Hydroxyl-radical production in physiological reactions A novel function of peroxidase 生理反应中产生的羟基自由基 过氧化物酶的新功能
Si-xue Chen and Peter Schopfer 陈思学和彼得-肖普费尔Institut für Biologie II der Universität Freiburg, Germany 德国弗莱堡大学第二生物学研究所
Abstract 摘要
Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H_(2)O_(2))\left(\mathrm{H}_{2} \mathrm{O}_{2}\right) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O_(2)\mathrm{O}_{2} to superoxide (O_(2)^(-))\left(\mathrm{O}_{2}{ }^{-}\right)and H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals ( OH ) from H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} in the presence of O_(2)^(-)\mathrm{O}_{2}{ }^{-}. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O_(2)^(--)\mathrm{O}_{2}{ }^{--}into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of ’ OH from O_(2)^(--)\mathrm{O}_{2}{ }^{--}and H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O_(2)^(--)\mathrm{O}_{2}{ }^{--}and H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The ^(@)OH{ }^{\circ} \mathrm{OH}-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O_(2)^(--)\mathrm{O}_{2}{ }^{--}and ^(@)OH{ }^{\circ} \mathrm{OH} scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-21-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high ^(@)OH-{ }^{\circ} \mathrm{OH}- producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the ^(@)OH{ }^{\circ} \mathrm{OH}-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously. 过氧化物酶在过氧化反应循环中催化过氧化氢 (H_(2)O_(2))\left(\mathrm{H}_{2} \mathrm{O}_{2}\right) 对各种酚类和内酚类底物的脱氢反应。此外,这些酶还表现出氧化酶活性,介导 O_(2)\mathrm{O}_{2} 被 NADH 或二羟富马酸等底物还原为超氧化物 (O_(2)^(-))\left(\mathrm{O}_{2}{ }^{-}\right) 和 H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 。在这里,我们证明辣根过氧化物酶还能催化第三种反应,即在 O_(2)^(-)\mathrm{O}_{2}{ }^{-} 存在的情况下,由 H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 生成羟自由基(OH)。我们提供的证据表明,要介导这一反应,辣根过氧化物酶的铁形态必须由 O_(2)^(--)\mathrm{O}_{2}{ }^{--} 转化为高铁形态(化合物 III),其中血红素铁可以呈现亚铁状态。由此得出结论,铁/高铁过氧化物酶偶联物是一种有效的生化催化剂,可以从 O_(2)^(--)\mathrm{O}_{2}{ }^{--} 和 H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 中产生 ' OH(铁催化的哈伯-魏斯反应)。这种反应可以通过苯甲酸的羟基化或脱氧核糖的降解来测量。 O_(2)^(--)\mathrm{O}_{2}{ }^{--} 和 H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 可由辣根过氧化物酶在 NADH 存在下的氧化酶反应产生。辣根过氧化物酶产生 ^(@)OH{ }^{\circ} \mathrm{OH} 的活性可被血红素铁的灭活剂或各种 O_(2)^(--)\mathrm{O}_{2}{ }^{--} 和 ^(@)OH{ }^{\circ} \mathrm{OH} 清除剂抑制。在等摩尔铁的基础上,辣根过氧化物酶的活性 1-21-2 比哈伯-魏斯反应的无机催化剂 Fe-EDTA 高出几个数量级。碱性辣根过氧化物酶同工酶和木质素酶型真菌过氧化物酶的 ^(@)OH-{ }^{\circ} \mathrm{OH}- 生成活性特别高,而乳过氧化物酶和大豆过氧化物酶的活性较低,髓过氧化物酶没有活性。 在 ^(@)OH{ }^{\circ} \mathrm{OH} 产生模式下,过氧化物酶可能是造成先前报道的活性氧众多破坏性和毒性作用的原因。
