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 $(H_{2} O_{2})$ of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of $O_{2}$ to superoxide $(O_{2}^{-})$ and $H_{2} O_{2}$ 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 $H_{2} O_{2}$ in the presence of $O_{2}^{-}$ . We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by $O_{2}^{- -}$ 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 $O_{2}^{- -}$ and $H_{2} O_{2}$ (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. $O_{2}^{- -}$ and $H_{2} O_{2}$ can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The $^{\circ} OH$ -producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various $O_{2}^{- -}$ and $^{\circ} OH$ scavengers. On an equimolar Fe basis, horseradish peroxidase is $1 - 2$ orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high $^{\circ} OH -$ 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 $^{\circ} OH$ -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 $(H_{2} O_{2})$ 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 $O_{2}$ 被 NADH 或二羟富马酸等底物还原为超氧化物 $(O_{2}^{-})$ 和 $H_{2} O_{2}$ 。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 $O_{2}^{-}$ 存在的情况下，由 $H_{2} O_{2}$ 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 $O_{2}^{- -}$ 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 $O_{2}^{- -}$ 和 $H_{2} O_{2}$ 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。 $O_{2}^{- -}$ 和 $H_{2} O_{2}$ 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 $^{\circ} OH$ 的活性可被血红素铁的灭活剂或各种 $O_{2}^{- -}$ 和 $^{\circ} OH$ 清除剂抑制。在等摩尔铁的基础上，辣根过氧化物酶的活性 $1 - 2$ 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 $^{\circ} OH -$ 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 $^{\circ} OH$ 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。

Keywords: Fenton reaction; hydrogen peroxide; hydroxyl radical; peroxidase; superoxide radical.
关键词芬顿反应；过氧化氢；羟自由基；过氧化物酶；超氧自由基。

The hydroxyl radical ( OH ) constitutes the chemically most reactive species of ‘activated oxygen’ formed by successive monovalent reduction of dioxygen

(O_{2})

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

^{\circ} 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}

under a variety of stress conditions and are involved in numerous cellular disorders such as inflammations [4], embryo teratogenesis [5], herbicide effects

[6, 7]

, cell death

[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

^{\circ} OH

of DNA [11], proteins [12], or membrane lipids [13]. It is generally assumed

[2, 12, 14]

that

^{\circ} OH

is generated in biological systems from

H_{2} 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})

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

^{\circ} 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}

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]

、细胞死亡

[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

^{\circ} OH

损伤。一般认为，

[2, 12, 14]

在生物系统中是由

H_{2} O_{2}

通过无机化学中已知的芬顿反应生成的：

{Fe}^{2 +} + H_{2} O_{2} \to {Fe}^{3 +} +^{\cdot} OH + {OH}^{-}

whereby

{Fe}^{2 +}

can be regenerated through the oxidation by the superoxide anion

(O_{2}^{- -})

:
其中

{Fe}^{2 +}

可以通过超氧阴离子

(O_{2}^{- -})

的氧化作用再生：

{Fe}^{3 +} + O_{2}^{- -} \to {Fe}^{2 +} + 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} + O_{2}^{- -} \overset{{Fe}^{2 +} / / {Fe}^{3 +}}{\to} OH + {OH}^{-} + O_{2}

H_{2} O_{2}

and

O_{2}^{- -}

are ubiquitously formed from

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 +}

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}

和

O_{2}^{- -}

是由

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 +}

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}

and are thus often regarded as antioxidant enzymes, protecting cells from the destructive influence of

H_{2} 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}

. It has been shown that this oxidative activity involves the formation of

O_{2}^{- -}

and

H_{2} O_{2}

as intermediates, and the conversion of the ferric form of the enzyme (

{Fe}^{3 +}

-peroxidase) into the labile perferryl form

({Fe}^{2 +} - O_{2} \cdot^{- -} {Fe}^{3 +} - O_{2}^{- -}

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}

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}

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}

。研究表明，这种氧化活性涉及作为中间产物的

O_{2}^{- -}

和

H_{2} O_{2}

的形成，以及铁形式的酶（

{Fe}^{3 +}

-过氧化物酶）向易失铁形式的

({Fe}^{2 +} - O_{2} \cdot^{- -} {Fe}^{3 +} - O_{2}^{- -}

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 +}

in the prosthetic haem group that can be easily converted to

{Fe}^{3 +}

, one can imagine that Compound III can act as a Fenton reagent in a manner similar to

