胞外ATP对AlCl3诱导的细胞死亡过程中胞内H2O2和Ca2+水平的影响及其生理机制分析

该实验以烟草悬浮细胞 BY 2 为材料,在烟草悬浮细胞中分别加入0.05、0.10、0.15、0.20 mmol·L^-1AlCl₃,以等体积去离子水处理的悬浮细胞液为对照,并依据前述实验结果选择0.15 mmol·L^-1 AlCl₃,分别添加5 mmol·L^-1 DMTU(H₂O₂ 抑制剂)、20 μmol·L^-1CaCl₂、15 μmol·L^-1 LaCl₃(Ca²⁺通道抑制剂)和50 μmol·L^-1 ATP设计多项处理,分析胞外ATP(eATP)对铝离子(Al³⁺)胁迫引起的植物细胞死亡及其胞内H₂O₂、Ca²⁺的影响,以揭示Al³⁺胁迫下植物调节细胞死亡的可能机制,进一步扩展对eATP功能的认知。结果显示：(1)随着 AlCl₃ 胁迫浓度的提高,细胞死亡水平和胞内H₂O₂水平上升,而胞内Ca²⁺和eATP水平则逐渐降低。(2)外援施加H₂O₂抑制剂 DMTU(二甲基硫脲)和Ca²⁺能够有效缓解AlCl₃诱导的细胞死亡水平的上升;而Ca²⁺通道抑制剂LaCl₃(三氯化镧)则加剧了AlCl₃胁迫下的细胞死亡。(3)在AlCl₃胁迫下对细胞添加外源ATP,能够缓解AlCl₃胁迫下胞内H₂O₂水平上升和Ca²⁺水平下降的同时,并显著降低AlCl₃胁迫导致的细胞死亡。研究表明, Al³⁺以剂量依赖的模式提升细胞死亡和细胞内H₂O₂的水平并降低胞内Ca²⁺和eATP水平,AlCl₃诱导的细胞死亡受到H₂O₂和Ca²⁺水平变化的调节,eATP可以通过调节H₂O₂与Ca²⁺水平缓解AlCl₃诱导的细胞死亡。推测Al³⁺胁迫可能通过抑制钙离子通道而破坏了细胞内H₂O₂和Ca²⁺之间的协同关系,外源ATP对Al³⁺诱导H₂O₂上升的缓解作用可能是由于其提升了细胞的抗氧化能力。     

H2O2－Fe3+所致人淋巴细胞DNA双链断裂损伤

采用脉冲电场凝胶电泳法检测H₂O₂－Fe³⁺体系产生的OH^·对人淋巴细胞DNA的双链断裂损伤．H₂O₂－Fe³⁺浓度与DNA双链断裂呈明显量效关系;随OH^·作用时间延长,细胞DNA双链断裂加重;过氧化氢酶对OH^·损伤有明显抑制作用．脉冲电场凝胶电泳法可检测到的H₂O₂和FeCl₃引起细胞DNA双链断裂的最低浓度为0.3 mmol/L和6 μmol/L．     

活性氧清除剂和钙离子通道阻断剂对人淋巴细胞化学发光的效应

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。     

羟自由基对培养细胞损伤作用的实验观察

将不同浓度的H₂O₂与V79细胞共同孵育24、48h后,结果显示SOD活性下降,LPO含量增加,细胞内GPT、GOT、LDH和CPK酶活性升高,其变化程度与培养时间和H₂O₂浓度成正比.     

植物叶片中过氧化氢含量测定方法的改进

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.     

