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1.
超氧阴离子诱导的叶绿素荧光猝灭   总被引:4,自引:0,他引:4  
分别通过黄嘌呤(X)与黄嘌呤氧化酶(XO)反应和甲基紫金(MV)的作用,观察了O·-2诱导莴苣叶绿体的叶绿素荧光猝灭过程.结果表明,O-·2的产生明显使光化学猝灭(qP)和非光化学猝灭(qN)增加.叶绿体内SOD被DDC抑制后,X+XO诱导的叶绿素荧光猝灭过程中,qP下降,qN上升;MV诱导的叶绿素荧光猝灭过程中,qP上升幅度不大,qN增加不明显.当碳代谢被碘乙酰胺(JAA)抑制后, qP下降,qN上升.解偶联剂NH4Cl增加质子跨类囊体膜的通透性,导致qP增加和qN降低,加入MV后qP和qN增加不明显.分析认为,-·2的产生和及时被清除对保持光合电子传递和增加跨膜ΔpH有很重要的作用,有利于叶绿体吸收的光能得到转化和耗散,在一定程度上减轻过量光能引起的光抑制损伤.  相似文献   

2.
本实验用 60Coγ线照射正常人新鲜全血,观察电离辐射对多形核白细胞(PMN)释放超氧化物阴离子自由基(O2-·)的影响,以及人血甲2巨球蛋白(α2M)制剂对辐射引起的PMN释放O2-·的作用。结果表明:正常人新鲜全血照射(5-20Gy)后1h,PMN释放O2-·的量较不照射组增高(P<0.01),红细胞中超氧化物歧化酶(SOD)的活力较不照射组降低(P<0.01)。照前1h加入人血α2M制剂(每ml全血中加入138.5单位)能有效地降低PMN的O2-·释放量,提高红细胞中SOD的活力。离体实验结果提示α2M治疗辐射损伤作用可能与其抑制过多的O2-·产生有关。  相似文献   

3.
本文报道了活性氧(ROS)清除剂——苯甲酸钠、维生素C、甘露醇、L-组氨酸、过氧化氢酶和超氧化物歧化酶对Con A诱导的人外周血淋巴细胞化学发光(Ly-CL)均有抑制效应,提示人Ly-CL与ROS的生成有关,参与人Ly-CL的ROS类型有·OH、1O2、H2O2和O2-·。钙通道阻断剂——Verapamil对人Ly-CL也有抑制效应,表明人Ly-CL依赖于人淋巴细胞内钙离子浓度的增加。  相似文献   

4.
植物叶片中过氧化氢含量测定方法的改进   总被引:28,自引:0,他引:28  
Ti(Ⅳ)-H2O2比色法因背景物质干扰而测得的植物叶片内H2O2含量偏高,5%三氯乙酸抽提,活性炭脱色,Ti(Ⅳ)-4-(2-吡啶偶氮)间苯二酚(PAR)比色法测得的H2O2含量偏低.萃取法有效地脱去丙酮提液中的色素,且H2O2的回收率在95%以上.用过氧化氢酶(CAT)处理作空白对照,利用H2O2与Ti(Ⅳ)-PAR的显色反应,建立了一种简便、快速、准确的植物叶片内的H2O2含量测定方法,H2O2的最低检测浓度为0.25 μmol·L-1.用该方法测得多种植物叶片中H2O2的含量在0.1~0.8 μmol·g-1.  相似文献   