{Fe}^{2 +}

-chelates, such as

{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

^{\circ} 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 +}

，可以很容易地转化为

{Fe}^{3 +}

，因此可以想象化合物 III 可以以类似于

{Fe}^{2 +}

-螯合物（如

{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

^{\circ} 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}^{- -}

and

H_{2} O_{2}

from

O_{2}

and NADH [26]. It was therefore of interest to find out whether this enzyme could mediate the production of

^{\circ} 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}

和 NADH 生成

O_{2}^{- -}

和

H_{2} 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

^{\circ} 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}

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}

solutions was standardized photometrically using an extinction coefficient of

39.4 m^{- 1} \cdot {cm}^{- 1}

240 nm . {Fe}^{3 +}

-EDTA was prepared by mixing equal concentrations of

{FeCl}_{3}

and

{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}

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}

溶液的浓度是用

39.4 m^{- 1} \cdot {cm}^{- 1}

在

240 nm . {Fe}^{3 +}

的消光系数

39.4 m^{- 1} \cdot {cm}^{- 1}

进行光度标准化的，

{FeCl}_{3}

和

{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}

和

{Na}_{2}

制备的。

Enzyme assays 酶测定

All incubations and assays were performed at

25^{\circ} C

in 10 mm sodium citrate buffer ( pH 6.0 , except where stated otherwise). Production of

^{\circ} OH

by peroxidase was determined with a fluorimetric method based on the hydroxylation of benzoate [27]. The reaction mixture ( 1 mL in a

5 \times 10 - 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

, 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 - 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^{\circ} C

进行。过氧化物酶产生的

^{\circ} OH

是用基于苯甲酸羟基化的荧光法测定的 [27]。反应混合物（

5 \times 10 - 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

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 - mL

等分
was mixed with 0.5 mL thiobarbituric-acid solution (

10 g \cdot L^{- 1}

in 50 mm NaOH ) and 0.5 mL trichloroacidic acid solution (

28 g \cdot 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 - 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（

10 g \cdot L^{- 1}

在 50 mm NaOH 中的浓度）和 0.5 mL 三氯酸溶液（

28 g \cdot L^{- 1}

）混合。在沸水浴中加热 20 分钟并在冰上冷却后，在

10 - 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

452 nm (Δ A_{452})

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

Δ A_{582}

[29]. Compound I was prepared by incubating

1 μ m

horseradish peroxidase with

1 μ M H_{2} O_{2}

for 5 min [19]. Compound II was similarly prepared by an incubation with

10 μ m H_{2} O_{2}

, or a mixture of

1 μ m H_{2} O_{2}

and

1 μ m K_{4} Fe (CN)_{6}

[30,31], and Compound III by an incubation with

200 μ 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 μ m

horseradish peroxidase with 1 mm dihydroxyfumarate for 15 min and passing the solution at

5^{\circ} C

through a column with 5 mL Sephadex G-25 previously equilibrated with buffer and precentrifuged (

1200 g, 1 \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

452 nm (Δ A_{452})

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

Δ 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

1 μ m

辣根过氧化物酶与

1 μ M H_{2} O_{2}

共孵育 5 分钟 [19]。化合物 II 同样是通过与

10 μ m H_{2} O_{2}

或

1 μ m H_{2} O_{2}

和

1 μ m K_{4} Fe (CN)_{6}

的混合物孵育制备的 [30,31]，化合物 III 是通过与

200 μ m

NADH 或二羟富马酸孵育制备的 [23,32]。生成的化合物 I、II 和 III 的分光光度特性与已发表的光谱一致 [29]。纯化化合物 III 的制备方法是：将 1 mL 辣根过氧化物酶与 1 mm 二羟基富马酸盐孵育 15 分钟，然后将溶液在