NO和H2O2诱导大豆根尖和边缘细胞耐铝反应的作用

 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。     

干旱及活性氧引起小麦膜脂过氧化与脱酯化

水分胁迫下随小麦叶片含水量下降,膜透性增大,两者高度负相关;同时叶片H₂O₂含量和叶片微粒体膜脂过氧化水平都上升,无论在干旱时或在外源O₂^-和·OH作用时,微粒体膜流动性均下降,外源O₂^-和·OH处理微粒体膜后,膜脂过氧化水平明显增高,脂肪酸不饱和度明显下降;同时磷脂减少,游离脂肪酸增加。结果表明干旱可能使植物体内活性氧增多,从而导致膜损伤,这种由活性氧引起的膜损伤是膜脂过氧化和脱酯化共同作用的结果     

Cr3+和Cd2+对普通小球藻生长及抗氧化酶活性的影响

[目的] 为探究重金属对淡水绿藻生长的影响。[方法] 选取对水质检测具有明显指示作用的普通小球藻（Chlorella vulgaris）为实验材料,CdCl₂·2H₂O和CrCl₃·7H₂O提供重金属离子,探究不同浓度Cr³⁺和Cd²⁺在单一和复合胁迫下对藻细胞浓度、叶绿素a及相关抗氧化酶活性的影响。[结果] 随着Cr³⁺和Cd²⁺浓度不断增加,藻细胞浓度呈先增长后下降趋势;叶绿素a含量呈现先下降后升高再下降的现象,浓度为1 mg/L的单一和复合胁迫下有最大值,且毒性作用表现为Cr³⁺ < Cd²⁺ < Cr³⁺+Cd²⁺;与藻细胞膜相关的丙二醛（MDA）和过氧化氢（H₂O₂）含量随着重金属离子浓度的增大而增长;重金属离子浓度低于10 mg/L时对藻细胞内抗氧化酶系统中的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）表现为促进作用,而大于10 mg/L时具有抑制作用。[结论] 结果表明在单一或复合重金属胁迫下,普通小球藻会充分调动与抗逆性相关的酶来维持自身的正常生长。     

呼吸链电子漏在细胞凋亡中的作用

实验证明细胞色素c具有很强的抗氧化功能,在线粒体中氧化态的细胞色素c直接清除O2·-,还原态的细胞色素c清除H2O2.由于呼吸链传递电子合成ATP的同时,总有少部分电子从呼吸链底物端的复合物Ⅰ和Ⅲ漏出,而且漏出的电子首先使氧分子还原成O2·- ,然后O2·-歧化成H2O2,所以细胞色素c清除O2·-和H2O2的功能使呼吸链出现了两条电子漏旁路.细胞色素c通过这两条电子漏路径实现其控制线粒体中O2·-和H2O2水平的功能.根据两条电子漏旁路都是O2·-代谢路径的事实,引进了线粒体自由基代谢的概念,并从自由基代谢失调的角度探讨了呼吸链电子漏在细胞凋亡中的作用.     

外源H2O2对盐胁迫下小白菜种子萌发和幼苗生理特性的影响

以小白菜"甜脆青"为试材,研究不同浓度（5、10、25、50和100 mmol/L）过氧化氢（H₂O₂）浸种处理对100 mmol/L NaCl胁迫下小白菜（Brassica chinensis L.）种子萌发、幼苗生长及生理特性的影响。结果表明：100 mmol/L NaCl胁迫明显抑制小白菜种子的萌发状况和幼苗生长,发芽势、发芽指数、活力指数及幼苗根和芽长度和鲜重均明显降低,根和芽中CAT的活性及K⁺含量明显受到抑制,渗透调节物质、活性氧和MDA含量显著增加。不同浓度H₂O₂浸种处理提高了NaCl胁迫下小白菜种子发芽势、发芽指数和活力指数,促进小白菜根和芽的生长,增强了NaCl胁迫下根和芽中SOD、CAT和APX的活性及K⁺含量,降低O₂^-·产生速率及H₂O₂和MDA含量,进一步促进脯氨酸和可溶性糖含量的增加,降低体内Na⁺含量。其中以10 mmol/L H₂O₂处理缓解盐胁迫效果最好,明显缓解NaCl胁迫对小白菜种子萌发和幼苗生长的抑制。     

Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in Escherichia coli Exposed to Hydrogen Peroxide

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O₂^·− and H₂O₂. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H₂O₂ but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (^·OH) as a consequence of the reaction of H₂O₂ with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H₂O₂ in the presence of an α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)–ethanol spin-trapping system, the 4-POBN–^·CH(CH₃)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating ^·OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H₂O₂ in a similar manner, the magnitude of ^·OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of ^·OH spin trapped. Exogenous SOD failed to inhibit ^·OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in ^·OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H₂O₂-mediated killing seen in these mutants lacking SOD.     