5.
NO和H2O2诱导大豆根尖和边缘细胞耐铝反应的作用   总被引:1,自引:0,他引:1       下载免费PDF全文
 NO和H2O2是参与植物抗非生物胁迫反应的重要信号分子, 为了确定NO和H2O2在大豆(Glycine max)根尖和根边缘细胞(root border cells, RBCs)耐铝反应中的作用及其相互关系, 以‘浙春3号’大豆为材料, 研究了铝毒胁迫下大豆根尖内源NO和H2O2的变化, 以及外源NO和H2O2诱导大豆根尖和RBCs的耐铝反应。结果表明, 50 μmol·L–1 Al处理48 h显著抑制大豆根的伸长, 提高Al在根尖的积累, 同时显著增加根尖内源NO和H2O2含量。施加0.25 mmol·L–1外源NO供体亚硝基铁氰化钠(Na2[Fe(CN)5NO]·2H2O, sodium nitroprusside, SNP)和0.1 mmol·L–1H2O2, 能有效地缓解Al对大豆根伸长的抑制、根尖Al积累和RBCs 的死亡, 该缓解作用可以被0.05 mmol·L–1 NO清除剂2-(4- 羧基苯)-4,4,5,5- 四甲基咪唑-1- 氧-3- 氧化物, 钾盐(C14H16N2O4·K, carboxy-PTIO, cPTIO)和150 U·mL–1 H2O2清除酶(catalase, CAT)逆转。并且外源NO能够显著促进根尖H2O2的积累, 而外源H2O2对根尖NO的含量无显著影响。这表明NO和H2O2是诱导大豆根尖及RBCs耐铝反应的两种信号分子, NO可能通过调控H2O2的形成, 进而诱导大豆根尖及RBCs的耐铝反应。  相似文献   

6.
竹红菌乙素敏化的人红细胞膜结构光损伤的Raman光谱特征   总被引:2,自引:0,他引:2  
采用Raman光谱从分子水平揭示了竹红菌乙素光敏损伤的人红细胞膜发生膜蛋白交联和膜脂脂质过氧化导致其功能变化 ;膜流动性和离子通透性增加的本质是竹红菌乙素产生的活性氧 ( 1O2 ,O2 -·和·OH等 )破坏了红细胞膜的有序结构 ,使膜蛋白主链结构的α 螺旋、β 折叠明显减少 ,无规卷曲增加并使其侧链结构的巯基基团、吲哚环、对羟苯基环、单基取代苯基环等也明显减少 .与此同时 ,随着光照时间的增加 ,膜脂的反式构象呈先增加后减少的趋势 ,它的扭曲构象则正好相反 .膜蛋白和膜脂构象不灵敏的CH2 和CH3弯曲振动谱线的明显下降 ,揭示它们有链的断裂 .  相似文献   

7.
用ESR方法研究光敏作用的动态过程   总被引:3,自引:0,他引:3  
通过动力学方法建立了光敏作用中1O2被TEMPONE捕捉产生的TAN自由基对时间的函数关系式,据此可求出光敏作用Ⅰ,Ⅱ型机制产生各种活性中间体的相对速率常数.并应用上述公式,结合实验结果,具体求算了3种无酵类光敏剂HA,HB,CP在DMF-H2O和DMSO-H2O体系中产生1O2,O2和PS的相对速率常数,进而探讨了苝醒光敏剂的构效关系和光敏机制与溶剂的关系.  相似文献   

8.
一种用分光光度计检测氧自由基的新方法   总被引:83,自引:0,他引:83  
报告检测过硫酸铵/N, N,N′,N′-四甲基乙二胺体系所产生的氧自由基的新方法.O-·2与羟胺溶液反应生成NO-2,NO-2经对氨基苯磺酸和α-萘胺显色在波长530 nm处有专一吸收峰,其颜色深浅与产生的O-·2呈量效关系.氧自由基清除剂抗坏血酸对O-·2的清除作用也呈明显的量效关系.  相似文献   