5^{\circ} C

温度下通过事先用缓冲液平衡过的 5 mL Sephadex G-25 色谱柱，并预离心（

1200 g, 1 \min

）以除去流动相。通过第二次相同的离心回收纯化的酶，无需稀释。它不含分光光度法-曲光检测到的二羟富马酸。

Statistical treatment of data
数据的统计处理

All experiments were repeated at least three times with similar results. The figures show either single representative results or means (

\pm

SE where appropriate).
所有实验至少重复三次，结果相似。图中显示的是单个代表性结果或平均值（

\pm

SE（如适用））。

RESULTS 结果

Previous investigations have led to the assertation that NADH, in contrast with dihydroxyfumarate, is an unsuitable substrate for 'OH production by horseradish peroxidase [21,23]. This conclusion, however, was obviously based on data obtained under inappropriate experimental conditions. The 'OH-producing activity of horseradish peroxidase in air-saturated buffer of pH 6.0 containing a suitable concentration of NADH could be readily demonstrated employing either the hydroxylation of benzoate [27] or the degradation of deoxyribose

[28, 33]

to detect

^{\circ} OH

formation (Figs 1 and 2, Table 1). The benzoate hydroxylation assay detects primarily the highly fluorescent 3-hydroxybenzoate formed, in addition to the more weakly fluorescent 4-hydroxybenzoate [27]. Fig. 1 identifies 3-hydroxybenzoate as the major fluorescent hydroxylation product of the horseradish peroxidase-catalyzed reaction. This assay produces
以往的研究表明，与二羟富马酸相比，NADH 是辣根过氧化物酶产生 OH 的不合适底物 [21,23]。然而，这一结论显然是基于在不适当的实验条件下获得的数据。在含有适当浓度 NADH 的 pH 值为 6.0 的空气饱和缓冲液中，辣根过氧化物酶产生'OH'的活性很容易通过苯甲酸盐的羟基化[27]或脱氧核糖

[28, 33]

的降解来证明，从而检测

^{\circ} OH

的形成（图 1 和图 2，表 1）。苯甲酸羟化测定主要检测形成的高荧光 3-羟基苯甲酸酯，此外还检测荧光较弱的 4-羟基苯甲酸酯 [27]。图 1 确定 3-羟基苯甲酸酯是辣根过氧化物酶催化反应的主要荧光羟化产物。这种检测方法会产生

Fig. 1. Fluorescence emission spectrum of reaction product obtained in the benzoate hydroxylation assay in the presence of horseradish peroxidase and NADH. (a) Authentic 3-hydroxybenzoate (

30 μ m

), (b) complete reaction mixture, © reaction mixture minus benzoate, (d) reaction mixture minus NADH, (e) reaction mixture minus horseradish peroxidase. Reaction mixtures (

200 μ M

NADH,

30 μ g \cdot {mL}^{- 1} = 0.7 μ M

horseradish peroxidase, 2 mm benzoate in 10 mm citrate buffer, pH 6.0 ) were incubated for 60 min . Remaining NADH was eliminated by adjusting to pH 3.2 with HCl . After 60 min the pH was adjusted to 5.5 with KOH . The fluorescence spectra were not affected by pH in the range of

pH 5.0 - 6.5

.
图 1.在辣根过氧化物酶和 NADH 的存在下，苯甲酸羟化反应产物的荧光发射光谱。 (a) 3-羟基苯甲酸正品 (

30 μ m

)，(b) 全部反应混合物，© 反应混合物减去苯甲酸盐，(d) 反应混合物减去 NADH，(e) 反应混合物减去辣根过氧化物酶。将反应混合物（

200 μ M

NADH、

30 μ g \cdot {mL}^{- 1} = 0.7 μ M

辣根过氧化物酶、2 mm 苯甲酸盐在 10 mm 柠檬酸缓冲液中，pH 6.0）培养 60 分钟。用盐酸调节 pH 值至 3.2，消除剩余的 NADH。60 分钟后，用 KOH 将 pH 值调至 5.5。在