Enzymatic radioiodination of phospholipids catalyzed by lactoperoxidase

Phospholipids were iodinated with iodide by a lactoperoxidase-catalyzed reaction in the presence of controlled amounts of H₂O₂ which were continuously supplied by glucose oxidase + glucose. Different molecular and ionic species of inorganic iodine present in the reaction mixture (i.e., I^?, I₂, I₃^?) were eliminated by thiosulfate reduction to I^? followed by gel filtration on Sephadex LH-20 which separated I^? from the phospholipids completely. Final separation and identification of individual phospholipids were done on a column of silica gel H using a single solvent mixture consisting of CHCl₃:CH₃OH:CH₃COOH:H₂O (25:15:4:2, by volume). Application of phospholipases A₂ and D or transesterification provided evidence to indicate a covalent iodination of the fatty acid moiety of the lipids by the enzymatic process, which apparently is substitution but could also proceed by addition to the double bonds, when present.     

Impact of SOD in eNOS uncoupling: a two-edged sword between hydrogen peroxide and peroxynitrite

In endothelial cell dysfunction, the uncoupling of eNOS results in higher superoxide (O₂^??) and lower NO production and a reduction in NO availability. Superoxide reacts with NO to form a potent oxidizing agent peroxynitrite (ONOO^?) resulting in nitrosative and nitroxidative stresses and dismutates to form hydrogen peroxide. Studies have shown superoxide dismutase (SOD) plays an important role in reduction of O₂^?? and ONOO^? during eNOS uncoupling. However, the administration or over-expression of SOD was ineffective or displayed deleterious effects in some cases. An understanding of interactions of the two enzyme systems eNOS and SOD is important in determining endothelial cell function. We analyzed complex biochemical interactions involving eNOS and SOD in eNOS uncoupling. A computational model of biochemical pathway of the eNOS-related NO and O₂^?? production and downstream reactions involving NO, O₂^??, ONOO^?, H₂O₂ and SOD was developed. The effects of SOD concentration on the concentration profiles of NO, O₂^??, ONOO^? and H₂O₂ in eNOS coupling/uncoupling were investigated. The results include (i) SOD moderately improves NO production and concentration during eNOS uncoupling, (ii) O₂^?? production rate is independent of SOD concentration, (iii) Increase in SOD concentration from 0.1 to 100 μM reduces O₂^?? concentration by 90% at all [BH₄]/[TBP] ratios, (iv) SOD reduces ONOO^? concentration and increases H₂O₂ concentration during eNOS uncoupling, (v) Catalase can reduce H₂O₂ concentration and (vi) Dismutation rate by SOD is the most sensitive parameter during eNOS uncoupling. Thus, SOD plays a dual role in eNOS uncoupling as an attenuator of nitrosative/nitroxidative stress and an augmenter of oxidative stress.     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

Interactions of tertiary phosphines with p-benzoquinones; X-ray structures of [HO(CH2)3]3PC6H2(O)(OH)(MeO) and Ph3PC6H3(O)(OH)

Phosphonium zwitterions of a known type were obtained in high yield via a 1:1 reaction of p-benzoquinone or methoxy-p-benzoquinone with the tertiary phosphines R₃P [R = (CH₂)₃OH, Ph, Et, Me] and Ph₂MeP, in acetone or benzene at room temperature. In all cases, attack of the P-atom occurs at a C-atom rather than at an O-atom. The products were characterized to various degrees by elemental analysis, ³¹P{¹H}, ¹H and ¹³C NMR spectroscopies, and mass spectrometry, and two of the zwitterions, the new [HO(CH₂)₃]₃P⁺C₆H₂(O⁻)(OH)(MeO) and the known Ph₃P⁺C₆H₃(O⁻)(OH), were structurally characterized by X-ray analysis. The PEt₃ reaction also produces small amounts of the ‘dimeric’, μ-oxo co-product Et₃P⁺C₆H₂(O⁻)(OH)-O-C₆H₃(O⁻)P⁺Et₃ that is tentatively characterized by 1D- and 2D-NMR data. 2,5-Di-tert-butyl- and 2,3,5,6-tetramethyl-p-benzoquinone do not react with [HO(CH₂)₃]₃P under the conditions noted above. Heating D₂O solutions of the water-soluble zwitterions R₃P⁺C₆H₃(O⁻)(OH) [R = (CH₂)₃OH, Et] at 90 °C for 72 h leads to complete H/D exchange of the H-atom in the position ortho to the phosphonium center.     