9.
利用自旋捕捉电子顺磁共振(ESR)的方法对从菠菜叶绿体中分离提纯的光系统Ⅱ(PSⅡ)颗粒产生O2-·的机理进行了直接检测.通过对样品充氧、加入超氧化物歧化酶(SOD)抑制剂四氰乙烯(TCNE)以及原位光照检测ESR信号等手段,在PSⅡ中检测到O2-·与DMPO加合物的特征ESR信号.而在没有SOD抑制剂的情况下,光照时PSⅡ中O2-·与DMPO加合物浓度显著下降.进一步实验发现PSⅡ中O2-·产率与氧分子浓度直接正相关.O2-·产率还具有pH值依赖性,在pH值为6.0~6.5范围内,O2-·产率最高,大于此范围时则呈显著下降趋势.而PSⅡ颗粒的Tris处理也将导致O2-·产率的急剧减少.以上结果证实水裂解放氧十分活跃的PSⅡ也是高等植物叶绿体在光照下产生活性O2-·的主要部位,通常大部分的O2-·能被内源SOD清除,且O2-·的生成与PSⅡ的电子传递活性密切相关.  相似文献   

10.
多形核白细胞产生的NO和O2-自由基主要形成 ONOO-   总被引:1,自引:0,他引:1  
用ESR自旋捕集技术研究了人多形核白细胞(PMN)受促癌剂佛波醇(PMA)刺激产生O2-和NO自由基的相互作用.发现加L-精氨酸使在PMA刺激PMN体系中捕捉到的O2-明显减少,加NG-甲基精氨酸(NGMA)使在PMA刺激PMN体系中捕捉的O2-明显增加.用黄嘌呤/黄嘌呤氧化酶和光照核黄素体系证明,加L-精氨酸使PMA刺激PMN产生NO-·自由基与O2-结合生成ONOO-是加入L-精氨酸使PMA刺激PMN体系捕捉的O2-减少的主要原因,并且推算了加入不同浓度L-精氨酸PMN产生NO-·自由基的量.用羟基自由基清除剂和ONOO-氧化DMPO和DMSO及其对pH的依赖关系,证明了ONOO-分解并没有直接生成羟基自由基.用依赖鲁咪诺的化学发光法研究了人多形核白细胞受促癌剂PMA刺激产生NO-·自由基动力学过程.另外,用化学合成的NO-·自由基和过氧亚硝基在模型体系研究了它们的ESR和化学发光特征.说明PMA刺激PMN生成的NO-·和O2-自由基反应形成的ONOO-是引起化学发光的主要形式.  相似文献   

11.
Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (λ>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (1O2) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of 1O2. In addition, the generation of 1O2 was verified further in the presence of D2O and specific singlet oxygen quencher — 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN3). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of 1O2 by PBPs are as follows: APC > C-PC > R-PE.  相似文献   

12.
In a chilling-sensitive plant, cucumber, chilling of leaves in the light results in irreversible damage to PSI. Recent in vitro studies suggested that hydroxyl radicals, which are formed in the presence of H2O2 and reduced Fe-S centers, are involved in the PSI inhibition. We therefore examined this possibility in vivo. Chilling of leaves at 5°C in the light caused a temporary increase in H2O2 concentration, which was probably due to the net H2O2 production in vivo. The activity, measured at 5°C, of the thylakoid ascorbate peroxidase (APX), a key enzyme of the H2O2-scavenging system, was about 20% of that measured at 25°C. The isolated thylakoids retaining high thylakoid APX activity did not show light-dependent net H2O2 production at 25°C. However, at 5°C, net production of H2O2 was observed. Since the rate of electron flow to molecular oxygen in the isolated thylakoids was ca 5 mmol e? mol?1 Chl s?1 at 5°C, the H2O2-scavenging capacity was below this level. When intact leaves were illuminated at 5°C at an irradiance of 100 µmol m?2 s?1, the rate of electron transport through PSII was ca 20 mmol e? mol?1 Chl s?1 and more than 80% of QA was in the reduced state. Since thylakoids are uncoupled in cucumber leaves at 5°C in the light. ATP is not formed and energy dissipation in the form of heat is suppressed. Therefore, the electron flow to molecular oxygen would be greater than 5 mmol e? mol?1 Chl s?1. Moreover, under such conditions, components in the electron transport chain, including Fe-S centers in PSI, were probably reduced. These features indicate that, when cucumber leaves are chilled in the light, hydroxyl radicals can be produced by the Fenton reaction and cause damage to PSI.  相似文献   

