用机理。
研究结果表明：PAAC在直接清除羟基自由(OH)的过程中,可能-OH与之发生了抽氢反应,引起了自身分子结构的变化,使得酚羟基和芳香伯氨基含量相对减少,羰基含量增加,这为进一步揭示PAAC清除羟基自由基的机制和抗氧化机理提供了实验依据和理论基础。
Hydroxyl radical (OH) is one of the most lively and reactive oxygen radicals, under high temperature and light environmental conditions, the plant is apt to cause a large amount of highly reactive hydroxyl radical. Once the formation of hydroxyl radical in organisms, due to its high oxidation activity, it can react efficiently and rapidly with biological macromolecules, resulting in the oxidative damage of cell membrane lipids and other biological macromolecules.
As a novel dune reed solute, PAAC is mainly distributed in the chloroplast. Through efficiently and directly scavenging of PAAC to OH and markedly suppressing the damage of lipid and protein, dune reed can adapt to high temperature and light environmental conditions. To further research the mechanism of PAAC scavenging OH, this experiment has been optimized to use UV photolysis of 10 mM initial concentration of H_2O_2 solution to provide foreign OH, we analysised the products of reaction system of PAAC in the UV photolysis of H_2O_2 by TLC,IR and MS to determine whether changes in its structure after PAAC scavenging OH. Then, we determined the changes of the hydroxyl and amino group content to explore the possible mechanism of PAAC scavenging OH with the results of infrared spectroscopy.
The results show that,in the process of directly scavenging OH,PAAC may have made a H-atom abstraction reaction with OH, causing changes in its molecular structure,making PAAC increase their carbonyl content and the phenolic hydroxyl and aromatic primary amino content are relatively less. This provides an experimental reference and theoretical basis for further reveal the mechanism of scavenging OH and the antioxidant mechanism.
PAAC作为沙丘芦苇的特有内含物,分布于叶绿体中,前人研究发现,PAAC在高温高光强的胁迫下主要是通过高效、直接清除-OH来减轻膜脂和蛋白的氧化伤害从而适应这种逆境的,为进一步研究PAAC直接清除-OH的可能机制,本实验经过优化,利用紫外光解初始浓度为10mM的H_2O_2溶液提供外源-OH,在水溶液中,对PAAC在紫外光解过氧化氢体系中反应形成的各产物通过紫外光谱分析、TLC分析来确定PAAC清除-OH后自身结构是否发生变化,然后通过比色法测定其羟基和氨基含量的变化,结合红外光谱分析来探究其与-OH可能的作用机理。
研究结果表明：PAAC在直接清除羟基自由(OH)的过程中,可能-OH与之发生了抽氢反应,引起了自身分子结构的变化,使得酚羟基和芳香伯氨基含量相对减少,羰基含量增加,这为进一步揭示PAAC清除羟基自由基的机制和抗氧化机理提供了实验依据和理论基础。
Hydroxyl radical (OH) is one of the most lively and reactive oxygen radicals, under high temperature and light environmental conditions, the plant is apt to cause a large amount of highly reactive hydroxyl radical. Once the formation of hydroxyl radical in organisms, due to its high oxidation activity, it can react efficiently and rapidly with biological macromolecules, resulting in the oxidative damage of cell membrane lipids and other biological macromolecules.
As a novel dune reed solute, PAAC is mainly distributed in the chloroplast. Through efficiently and directly scavenging of PAAC to OH and markedly suppressing the damage of lipid and protein, dune reed can adapt to high temperature and light environmental conditions. To further research the mechanism of PAAC scavenging OH, this experiment has been optimized to use UV photolysis of 10 mM initial concentration of H_2O_2 solution to provide foreign OH, we analysised the products of reaction system of PAAC in the UV photolysis of H_2O_2 by TLC,IR and MS to determine whether changes in its structure after PAAC scavenging OH. Then, we determined the changes of the hydroxyl and amino group content to explore the possible mechanism of PAAC scavenging OH with the results of infrared spectroscopy.
The results show that,in the process of directly scavenging OH,PAAC may have made a H-atom abstraction reaction with OH, causing changes in its molecular structure,making PAAC increase their carbonyl content and the phenolic hydroxyl and aromatic primary amino content are relatively less. This provides an experimental reference and theoretical basis for further reveal the mechanism of scavenging OH and the antioxidant mechanism.
引文
[1].Haslam SM.Variation of population types in Phragmites communis Trin.Ann Bot,1970,34:1 47-158.