Solubility and reactivity of caseins and β-lactoglobulin in protic solvents

The study of the solubility of unstructured proteins (α_S1-, β-, and κ-casein) and well-structured globulin (β-lactoglobulin) in low water binary solvent systems demonstrated the crucial importance of solvent polarity and neutralization of protein polar functions on the final outcome of solubility experiments. The solubilities up to 38, 56, and 96% in CHCl₃/CH₃OH (1/1, v/v) acidified with HCl and up to 5, 10, and 25% in CHCl₃/CH₃OH (1/1, v/v) in the presence of triethylamine (TEA) were obtained for κ-, α_S1-, and β-casein, respectively. The importance of protein charge neutralization was apparent when the solubilization was performed in basified CHCl₃/CH₃OH media, giving the optimal results when the studied proteins were brought before to their isoionic point. The maximum solubility of β-casein at its pI in 30–70% methanol in CHCl₃ was reaching 50–60% with triethylamine (TEA) added. β-lactoglobulin could be solubilized up to 70% in CHCl₃/CH₃OH (7/3, v/v) acidified with HCl and up to 40% in CHCl₃/CH₃OH (3/7, v/v) in the presence of TEA. The observed yield of reductive alkylation of β-lactoglobulin was much higher (98%) when performed in studied solvent system than in aqueous conditions (75%). Apparently, steric hindrance of the well-folded β-barrel (in aqueous conditions) structure masks the portion of ε-NH₂ groups. In the case of unstructured aqueous media β-casein, 90% alkylation yields were obained in organic and aqueous conditions.     

Novel dioxomolybdenum(VI) and oxomolybdenum(V) complexes with pyridoxal thiosemicarbazone ligands: Synthesis and structural characterisation

New molybdenum complexes were prepared by the reaction of [Mo^VIO₂(acac)₂] or (NH₄)₂[Mo^VOCl₅] with different N-substituted pyridoxal thiosemicarbazone ligands (H₂L¹ = pyridoxal 4-phenylthiosemicarbazone; H₂L² = pyridoxal 4-methylthiosemicarbazone, H₂L³ = pyridoxal thiosemicarbazone). The investigation of monomeric [MoO₂L¹(CH₃OH)] or polymeric [MoO₂L^1-3] molybdenum(VI) complexes revealed that molybdenum is coordinated with a tridentate doubly-deprotonated ligand. In the oxomolybdenum(V) complexes [MoOCl₂(HL^1-3)] the pyridoxal thiosemicarbazonato ligands are tridentate mono-deprotonated. Crystal and molecular structures of molybdenum(VI) [MoO₂L¹(CH₃OH)]·CH₃OH, and molybdenum(V) complexes [MoOCl₂(HL¹)]·C₂H₅OH, as well as of the pyridoxal thiosemicarbazone ligand methanol solvate H₂L³·MeOH, were determined by the single crystal X-ray diffraction method.     