pH 5.0 - 6.5

的范围内，荧光光谱不受 pH 值的影响。
approximately linear reaction kinetics for at least 30 min and can be used for kinetically testing the effects of inhibitors such as superoxide dismutase (Fig. 2); it was therefore used in most of the subsequent experiments. Both assay reactions were absolutely dependent on the presence of native peroxidase and a reductant such as NADH. Measurements with different horseradish peroxidase preparations revealed significant differences in activity in the standard benzoate hydroxylation assay, based on equal amounts of enzyme protein. The data reported in this paper were obtained with a single batch of Boehringer horseradish peroxidase and are therefore directly comparable.
至少 30 分钟的近似线性反应动力学，可用于对超氧化物歧化酶等抑制剂的影响进行动力学测试（图 2）；因此在随后的大多数实验中都使用了这种方法。两种检测反应都绝对依赖于原生过氧化物酶和还原剂（如 NADH）的存在。使用不同的辣根过氧化物酶制剂进行测定发现，在等量酶蛋白的基础上，标准苯甲酸酯羟化测定中的活性存在显著差异。本文报告的数据是用一批勃林格辣根过氧化物酶获得的，因此具有直接可比性。

As shown previously [18], horseradish peroxidase can be converted to Compound III in a reaction started by NADH in the presence of

O_{2}

. The conversion of the haem group from the ferric form into the perferryl form must involve

O_{2}^{- -}

as a reductant, as it can be prevented by superoxide dismutase (Fig. 3). Other

O_{2}^{- -}

-scavenging reagents (

100 μ m

Tiron or

10 μ M {CuCl}_{2}

) were as effective as superoxide dismutase, while catalase

(10 μ g \cdot {mL}^{- 1})

only slightly impaired Compound-III formation (data not shown). The interconversion of ferriperoxidase into Compound III can be photometrically determined without interference from other forms of the enzyme, by measuring the increase in absorbance at 452 nm [29]. Employing this method, Fig. 4A shows that Compound-III formation demonstrates a broad plateau in the range of

pH 4.5 - 6.0

. Benzoate hydroxylation and deoxyribose degradation used for determining

^{\circ} OH

production under these conditions showed optimal reaction rates in the same pH range, although the curves
如前文[18]所示，辣根过氧化物酶可以在

O_{2}

存在下通过 NADH 启动的反应转化为化合物 III。血红素基团从铁形式转化为铁形式必须有

O_{2}^{- -}

作为还原剂，因为超氧化物歧化酶可以阻止这种转化（图 3）。其他

O_{2}^{- -}

清除试剂（

100 μ m

铁龙或

10 μ M {CuCl}_{2}

）与超氧化物歧化酶一样有效，而过氧化氢酶

(10 μ g \cdot {mL}^{- 1})

只能轻微阻碍化合物-III 的形成（数据未显示）。铁过氧化物酶相互转化为化合物 III 的过程可通过光度计测定，不受其他形式酶的干扰，方法是测量 452 纳米波长处吸光度的增加 [29]。利用这种方法，图 4A 显示化合物 III 的形成在

pH 4.5 - 6.0

范围内呈现出一个宽广的高原。在这些条件下，用于确定

^{\circ} OH

生成量的苯甲酸羟基化和脱氧核糖降解在相同的 pH 值范围内显示出最佳反应速率，尽管曲线

Fig. 2. Reaction kinetics of OH production by horseradish peroxidase in the presence of NADH determined by benzoate hydroxylation. NADH

(200 μ m)

, horseradish peroxidase

(60 μ g \cdot {mL}^{- 1} = 1.4 μ m)

and superoxide dismutase (

50 μ g \cdot {mL}^{- 1}

) were added as indicated.