[2].Zheng WJ, zheng XP, zhang CL.A survey of photosynthetic carbon metabolism in 4 ecotypes of Phragmites communis in northwest China:leaf anatomy, ultrastructure, and activities of ribulose 1,5-bisphosphate carboxylase, phosphoenolpyruvate carboxylase and glycollate oxidase.Physiol Plant,2000,110:201-208.
[3].《中国沙漠植物志》编辑组.中国沙漠植物志(第一卷).北京：科学出版社,1985.34.
[4]. Bohnert HJ, Jensen RG. Strategies for engineering water-stress tolerance in plants[J]. Trends Biotechnol 1996,14:89-97.
[5]. Hare PD, Cress WA, Staden JV. Dissecting the role of osmolyte accumulation during stress. Plant Cell and Environ.,1998,21:535-553.
[6].浦铜良,程佑发,张承烈.沙丘芦苇特有小分子化合物及其对叶绿体的逆境保护效应[J].科学通报,2000,45(12)：1308-1313.
[7].惠磊,张安龙.紫外光谱应用于造纸废水的检测[J].分析检验,2010,29(6)：50-51.
[8].刘杰,韩卿.红外光谱技术在木素结构研究中的应用[J].江苏造纸,2010,9(3)：25-26.
[9].汪小兰.有机化学(第四版)[M].北京：高等教育出版社,2005.
[10].余瑞元等.生物化学实验原理和方法(第二版)[M].北京：高等教育出版社,2005.
[11].张启峰,张世平,张启涛.有机分析教程[M]北京：中国标准出版社,1996
[12].刘新生,王永录.质谱技术在抗原表位研究中的应运[J].江西农业学报,2010,22(5)：152-154.
[13].林静.质谱联用技术及其在临床上的应用[J].医学综述,2010,16(4)：628.
[14].陈培榕,邓勃.现代仪器分析实验室与技术[M].北京：清华大学出版社,1999.
[15].Lee MS, Kerns EH.LC/MS applications in drug development[M]. Mass Spec.Rev,1999,1 8:187-279
[16].扬芬等.羟基自由基的产生、捕集及检测方法[J],药学学报,2007,42(7)：692.
[17].郑荣梁,黄中洋.自由基生物学(第三版)[M].北京：高等教育出版社,2007.
[18]. L. W.Ehrlich, A modified Newto method for polynomials CommACM[J],1967,10:107-108
[19].孙贤波,赵庆祥,曹国民等.高级氧化法的特性及其应用[J].中国给水排水,2002,18(5)：33-35.
[20].李明波,申欢等.水处理中的羟基自由基·OH[J].陕西环境,2003,10(4)：20
[21].陈建孟,潘伟伟,刘臣亮.电化学体系中羟基自由基产生机理与检测的研究进展[J].浙江工业大学学报,2008,36(4)：417-418.
[22].邓淑芳,白敏冬,白希尧,刘兴旺.羟基自由基特性及其化学反应[J].大连海事大学学报,2004,30(3)：63-64.
[23].李灵香玉,马香娟.羟基自由基(·OH)的特性及其在光化学氧化中的反应机理[J].化工技术与开发,2006,35(8)：27-28.
[24].Jurva U, Wik strom HV, Bruins AP. Electrochemically assisted Fenton reaction of hydrox y1 radicals with xenobiotics followed by on-line analysis with high performance liquid ch romatography tandem mass spectrometry[J].Rapid Commun. Mass Spectrom.,2002,16:19 34-1940
[25].Cheng SA,Fung WK, Chan KY, e tal. Optimizing electron spin resonance detection of hydroxyl radical in water [J].Chemosphere,2003,52:1797-1805.
[26].何杰等.UV-H2O2法去除亚硝酸盐氮和氨氮的研究[J].环境科学研究,1999：12(1)：15-16
[27]. Harrison CC et al. The UV-enhanced decomposition of aqueous ammonium nitrite[J]. Photochem. Photobiol A.1995:89(3)215-219.
[28]. Choi CW, Kim SC, HWang SS, et al Antioxidant activity and free radical scavenging capacity between Korean medicinal plants and flavonoids by assay-quided comparison [J].Plant Sci,2002,163:1161-1168
[29].RussoA, Cardile V, Lombardo L, et al Antioxidant activity and antiproliferative action of methanolic extract of Geum quellyon Sweet roots in human tumor cell lines [J].Ethnopharmacol,2005,100:323-332
[30].Ince NH, Tezcanli G, Belen RK, et al.Ultrasound as a catalyzer of aqueous reaction sy stems:he state of the art and environment application[J].Applied catalysis B:Environme ntal,2001,29:167-176.
[31]James DO. Enviromental application of ultrasound [D].Urbana Illinois, USA:Univ of Illionis at Urbana-Champaign,2003.