Synthesis and X-ray structures of rhenium(I) carbonyl aminoalkoxide and aminocarboxylate complexes

The complexes [Re{MeN(CH₂CH₂O)(CH₂CH₂OH)-κ³N,O,O}(CO)₃] (1), [Re{N(CH₂CH₂O)(CH₂CH₂OH)₂-κ³N,O,O}(CO)₃] (2), [Me₃NH]₂[(OC)₃Re{N(CH₂CO₂)₂-κ³N,O,O}CH₂CH₂{N(CH₂CO₂)₂-κ³N,O,O}Re(CO)₃] (3), [Me₃NH]₂[Re₂{μ₂-2,6-(O₂C)₂(C₅H₃N)-κ³N,O,O}₂(CO)₆] (4) and [Re₂{μ₂-2,6-(OCH₂)(C₅H₃N)(CH₂OH)-κ²N,O}₂(CO)₆] (5) were synthesized in high yields via the reactions of [Re₂(CO)₁₀] and Me₃NO with MeN(CH₂CH₂OH)₂, N(CH₂CH₂OH)₃, EDTA, pyridine-2,6-dicarboxylic acid and pyridine-2,6-dimethanol, respectively. Complexes 1-5 were characterized by IR and ¹H NMR spectroscopy, elemental analysis and X-ray crystallography.     

Mitochondria-derived Hydrogen Peroxide Selectively Enhances T Cell Receptor-initiated Signal Transduction

T cell receptor (TCR)-initiated signal transduction is reported to increase production of intracellular reactive oxygen species, such as superoxide (O₂^⨪) and hydrogen peroxide (H₂O₂), as second messengers. Although H₂O₂ can modulate signal transduction by inactivating protein phosphatases, the mechanism and the subcellular localization of intracellular H₂O₂ as a second messenger of the TCR are not known. The antioxidant enzyme superoxide dismutase (SOD) catalyzes the dismutation of highly reactive O₂^⨪ into H₂O₂ and thus acts as an intracellular generator of H₂O₂. As charged O₂^⨪ is unable to diffuse through intracellular membranes, cells express distinct SOD isoforms in the cytosol (Cu,Zn-SOD) and mitochondria (Mn-SOD), where they locally scavenge O₂^⨪ leading to production of H₂O₂. A 2-fold organelle-specific overexpression of either SOD in Jurkat T cell lines increases intracellular production of H₂O₂ but does not alter the levels of intracellular H₂O₂ scavenging enzymes such as catalase, membrane-bound peroxiredoxin1 (Prx1), and cytosolic Prx2. We report that overexpression of Mn-SOD enhances tyrosine phosphorylation of TCR-associated membrane proximal signal transduction molecules Lck, LAT, ZAP70, PLCγ1, and SLP76 within 1 min of TCR cross-linking. This increase in mitochondrial H₂O₂ specifically modulates MAPK signaling through the JNK/cJun pathway, whereas overexpressing Cu,Zn-SOD had no effect on any of these TCR-mediated signaling molecules. As mitochondria translocate to the immunological synapse during TCR activation, we hypothesize this translocation provides the effective concentration of H₂O₂ required to selectively modulate downstream signal transduction pathways.     

Assay and Electrophoresis of Superoxide Dismutase from Red Spruce (Picea rubens Sarg.), Loblolly Pine (Pinus taeda L.), and Scotch Pine (Pinus sylvestris L.) : A Method for Biomonitoring

This paper reports a method for extracting the antioxidant enzyme superoxide dismutase (SOD) from the needles of red spruce (Picea rubens Sarg.), loblolly pine (Pinus taeda L.), and scotch pine (Pinus sylvestris L.) with high efficiency and free from interfering compounds. The extraction employs phosphate buffer with polyvinylpolypyrrolidone and Triton X-100 followed by dialysis overnight. The isozymes of SOD in each species were separated electrophoretically and tested for their sensitivity to KCN and H₂O₂. An isozyme resistant to these inhibitors was found in the spruce but not the pine needles. The isozymes from the spruce needles were examined for individual responses to aging and H₂O₂ inhibition. Four of the five CuZn isozymes in spruce were found to have increased significantly but equally by October of their first year and two of those four isozymes were found to be more sensitive to H₂O₂. The response of the SOD isozymes in loblolly pine seedlings to O₃ was also examined and the isozymes were found to be induced equally. Because the SOD activity in the young pine needles was too low to electrophorese, the SOD activity from the pines in the O₃ experiment had to be partially purified using CHCl₃ and ethanol, then concentrated.