Δ F_{407} = 1

corresponds to a 3-hydroxybenzoate concentration of

3.8 μ M

in buffer (

9.5 μ M

in the presence of

200 μ mADH

). HRP, horseradish peroxidase; SOD, superoxide dismutase.
图 2.通过苯甲酸羟化测定辣根过氧化物酶在 NADH 存在下产生 OH 的反应动力学。NADH

(200 μ m)

、辣根过氧化物酶

(60 μ g \cdot {mL}^{- 1} = 1.4 μ m)

和超氧化物歧化酶 (

50 μ g \cdot {mL}^{- 1}

) 的添加如图所示。

Δ F_{407} = 1

相当于缓冲液中 3-羟基苯甲酸浓度

3.8 μ M

（

9.5 μ M

存在

200 μ mADH

时）。HRP：辣根过氧化物酶；SOD：超氧化物歧化酶。
differed significantly, indicating different pH requirements of these reactions (Fig. 4B). The background fluorescence change of the reaction mixture in the absence of benzoate increased markedly because of NADH degradation at

pH < 6.0

. To minimize this effect and to approximate physiological conditions, a pH of 6.0 was used for all further experiments.
差异很大，表明这些反应对 pH 值的要求不同（图 4B）。由于 NADH 在

pH < 6.0

时发生降解，在没有苯甲酸盐的情况下，反应混合物的背景荧光变化明显增加。为了尽量减少这种影响并接近生理条件，所有进一步的实验都使用了 6.0 的 pH 值。

In order to scrutinize the involvement of OH and other components of Eqn (3) in the peroxidase-catalyzed hydroxylation of benzoate and degradation of deoxyribose, the effects of various diagnostic inhibitors were investigated. Table 1 shows that both assay reactions can be inhibited with established

^{\circ} OH

scavengers such as mannitol, formate and thiourea, in a concentration-dependent manner. In addition, these reactions can be inhibited by removing

O_{2}^{- -}

with

{Cu}^{2 +}

[34] or superoxide dismutase, or by removing

H_{2} O_{2}

with catalase. Inactivation of the haem iron of peroxidase by desferrioxamine [35],

{NaN}_{3}

or KCN likewise inhibited both reactions, although the effects of desferrioxamine and

{NaN}_{3}

may also be the result of

^{\circ} OH -

and

O_{2}^{-^{-} -scavenging actions that have previously been attributed to}

these reagents [14]. The NADPH-oxidase inhibitor diphenyleneiodonium has recently been shown to interfere with the NADH-dependent

O_{2}^{- -}

and

H_{2} O_{2}

-producing activity of horseradish peroxidase [36]. The differences in sensitivity of benzoate hydroxylation and deoxyribose degradation towards most of the inhibitors are difficult to interpret as these reactions are chemically complex and different radical
为了仔细研究在过氧化物酶催化的苯甲酸羟基化和脱氧核糖降解过程中，OH 和公式（3）中的其他成分的参与情况，研究了各种诊断抑制剂的效果。表 1 显示，甘露醇、甲酸盐和硫脲等已有的

^{\circ} OH

清除剂可以以浓度依赖性的方式抑制这两种检测反应。此外，用

{Cu}^{2 +}

[34] 或超氧化物歧化酶去除

O_{2}^{- -}

或用过氧化氢酶去除

H_{2} O_{2}

也能抑制这些反应。去铁胺[35]、

{NaN}_{3}

或 KCN 使过氧化物酶的血红素铁失活，同样也会抑制这两种反应，尽管去铁胺和

{NaN}_{3}

的作用也可能是

^{\circ} OH -

和

O_{2}^{-^{-} -scavenging actions that have previously been attributed to}

这些试剂的结果[14]。最近的研究表明，NADPH-氧化酶抑制剂二苯基碘鎓会干扰辣根过氧化物酶依赖于 NADH 的

O_{2}^{- -}

和

H_{2} O_{2}

生成活性 [36]。苯甲酸羟化和脱氧核糖降解对大多数抑制剂的敏感性差异很难解释，因为这些反应的化学性质复杂，不同的自由基对它们的敏感性也不同。

Table 1. Effects of various inhibitors on

O H

production by horseradish peroxidase assayed either by benzoate hydroxylation or deoxyribose degradation. In the benzoate assay, the test substances were added to the reaction mixture 15 min after starting the reaction with horseradish peroxidase