The hydroxyl radical ( OH ) constitutes the chemically most reactive species of ‘activated oxygen’ formed by successive monovalent reduction of dioxygen (O_(2))\left(\mathrm{O}_{2}\right) in cell metabolism, and is primarily responsible for the cytotoxic effects of oxygen in plants, animals and micro-organisms, living in an oxygenic atmosphere [1,2]. The short-lived ^(@)OH{ }^{\circ} \mathrm{OH} molecule unspecifically attacks biomolecules in a diffusion-limited reaction and is thus able to crack, for instance, polysaccharides, proteins and nucleic acids located less than a few nanometres from its site of generation [3]. Hydroxyl radicals can be produced from O_(2)\mathrm{O}_{2} under a variety of stress conditions and are involved in numerous cellular disorders such as inflammations [4], embryo teratogenesis [5], herbicide effects [6,7][6,7], cell death [8,9][8,9] and killing of micro-organisms in pathogen-defence reactions [10]. There is evidence that these toxic effects can be traced back to damage by ^(@)OH{ }^{\circ} \mathrm{OH} of DNA [11], proteins [12], or membrane lipids [13]. It is generally assumed [2,12,14][2,12,14] that ^(@)OH{ }^{\circ} \mathrm{OH} is generated in biological systems from H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} by the Fenton reaction, known from inorganic chemistry: 羟自由基(OH)是细胞新陈代谢中二氧 (O_(2))\left(\mathrm{O}_{2}\right) 连续一价还原所形成的化学活性最强的 "活化氧 "物种,是氧对生活在含氧环境中的植物、动物和微生物产生细胞毒性作用的主要原因 [1,2]。这种寿命很短的 ^(@)OH{ }^{\circ} \mathrm{OH} 分子在扩散受限的反应中非特异性地攻击生物大分子,因此能够裂解距离其产生地点不到几纳米的多糖、蛋白质和核酸等物质 [3]。在各种压力条件下, O_(2)\mathrm{O}_{2} 可产生羟自由基,羟自由基与许多细胞疾病有关,如炎症[4]、胚胎致畸[5]、除草剂效应 [6,7][6,7] 、细胞死亡 [8,9][8,9] 和病原体防御反应中的微生物杀灭[10]。有证据表明,这些毒性作用可追溯到 DNA[11]、蛋白质[12]或膜脂质[13]的 ^(@)OH{ }^{\circ} \mathrm{OH} 损伤。一般认为, [2,12,14][2,12,14] 在生物系统中是由 H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 通过无机化学中已知的芬顿反应生成的:
whereby Fe^(2+)\mathrm{Fe}^{2+} can be regenerated through the oxidation by the superoxide anion (O_(2)^(--))\left(\mathrm{O}_{2}{ }^{--}\right): 其中 Fe^(2+)\mathrm{Fe}^{2+} 可以通过超氧阴离子 (O_(2)^(--))\left(\mathrm{O}_{2}{ }^{--}\right) 的氧化作用再生: Fe^(3+)+O_(2)^(--)rarrFe^(2+)+O_(2)\mathrm{Fe}^{3+}+\mathrm{O}_{2}^{--} \rightarrow \mathrm{Fe}^{2+}+\mathrm{O}_{2}
The combination of Eqns (1) and (2) is referred to as the ironcatalyzed Haber-Weiss reaction [15] shown below: 公式 (1) 和 (2) 的组合称为铁催化的哈伯-魏斯反应 [15] ,如下图所示:
H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} and O_(2)^(--)\mathrm{O}_{2}{ }^{--}are ubiquitously formed from O_(2)\mathrm{O}_{2} as byproducts of electron transport process and flavin-catalyzed oxidase reactions and are thus potentially available in all aerobic cells. In contrast, the catalytic role of Fe^(2+)//Fe^(3+)\mathrm{Fe}^{2+} / \mathrm{Fe}^{3+} in the production of 'OH by biological systems has not yet been demonstrated directly and the ‘biological Haber-Weiss reaction’ is so far an extrapolation from inorganic chemistry rather than an experimentally proven fact. H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 和 O_(2)^(--)\mathrm{O}_{2}{ }^{--} 是由 O_(2)\mathrm{O}_{2} 作为电子传递过程和黄素催化的氧化酶反应的副产物普遍形成的,因此可能存在于所有有氧细胞中。相比之下, Fe^(2+)//Fe^(3+)\mathrm{Fe}^{2+} / \mathrm{Fe}^{3+} 在生物系统产生'OH'过程中的催化作用尚未得到直接证明,'生物哈伯-魏斯反应'迄今为止只是从无机化学中推断出来的,而不是实验证明的事实。