13.
Cucumber (Cucumis sativus L., cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid and then incubated in the dark for 14 hours. The intact chloroplasts were isolated from the above plants in the dark and were exposed to weak light (250 micromoles per square meter per second). Within 30 minutes, photosystem II activity was reduced by 50%. The singlet oxygen (1O2) scavengers, histidine and sodium azide (NaN3) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of 1O2 monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the 1O2 quencher, NaN3. Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN3 and histidine. Protochlorophyllide was the most abundant pigment accumulated in intact chloroplasts isolated from 5-aminolevulinic acid-treated plants and was probably acting as type II photosensitizer.  相似文献   

14.
Oxygen radical scavengers have been shown to prevent the development of ischemic preconditioning, suggesting that reactive oxygen species (ROS) might be involved in this phenomenon. In the present study, we have investigated whether direct exposure to ROS produced by photoactivated Rose Bengal (RB) could mimic the protective effects of ischemic preconditioning.

Methods In vitro generation of ROS from photoactivated RB in a physiological buffer was first characterised by ESR spectroscopy in the presence of 2,2,6,6-tetramethyl-1-piperidone (oxoTEMP) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In a second part of the study, isolated rat hearts were exposed for 2.5 min to photoactivated RB. After 5 min washout, hearts underwent 30 min no-flow normothermic ischemia followed by 30 min of reperfusion.

Results and Conclusions The production of singlet oxygen (1O2) by photoactivated RB in the perfusion medium was evidenced by the ESR detection of the nitroxyl radical oxoTEMPO. Histidine completely inhibited oxoTEMPO formation. In addition, the use of DMPO has indicated that (i) superoxide anions (O·-2) are produced directly and (ii) hydroxyl radicals (HO·) are formed indirectly from the successive O·-2 dismutation and the Fenton reaction. In the perfusion experiments, myocardial post-ischemic recovery was dramatically impaired in hearts previously exposed to the ROS produced by RB photoactivation (1O2, O·-2, H2O2 and HO·) as well as when 1O2 was removed by histidine (50 mM) addition. However, functional recovery was significantly improved when hearts were exposed to photoactivated RB in presence of superoxide dismutase (105 IU/L) and catalase (106 IU/L).

Further studies are now required to determine whether the cardioprotective effects of Rose Bengal in presence of O·-2 and H2O2 scavengers are due to singlet oxygen or to other species produced by Rose Bengal degradation.  相似文献   

15.
The possible protective role of endogenous isoprene against oxidative stress caused by singlet oxygen (1O2) was studied in the isoprene‐emitting plant Phragmites australis. Leaves emitting isoprene and leaves in which isoprene synthesis was inhibited by fosmidomycin were exposed to increasing concentrations of 1O2 generated by Rose Bengal (RB) sensitizer at different light intensities. In isoprene‐emitting leaves, photosynthesis and H2O2 and malonyldialdehyde (MDA) contents were not affected by low to moderate 1O2 concentrations generated at light intensities of 800 and 1240 µmol m?2 s?1, but symptoms of damage and reactive oxygen accumulation started to be observed when high levels of 1O2 were generated by very high light intensity (1810 µmol m?2 s?1). A dramatic decrease in photosynthetic performance and an increase in H2O2 and MDA levels were measured in isoprene‐inhibited RB‐fed leaves, but photosynthesis was not significantly inhibited in leaves in which the isoprene leaf pool was reconstituted by fumigating exogenous isoprene. The inhibition of photosynthesis in isoprene‐inhibited leaves was linearly associated with the light intensity and with the consequently formed 1O2. Hence, physiological levels of endogenous isoprene may supply protection against 1O2. The protection mechanisms may involve a direct reaction of isoprene with 1O2. Moreover, as it is a small lipophilic molecule, it may assist hydrophobic interactions in membranes, resulting in their stabilization. The isoprene‐conjugated double bond structure may also quench 1O2 by facilitating energy transfer and heat dissipation. This action is typical of other isoprenoids, but we speculate that isoprene may provide a more dynamic protection mechanism as it is synthesized promptly when high light intensity produces 1O2.  相似文献   