[32].宋卫峰,吴斌等.电解法降解有机污染物机理及动力学的研究[J].化工环保,2001,3：131-135
[33].刘烈炜,赵志祥等.电解法降解水中有机污染物的研究进展[J].环境科学与技术,2003,5：60-63
[34].陈震,郑曦等.电化学方法生成羟基自由基及其在酸性络蓝降解脱色中的应用[J].环境化学,2001,5：275-280.
[35].林植芳,李双顺,李桂珠等.衰老叶片和叶绿体中的H2O2积累与膜脂过氧化的关系[J].植物生理学报,1988,14：16
[36].Epperlein MH,Noronha-Dutra AA,Strange RN.Involvement of the hydroxyl radiacal in the abiotic elicitation of phytoalexins in legumes[J].Physiol Molplant Pathol,1986,28;67
[37].Thompson JE, Legge RL,Barber RF.The role of free radicals in senescence and woundin g[J].New phytol,1987,105:317
[38].Pu TL,cheng YF,zhang CL.A novel small compound specified in dune reed and its pos sible role as a protectant on the chloroplasts under stress.Chinese Science Bulletin,2000,4 5(12):1308-1313.
[39].马淑慧,高吉刚,周杰.羟基自由基与鸟苷作用分子机理的荧光分析研究[J].分析实验室,2008,27(增刊)：239-241.
[40].徐向荣,王文华,李华斌.比色法测定Fenton反应产生的羟自由基及其应用[J].生物化学与生物物理进展,1996,26(1)：67-68.
[41].Anderson. B.L & Berry, RW. Anal Biochem,1983,132-365.
[42].段丽菊,刘英帅等DNPH比色法：一种简单的蛋白质羰基含量测定方法[J].毒理学杂志,2005,19(4)：320-321.
[43].赖玉荣,张曾等.FC法测定木素和纸浆中的酚羟基[J].中国造纸学报,2007,22(1)：54-55
[44].杨晓芬,赵美萍等.水中苯胺类化合物的分光光度法测定[J].分析化学,2002,30(5)：540-543
[45].谭艳华.甘露醇对小儿心肌酶的影响[J].西部医学,2009,21(5)：742-743.
[2].Zheng WJ, zheng XP, zhang CL.A survey of photosynthetic carbon metabolism in 4 ecotypes of Phragmites communis in northwest China:leaf anatomy, ultrastructure, and activities of ribulose 1,5-bisphosphate carboxylase, phosphoenolpyruvate carboxylase and glycollate oxidase.Physiol Plant,2000,110:201-208.
[3].《中国沙漠植物志》编辑组.中国沙漠植物志(第一卷).北京：科学出版社,1985.34.
[4]. Bohnert HJ, Jensen RG. Strategies for engineering water-stress tolerance in plants[J]. Trends Biotechnol 1996,14:89-97.
[5]. Hare PD, Cress WA, Staden JV. Dissecting the role of osmolyte accumulation during stress. Plant Cell and Environ.,1998,21:535-553.
[6].浦铜良,程佑发,张承烈.沙丘芦苇特有小分子化合物及其对叶绿体的逆境保护效应[J].科学通报,2000,45(12)：1308-1313.
[7].惠磊,张安龙.紫外光谱应用于造纸废水的检测[J].分析检验,2010,29(6)：50-51.
[8].刘杰,韩卿.红外光谱技术在木素结构研究中的应用[J].江苏造纸,2010,9(3)：25-26.
[9].汪小兰.有机化学(第四版)[M].北京：高等教育出版社,2005.
[10].余瑞元等.生物化学实验原理和方法(第二版)[M].北京：高等教育出版社,2005.
[11].张启峰,张世平,张启涛.有机分析教程[M]北京：中国标准出版社,1996
[12].刘新生,王永录.质谱技术在抗原表位研究中的应运[J].江西农业学报,2010,22(5)：152-154.
[13].林静.质谱联用技术及其在临床上的应用[J].医学综述,2010,16(4)：628.
[14].陈培榕,邓勃.现代仪器分析实验室与技术[M].北京：清华大学出版社,1999.
[15].Lee MS, Kerns EH.LC/MS applications in drug development[M]. Mass Spec.Rev,1999,1 8:187-279
[16].扬芬等.羟基自由基的产生、捕集及检测方法[J],药学学报,2007,42(7)：692.
[17].郑荣梁,黄中洋.自由基生物学(第三版)[M].北京：高等教育出版社,2007.