Peroxidases constitute a class of haem-containing enzymes ubiquitously present in prokaryotic and eukaryotic organisms [16] which catalyze the dehydrogenation of structurally diverse phenolic and endiolic substrates by H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} and are thus often regarded as antioxidant enzymes, protecting cells from the destructive influence of H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} and derived oxygen species [17]. However, in addition to this peroxidatic activity, peroxidases possess an oxidase activity by which electrons can be transferred from reducing substrates such as NADH to O_(2)\mathrm{O}_{2}. It has been shown that this oxidative activity involves the formation of O_(2)^(--)\mathrm{O}_{2}{ }^{--} and H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} as intermediates, and the conversion of the ferric form of the enzyme ( Fe^(3+)\mathrm{Fe}^{3+}-peroxidase) into the labile perferryl form (Fe^(2+)-O_(2)*^(--)Fe^(3+)-O_(2)^(--):}\left(\mathrm{Fe}^{2+}-\mathrm{O}_{2} \cdot{ }^{--} \mathrm{Fe}^{3+}-\mathrm{O}_{2}{ }^{--}\right.peroxidase) also designated as Compound III [18]. Compound III is generally considered as enzymatically 过氧化物酶是一类普遍存在于原核生物和真核生物体内的含血酶 [16],可催化 H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 对结构多样的酚类和内酚类底物的脱氢反应,因此常被视为抗氧化酶,可保护细胞免受 H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 和衍生氧物种的破坏性影响 [17]。然而,除了过氧化活性外,过氧化物酶还具有氧化酶活性,可将电子从还原底物(如 NADH)转移到 O_(2)\mathrm{O}_{2} 。研究表明,这种氧化活性涉及作为中间产物的 O_(2)^(--)\mathrm{O}_{2}{ }^{--} 和 H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 的形成,以及铁形式的酶( Fe^(3+)\mathrm{Fe}^{3+} -过氧化物酶)向易失铁形式的 (Fe^(2+)-O_(2)*^(--)Fe^(3+)-O_(2)^(--):}\left(\mathrm{Fe}^{2+}-\mathrm{O}_{2} \cdot{ }^{--} \mathrm{Fe}^{3+}-\mathrm{O}_{2}{ }^{--}\right. 过氧化物酶(也称为化合物 III)的转化 [18]。化合物 III 通常被认为是酶
inactive although it has been implicated in the oxidation of indole-3-acetic acid [19]. As Compound III contains Fe^(2+)\mathrm{Fe}^{2+} in the prosthetic haem group that can be easily converted to Fe^(3+)\mathrm{Fe}^{3+}, one can imagine that Compound III can act as a Fenton reagent in a manner similar to Fe^(2+)\mathrm{Fe}^{2+}-chelates, such as Fe^(2+)\mathrm{Fe}^{2+}-EDTA [20]. This idea was fostered by the fact that the unphysiological substrate dihydroxyfumarate converts horseradish peroxidase into Compound III and mediates the hydroxylation of aromatic compounds via the generation of ^(@)OH[21-23]{ }^{\circ} \mathrm{OH}[21-23]. To our knowledge, the obvious possibility that the Compound III/Ferr-peroxidase couple functions as a biological catalyst of the Haber-Weiss reaction has not yet been examined rigorously. 虽然化合物 III 与吲哚-3-乙酸的氧化作用有关 [19],但它不具有活性。由于化合物 III 的人工血红素基团中含有 Fe^(2+)\mathrm{Fe}^{2+} ,可以很容易地转化为 Fe^(3+)\mathrm{Fe}^{3+} ,因此可以想象化合物 III 可以以类似于 Fe^(2+)\mathrm{Fe}^{2+} -螯合物(如 Fe^(2+)\mathrm{Fe}^{2+} -EDTA)的方式充当芬顿试剂 [20]。促进这一想法的事实是,非生理学底物富马酸二羟酯可将辣根过氧化物酶转化为化合物 III,并通过生成 ^(@)OH[21-23]{ }^{\circ} \mathrm{OH}[21-23] 来介导芳香族化合物的羟基化。据我们所知,化合物 III/铁-过氧化物酶偶联物作为哈伯-魏斯反应的生物催化剂的明显可能性尚未得到严格研究。