16.
The study of the reaction of p-nitrophenyl acetate (PNPA) with histidine and certain derivatives showed that the species in which the amino group is unprotonated (R(NH2)Im) react with second-order rate constants ( ) that are higher than predicted by a Brønsted relation for a series of neutral amino acids. The reason for this behavior was investigated through an analysis of the kinetics of the reaction of PNPA with these compounds in order to assess the reactivities of the amino and imidazole groups in the two species . The rate constant for the reaction with the imidazole group ( ) of Nπ-methyl histidine agrees with the value predicted by a Brønsted relation obtained from a series of model imidazole compounds. Nτ-Methyl histidine, however, is unreactive, indicating that Nτ is the reactive nitrogen in the imidazole ring of histidine. The values found for histidine, histidine methyl ester, and Nα-dimethyl histidine are lower than predicted by the Brønsted relation. This behavior was found to be due to low reactivity of the
. The evidence presented suggests that the lower reactivity of is due to an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring, which displaces the tautomeric equilibrium toward the unreactive Nτ-H form. The higher reactivity of the imidazole group in the species R(NH2)Im, relative to that in , is responsible for the observed high values for histidine, for histidine methyl ester, for Nτ-methyl histidine, and for Nα-dimethyl histidine, in contrast with the normal value found for Nτ-methyl histidine. The conclusions from this study of histidine and its derivatives support the proposal of an interaction between the protonated N-terminal amino group and the imidazole ring of His6 in the octapeptide hormone angiotensin.  相似文献   

17.
AimsProtective effects of edaravone, an approved medicine for acute brain infarction in Japan, on cell death induced by singlet oxygen (1O2) were examined.Main methodThe 1O2 scavenging activity was examined by direct analysis of near-infrared luminescence in a cell-free system and by fluorospectrometry in the presence of cells. The protective effects of edaravone on 1O2-induced cell death were examined, using rat neuronal B50 cells. Cell death was evaluated by mitochondrial respiration (MTT assay), confocal microscopy and time-lapse imaging. The chemical reaction of edaravone with 1O2 was examined by production analysis using high performance liquid chromatography (HPLC).Key findingsWhen rose Bengal (RB) in D2O was irradiated by a 514 nm laser beam, the signal of 1O2 was observed. Edaravone suppressed the 1O2 signal more potently than azide, a 1O2 scavenger. When B50 cells were irradiated by 525 nm green light in the RB solution, production of 1O2 and induction of cell death were observed. The fluorospectrometric study and the MTT assay revealed that 100–400 µM edaravone suppressed the 1O2 production and attenuated cell death in a concentration-dependent manner. Confocal microscopy and the time-lapse imaging revealed that edaravone prevented the impairment of membrane integrity and the progression of cell death induced by 1O2. The HPLC study revealed that edaravone chemically reacted with 1O2 and changed another compound.SignificanceSince 1O2 is possibly involved in post-ischemic neuronal damage, the clinically approved curative effects of edaravone on acute brain infarction might be attributed to its potent 1O2 scavenging activity.  相似文献   

18.
Immobilization of lettuce (Lactuca sativa) thylakoids has been performed by using glutaraldehyde and bovine serum albumin. Confirming previous reports, a stabilization of the O2 evolution activity of the photosystem II (PSII) under storage and functional conditions has been observed. The present work is devoted to the role played by mono-and divalent cations, during the immobilization process itself, on the O2 production. Four types of measurements have been employed: kinetic measurements, low temperature (77 K) fluorescence emission, photoacoustic (PA) spectroscopy, and electron microscopy observations. We show that the effect of glutaraldehyde is complex because it acts as an inhibitor, a stabilizing agent, and a cross-linking reactive. In the present studies, the thylakoids are immobilized within a polymeric insoluble albumin matrix. The highest activity yield and the best storage conditions are obtained when 0.15 mm Na+ (or K+), 1 mm Mg2+, and 0.1 mm Mn2+ are present in the resuspending media before the immobilization. Due to modifications of the ionic content during such a process, structural differences are observed on the stacking degree of thylakoids. No modification of the fluorescence and PA spectra after the immobilization are found. Furthermore, a correlation between activities and spectral changes have been shown: when the activities increase, the F735 to F695 ratio increases and the PA676 to PA440 ratio decreases.  相似文献   