(1.4 μ m)

, and the change in rate determined during the following 10 min as shown in Fig. 2. In the deoxyribose assay, the test substances were added before starting the reaction with horseradish peroxidase, and the increase in reaction product determined after a period of 60 min. ND, not determined.
表 1.各种抑制剂对辣根过氧化物酶通过苯甲酸酯羟化或脱氧核糖降解测定的

O H

生成的影响。在苯甲酸酯试验中，在辣根过氧化物酶

(1.4 μ m)

开始反应 15 分钟后，将试验物质加入反应混合物中，并测定随后 10 分钟内的速率变化，如图 2 所示。在脱氧核糖测定中，在开始与辣根过氧化物酶反应之前加入试验物质，60 分钟后测定反应产物的增加量。ND，未测定。

Reagent added 添加试剂

Benzoate hydroxylation

(Δ F_{407} \cdot 10 \min^{- 1})

苯甲酸羟化

(Δ F_{407} \cdot 10 \min^{- 1})

Inhibition (%) 抑制率 (%)

Deoxyribose degradation 脱氧核糖降解

(Δ A_{532} \cdot 60 \min^{- 1} \cdot 10^{3})

抑制率 (%)

Inhibition

(%)

None 无

1.10 \pm 0.04

23.8 \pm 0.8

Sodium benzoate 苯甲酸钠

10 mm 10 毫米

15.1 \pm 0.6

20 mm 20 毫米

11.4 \pm 1.2

Mannitol 甘露醇

20 mm 20 毫米

0.90 \pm 0.04

20.7 \pm 0.7

100 mm 100 毫米

0.65 \pm 0.05

16.7 \pm 1.0

250 mm 250 毫米

0.38 \pm 0.05

12.9 \pm 0.8

Na-formate 甲酸纳

10 mm 10 毫米

0.93 \pm 0.07

16.9 \pm 0.4

50 mm 50 毫米

0.50 \pm 0.04

11.2 \pm 0.8

200 mm 200 毫米

0.22 \pm 0.02

Thiourea 硫脲

5 μ m

0.84 \pm 0.02

21.7 \pm 0.9

100 μ M

0.47 \pm 0.02

16.7 \pm 0.6

1 mm 1 毫米

0.22 \pm 0.03

13.0 \pm 0.5

{CuCl}_{2}

100 nm 100 纳米

1.02 \pm 0.07

18.3 \pm 0.5

500 nm 500 纳米

0.51 \pm 0.07

15.4 \pm 0.7

10 μ m

0.10 \pm 0.01

1.8 \pm 0.7

Superoxide dismutase 超氧化物歧化酶

0.2 μ g \cdot {mL}^{- 1}

0.73 \pm 0.02

23.2 \pm 0.3

2 μ g \cdot {mL}^{- 1}

0.50 \pm 0.04

22.7 \pm 0.2

20 μ g \cdot {mL}^{- 1}

0.23 \pm 0.03

11.3 \pm 0.4

100 μ g \cdot {mL}^{- 1}

0.13 \pm 0.03

Catalase 过氧化氢酶

0.4 μ g \cdot {mL}^{- 1}

0.71 \pm 0.05

23.2 \pm 0.5

2 μ g \cdot {mL}^{- 1}

0.46 \pm 0.04

20.1 \pm 1.1

10 μ g \cdot {mL}^{- 1}

13.5 \pm 0.2

50 μ g \cdot {mL}^{- 1}

0.04 \pm 0.02

Desferrioxamine 去铁胺

10 μ m

0.82 \pm 0.05

20.6 \pm 0.3

100 μ M

0.53 \pm 0.08

17.4 \pm 0.6

1 mm 1 毫米

0.38 \pm 0.04

13.0 \pm 0.3

{NaN}_{3}

1 μ M

0.96 \pm 0.03

23.0 \pm 0.8

100 μ M

0.46 \pm 0.03

16.8 \pm 0.5

500 μ m

0.14 \pm 0.03

11.6 \pm 0.1

KCN

10 μ m