In the course of a research program aimed at the elucidation of the biochemical mechanism of cell-wall loosening during auxin-dependent elongation growth of plant organs [24] we consider the possibility that this process is mediated by a sitespecific production of OH in the cell wall, resulting in the cleavage of load-bearing bonds within wall polymers. Peroxidase is generally present abundantly in the walls of growing plant cells [25] and has been implicated in the apoplastic generation of O_(2)^(--)\mathrm{O}_{2}{ }^{--}and H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} from O_(2)\mathrm{O}_{2} and NADH [26]. It was therefore of interest to find out whether this enzyme could mediate the production of ^(@)OH{ }^{\circ} \mathrm{OH} under these conditions. 我们的研究计划旨在阐明植物器官在依赖助长素的伸长生长过程中细胞壁松弛的生化机制 [24],在这一研究过程中,我们考虑了这样一种可能性,即这一过程是由细胞壁中特定位点产生的 OH 介导的,从而导致细胞壁聚合物中的承重键裂解。过氧化物酶通常大量存在于生长中的植物细胞壁中 [25],并与 O_(2)\mathrm{O}_{2} 和 NADH 生成 O_(2)^(--)\mathrm{O}_{2}{ }^{--} 和 H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 的凋亡过程有关 [26]。因此,我们有兴趣了解这种酶是否能在这些条件下介导 ^(@)OH{ }^{\circ} \mathrm{OH} 的产生。
MATERIALS AND METHODS 材料和方法
Chemicals 化学品
Horseradish peroxidase (grade I, mixture of basic and acidic forms in 3.2 m ammonium sulfate), catalase (from bovine liver), superoxide dismutase (from bovine erythrocytes), NADH and NADPH were from Boehringer (Mannheim, Germany); sodium benzoate, sodium formate, thiourea, Tiron, desferrioxamine mesylate, dihydroxyfumarate, Chelex 100 chelating resin and other types of peroxidase were from Sigma (Deisenhofen, Germany); diphenyleneiodonium chloride (dissolved in dimethylsulfoxide) was from Biomol (Hamburg, Germany); ascorbate was from Merck (Darmstadt, Germany), H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} was from Fluka (Buchs, Switzerland); 2-deoxy-D-ribose was from Serva (Heidelberg, Germany). All other reagents were of analytical grade. The concentration of H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} solutions was standardized photometrically using an extinction coefficient of 39.4m^(-1)*cm^(-1)39.4 \mathrm{~m}^{-1} \cdot \mathrm{~cm}^{-1} at 240nm.Fe^(3+)240 \mathrm{~nm} . \mathrm{Fe}^{3+}-EDTA was prepared by mixing equal concentrations of FeCl_(3)\mathrm{FeCl}_{3} and Na_(2)\mathrm{Na}_{2}-EDTA in 10 mm sodium citrate buffer ( pH 6.0 ). 辣根过氧化物酶(I 级,在 3.2 m 硫酸铵中的碱性和酸性混合物)、过氧化氢酶(来自牛肝)、超氧化物歧化酶(来自牛红细胞)、NADH 和 NADPH 来自 Boehringer 公司(德国曼海姆);苯甲酸钠、甲酸钠、硫脲、Tiron、甲磺酸去铁胺、二羟富马酸盐、Chelex 100 螯合树脂和其他类型的过氧化物酶来自 Sigma 公司(德国代森霍芬);二苯基碘氯铵(溶于二甲基亚砜)来自 Biomol 公司(德国汉堡);抗坏血酸来自 Merck 公司(德国达姆施塔特); H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 来自 Fluka 公司(瑞士布赫斯);2-脱氧-D-核糖来自 Serva 公司(德国海德堡)。