[18]. L. W.Ehrlich, A modified Newto method for polynomials CommACM[J],1967,10:107-108
[19].孙贤波,赵庆祥,曹国民等.高级氧化法的特性及其应用[J].中国给水排水,2002,18(5)：33-35.
[20].李明波,申欢等.水处理中的羟基自由基·OH[J].陕西环境,2003,10(4)：20
[21].陈建孟,潘伟伟,刘臣亮.电化学体系中羟基自由基产生机理与检测的研究进展[J].浙江工业大学学报,2008,36(4)：417-418.
[22].邓淑芳,白敏冬,白希尧,刘兴旺.羟基自由基特性及其化学反应[J].大连海事大学学报,2004,30(3)：63-64.
[23].李灵香玉,马香娟.羟基自由基(·OH)的特性及其在光化学氧化中的反应机理[J].化工技术与开发,2006,35(8)：27-28.
[24].Jurva U, Wik strom HV, Bruins AP. Electrochemically assisted Fenton reaction of hydrox y1 radicals with xenobiotics followed by on-line analysis with high performance liquid ch romatography tandem mass spectrometry[J].Rapid Commun. Mass Spectrom.,2002,16:19 34-1940
[25].Cheng SA,Fung WK, Chan KY, e tal. Optimizing electron spin resonance detection of hydroxyl radical in water [J].Chemosphere,2003,52:1797-1805.
[26].何杰等.UV-H2O2法去除亚硝酸盐氮和氨氮的研究[J].环境科学研究,1999：12(1)：15-16
[27]. Harrison CC et al. The UV-enhanced decomposition of aqueous ammonium nitrite[J]. Photochem. Photobiol A.1995:89(3)215-219.
[28]. Choi CW, Kim SC, HWang SS, et al Antioxidant activity and free radical scavenging capacity between Korean medicinal plants and flavonoids by assay-quided comparison [J].Plant Sci,2002,163:1161-1168
[29].RussoA, Cardile V, Lombardo L, et al Antioxidant activity and antiproliferative action of methanolic extract of Geum quellyon Sweet roots in human tumor cell lines [J].Ethnopharmacol,2005,100:323-332
[30].Ince NH, Tezcanli G, Belen RK, et al.Ultrasound as a catalyzer of aqueous reaction sy stems:he state of the art and environment application[J].Applied catalysis B:Environme ntal,2001,29:167-176.
[31]James DO. Enviromental application of ultrasound [D].Urbana Illinois, USA:Univ of Illionis at Urbana-Champaign,2003.
[32].宋卫峰,吴斌等.电解法降解有机污染物机理及动力学的研究[J].化工环保,2001,3：131-135
[33].刘烈炜,赵志祥等.电解法降解水中有机污染物的研究进展[J].环境科学与技术,2003,5：60-63
[34].陈震,郑曦等.电化学方法生成羟基自由基及其在酸性络蓝降解脱色中的应用[J].环境化学,2001,5：275-280.
[35].林植芳,李双顺,李桂珠等.衰老叶片和叶绿体中的H2O2积累与膜脂过氧化的关系[J].植物生理学报,1988,14：16
[36].Epperlein MH,Noronha-Dutra AA,Strange RN.Involvement of the hydroxyl radiacal in the abiotic elicitation of phytoalexins in legumes[J].Physiol Molplant Pathol,1986,28;67
[37].Thompson JE, Legge RL,Barber RF.The role of free radicals in senescence and woundin g[J].New phytol,1987,105:317
[38].Pu TL,cheng YF,zhang CL.A novel small compound specified in dune reed and its pos sible role as a protectant on the chloroplasts under stress.Chinese Science Bulletin,2000,4 5(12):1308-1313.
[39].马淑慧,高吉刚,周杰.羟基自由基与鸟苷作用分子机理的荧光分析研究[J].分析实验室,2008,27(增刊)：239-241.
[40].徐向荣,王文华,李华斌.比色法测定Fenton反应产生的羟自由基及其应用[J].生物化学与生物物理进展,1996,26(1)：67-68.
[41].Anderson. B.L & Berry, RW. Anal Biochem,1983,132-365.
[42].段丽菊,刘英帅等DNPH比色法：一种简单的蛋白质羰基含量测定方法[J].毒理学杂志,2005,19(4)：320-321.
[43].赖玉荣,张曾等.FC法测定木素和纸浆中的酚羟基[J].中国造纸学报,2007,22(1)：54-55
[44].杨晓芬,赵美萍等.水中苯胺类化合物的分光光度法测定[J].分析化学,2002,30(5)：540-543
[45].谭艳华.甘露醇对小儿心肌酶的影响[J].西部医学,2009,21(5)：742-743.