19.
The objective of the present study is to delineate the role of active site arginine and histidine residues of horseradish peroxidase (HRP) in controlling iodide oxidation using chemical modification technique. The arginine specific reagent, phenylglyoxal (PGO) irreversibly blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 25.12 min-1 M-1. Radiolabelled PGO incorporation studies indicate an essential role of a single arginine residue in enzyme inactivation. The enzyme can be protected both by iodide and an aromatic donor such as guaiacol. Moreover, guaiacol-protected enzyme can oxidise iodide and iodide-protected enzyme can oxidise guaiacol suggesting the regulatory role of the same active site arginine residue in both iodide and guaiacol binding. The protection constant (Kp) for iodide and guaiacol are 500 and 10 M respectively indicating higher affinity of guaiacol than iodide at this site. Donor binding studies indicate that guaiacol competitively inhibits iodide binding suggesting their interaction at the same binding site. Arginine-modified enzyme shows significant loss of iodide binding as shown by increased Kd value to 571 mM from the native enzyme (Kd = 150 mM). Although arginine-modified enzyme reacts with H2O2 to form compound II presumably at a slow rate, the latter is not reduced by iodide presumably due to low affinity binding.The role of the active site histidine residue in iodide oxidation was also studied after disubstitution reaction of the histidine imidazole nitrogens with diethylpyrocarbonate (DEPC), a histidine specific reagent. DEPC blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 0.66 min-1 M-1. Both the nitrogens (, ) of histidine imidazole were modified as evidenced by the characteristic peak at 222 nm. The enzyme is not protected by iodide suggesting that imidazolium ion is not involved in iodide binding. Moreover, DEPC-modified enzyme binds iodide similar to the native enzyme. However, the modified enzyme does not form compound II but forms compound I only with higher concentration of H2O2 suggesting the catalytic role of this histidine in the formation and autoreduction of compound I. Interestingly, compound I thus formed is not reduced by iodide indicating block of electron transport from the donor to the compound I. We suggest that an active site arginine residue regulates iodide binding while the histidine residue controls the electron transfer to the heme ferryl group during oxidation.  相似文献   

20.
When leaflets of bean and leaves of other species of C3 plants are illuminated in the absence of CO2 and at low O2 partial pressure, the capacity for CO2 assimilation at saturating light and its efficiency at low light intensities are inhibited. This photoinhibition is dependent on leaflet age and period of illumination. In young leaflets and following short exposure to these photoinhibitory conditions, some recovery of CO2 assimilation capacity is observed immediately after treatment. Following substantial (70 to 80%) photoinhibition of CO2 assimilation, recovery in fully expanded leaflets is observed only after 48 hours in normal air. The photoinhibition is largely prevented by providing CO2 at partial pressures equivalent to the CO2 compensation point, or by >210 millibars O2 which permits internal CO2 production by photorespiration. If leaflets are illuminated in 60 microbars CO2 and 210 millibars O2 (the CO2 compensation point in air), no photoinhibition is observed. Electron transport processes and fluorescence emission associated with photosystem II are inhibited in chloroplast thylakoids isolated from leaflets after illumination in zero CO2 and 10 millibars O2. These studies support the hypothesis that CO2 recycling through photorespiration is one means of effectively dissipating excess photochemical energy when CO2 supply to illuminated leaves is limited.  相似文献   

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