所有其他试剂均为分析级。 H_(2)O_(2)\mathrm{H}_{2} \mathrm{O}_{2} 溶液的浓度是用 39.4m^(-1)*cm^(-1)39.4 \mathrm{~m}^{-1} \cdot \mathrm{~cm}^{-1} 在 240nm.Fe^(3+)240 \mathrm{~nm} . \mathrm{Fe}^{3+} 的消光系数 39.4m^(-1)*cm^(-1)39.4 \mathrm{~m}^{-1} \cdot \mathrm{~cm}^{-1} 进行光度标准化的, FeCl_(3)\mathrm{FeCl}_{3} 和 Na_(2)\mathrm{Na}_{2} -EDTA是在 10 mm 柠檬酸钠缓冲液(pH 6.0)中混合等浓度的 FeCl_(3)\mathrm{FeCl}_{3} 和 Na_(2)\mathrm{Na}_{2} 制备的。
Enzyme assays 酶测定
All incubations and assays were performed at 25^(@)C25^{\circ} \mathrm{C} in 10 mm sodium citrate buffer ( pH 6.0 , except where stated otherwise). Production of ^(@)OH{ }^{\circ} \mathrm{OH} by peroxidase was determined with a fluorimetric method based on the hydroxylation of benzoate [27]. The reaction mixture ( 1 mL in a 5xx10-mm5 \times 10-\mathrm{mm} cell, type OS, Hellma, Germany) contained 2 mm benzoate and additions as indicated in legends. The reaction was normally started by adding enzyme and the increase in fluorescence (excitation 305 nm , emission 407 nm ), mainly a result of the formation of 3-hydroxybenzoate, was recorded for up to 30 min . If, e.g. at pH < 6\mathrm{pH}<6, the reaction demonstrated a significant background fluorescence change in the absence of benzoate, this had to be subtracted from the rate measured in the presence of benzoate. Alternatively, OH production was determined with a photometric method based on the degradation of deoxyribose [28]. Reaction mixtures as above, but containing 2 mm deoxyribose instead of benzoate, were incubated for 1 h . A 0.6-mL0.6-\mathrm{mL} aliquot 除另有说明外,所有培养和检测均在 10 mm 柠檬酸钠缓冲液(pH 6.0)中 25^(@)C25^{\circ} \mathrm{C} 进行。过氧化物酶产生的 ^(@)OH{ }^{\circ} \mathrm{OH} 是用基于苯甲酸羟基化的荧光法测定的 [27]。反应混合物( 5xx10-mm5 \times 10-\mathrm{mm} 池中的 1 mL,OS 型,德国 Hellma 公司)含有 2 mm 苯甲酸盐和图例中标明的添加物。反应通常是通过加入酶开始的,荧光(激发波长 305 nm,发射波长 407 nm)的增加主要是 3-羟基苯甲酸酯形成的结果,记录时间长达 30 分钟。如果在 pH < 6\mathrm{pH}<6 处,反应在没有苯甲酸盐的情况下显示出明显的背景荧光变化,则必须将其从苯甲酸盐存在时测得的速率中减去。另一种方法是使用基于脱氧核糖降解的光度法测定 OH 的产生[28]。将含有 2 mm 脱氧核糖而不是苯甲酸盐的上述反应混合物培养 1 小时。取 0.6-mL0.6-\mathrm{mL} 等分
was mixed with 0.5 mL thiobarbituric-acid solution ( 10g*L^(-1)10 \mathrm{~g} \cdot \mathrm{~L}^{-1} in 50 mm NaOH ) and 0.5 mL trichloroacidic acid solution ( 28g*L^(-1)28 \mathrm{~g} \cdot \mathrm{~L}^{-1} ). After heating in a boiling water bath for 20 min and cooling on ice, the absorbance of the pink thiobarbituric-acid adduct was measured at 532 nm in a 10-mm10-\mathrm{mm} cell against blanks without enzyme. Turbid solutions were extracted with an equal volume of butan-1-ol and the absorbance measured in the extract. The presence of contaminating iron in the reagents was checked by passing buffer, etc. through a column of Chelex 100 chelating resin and horseradish peroxidase solutions though Sephadex G-25 (Pharmacia). As these precautions did not significantly affect the results, they were omitted in later experiments. 与 0.5 mL 硫代巴比妥酸溶液( 10g*L^(-1)10 \mathrm{~g} \cdot \mathrm{~L}^{-1} 在 50 mm NaOH 中的浓度)和 0.5 mL 三氯酸溶液( 28g*L^(-1)28 \mathrm{~g} \cdot \mathrm{~L}^{-1} )混合。在沸水浴中加热 20 分钟并在冰上冷却后,在 10-mm10-\mathrm{mm} 波长 532 nm 处测量粉红色硫代巴比妥酸加合物的吸光度,与不含酶的空白对照。用等体积的丁-1-醇萃取浑浊溶液,并测量萃取液的吸光度。将缓冲液等通过 Chelex 100 螯合树脂柱,辣根过氧化物酶溶液通过 Sephadex G-25 (Pharmacia),检查试剂中是否存在污染铁。由于这些预防措施对结果影响不大,因此在后来的实验中省略了。
Preparation of different redox states of horseradish peroxidase 制备不同氧化还原状态的辣根过氧化物酶
Compounds I, II and III were obtained following published procedures and identified by their absorption spectra [29]. As all states of the enzyme except Compound III (and ferroperoxidase) show an isosbestic point at 452 nm [29], the change in absorbance at 452nm(DeltaA_(452))452 \mathrm{~nm}\left(\Delta \mathrm{~A}_{452}\right) can be used to measure Compound-III formation against the background of other forms of horseradish peroxidase. The conversion of ferri-peroxidase to Compound III and vice versa can be determined by measuring DeltaA_(582)\Delta \mathrm{A}_{582} [29]. Compound I was prepared by incubating 1mum1 \mu \mathrm{~m} horseradish peroxidase with 1muMquadH_(2)O_(2)1 \mu \mathrm{M} \quad \mathrm{H}_{2} \mathrm{O}_{2} for 5 min [19]. Compound II was similarly prepared by an incubation with 10 mumH_(2)O_(2)10 \mu \mathrm{~m} \mathrm{H}_{2} \mathrm{O}_{2}, or a mixture of 1mumH_(2)O_(2)1 \mu \mathrm{~m} \mathrm{H}_{2} \mathrm{O}_{2} and 1mumK_(4)Fe(CN)_(6)1 \mu \mathrm{~m} \mathrm{~K}_{4} \mathrm{Fe}(\mathrm{CN})_{6} [30,31], and Compound III by an incubation with 200 mum200 \mu \mathrm{~m} NADH or dihydroxyfumarate [23,32]. The spectrophotometric characteristics of Compounds I, II, III generated were in agreement with published spectra [29]. Purified Compound III was prepared by incubating 1 mL of 50 mum50 \mu \mathrm{~m} horseradish peroxidase with 1 mm dihydroxyfumarate for 15 min and passing the solution at 5^(@)C5{ }^{\circ} \mathrm{C} through a column with 5 mL Sephadex G-25 previously equilibrated with buffer and precentrifuged ( 1200g,1min1200 \mathrm{~g}, 1 \mathrm{~min} ) to remove the mobile phase. The purified enzyme was recovered without dilution by a second, identical centrifugation. It was free of spectrophotome-trically-detectable dihydroxyfumarate. 化合物 I、II 和 III 按照已公布的程序获得,并通过其吸收光谱进行鉴定 [29]。由于除化合物 III(和铁过氧化物酶)之外的所有酶的状态都在 452 纳米波长处显示等基点 [29],因此 452nm(DeltaA_(452))452 \mathrm{~nm}\left(\Delta \mathrm{~A}_{452}\right) 处的吸光度变化可用于在其他形式辣根过氧化物酶的背景下测量化合物 III 的形成。通过测量 DeltaA_(582)\Delta \mathrm{A}_{582} 可以确定铁过氧化物酶向化合物 III 的转化,反之亦然 [29]。化合物 I 的制备方法是将 1mum1 \mu \mathrm{~m} 辣根过氧化物酶与 1muMquadH_(2)O_(2)1 \mu \mathrm{M} \quad \mathrm{H}_{2} \mathrm{O}_{2} 共孵育 5 分钟 [19]。化合物 II 同样是通过与 10 mumH_(2)O_(2)10 \mu \mathrm{~m} \mathrm{H}_{2} \mathrm{O}_{2} 或 1mumH_(2)O_(2)1 \mu \mathrm{~m} \mathrm{H}_{2} \mathrm{O}_{2}