0.95 \pm 0.02

19.6 \pm 0.3

1 mm 1 毫米

0.48 \pm 0.04

14.1 \pm 0.6

10 mm 10 毫米

0.28 \pm 0.03

11.5 \pm 1.4

Diphenyleneiodonium

^{a}

二苯基碘

^{a}

20 nM 20 毫微米

0.99 \pm 0.05

100 nM 100 毫微米

0.50 \pm 0.07

17.5 \pm 0.6

1 μ M

0.07 \pm 0.02

11.8 \pm 0.2

| Reagent added | Benzoate hydroxylation $\left(\Delta \mathrm{F}_{407} \cdot 10 \mathrm{~min}^{-1}\right)$ | Inhibition (%) | Deoxyribose degradation $\left(\Delta \mathrm{A}_{532} \cdot 60 \mathrm{~min}^{-1} \cdot 10^{3}\right)$ | Inhibition <br> (%) | | :---: | :---: | :---: | :---: | :---: | | None | $1.10 \pm 0.04$ | 0 | $23.8 \pm 0.8$ | 0 | | Sodium benzoate | | | | | | 10 mm | ND | - | $15.1 \pm 0.6$ | 37 | | 20 mm | ND | - | $11.4 \pm 1.2$ | 52 | | Mannitol | | | | | | 20 mm | $0.90 \pm 0.04$ | 22 | $20.7 \pm 0.7$ | 13 | | 100 mm | $0.65 \pm 0.05$ | 41 | $16.7 \pm 1.0$ | 30 | | 250 mm | $0.38 \pm 0.05$ | 65 | $12.9 \pm 0.8$ | 46 | | Na-formate | | | | | | 10 mm | $0.93 \pm 0.07$ | 15 | $16.9 \pm 0.4$ | 29 | | 50 mm | $0.50 \pm 0.04$ | 55 | $11.2 \pm 0.8$ | 53 | | 200 mm | $0.22 \pm 0.02$ | 80 | ND | - | | Thiourea | | | | | | $5 \mu \mathrm{~m}$ | $0.84 \pm 0.02$ | 24 | $21.7 \pm 0.9$ | 9 | | $100 \mu \mathrm{M}$ | $0.47 \pm 0.02$ | 57 | $16.7 \pm 0.6$ | 30 | | 1 mm | $0.22 \pm 0.03$ | 80 | $13.0 \pm 0.5$ | 45 | | $\mathrm{CuCl}_{2}$ | | | | | | 100 nm | $1.02 \pm 0.07$ | 7 | $18.3 \pm 0.5$ | 23 | | 500 nm | $0.51 \pm 0.07$ | 54 | $15.4 \pm 0.7$ | 35 | | $10 \mu \mathrm{~m}$ | $0.10 \pm 0.01$ | 91 | $1.8 \pm 0.7$ | 93 | | Superoxide dismutase | | | | | | $0.2 \mu \mathrm{~g} \cdot \mathrm{~mL}^{-1}$ | $0.73 \pm 0.02$ | 34 | $23.2 \pm 0.3$ | 3 | | $2 \mu \mathrm{~g} \cdot \mathrm{~mL}^{-1}$ | $0.50 \pm 0.04$ | 54 | $22.7 \pm 0.2$ | 5 | | $20 \mu \mathrm{~g} \cdot \mathrm{~mL}^{-1}$ | $0.23 \pm 0.03$ | 79 | $11.3 \pm 0.4$ | 53 | | $100 \mu \mathrm{~g} \cdot \mathrm{~mL}^{-1}$ | $0.13 \pm 0.03$ | 88 | ND | - | | Catalase | | | | | | $0.4 \mu \mathrm{~g} \cdot \mathrm{~mL}^{-1}$ | $0.71 \pm 0.05$ | 35 | $23.2 \pm 0.5$ | 3 | | $2 \mu \mathrm{~g} \cdot \mathrm{~mL}^{-1}$ | $0.46 \pm 0.04$ | 58 | $20.1 \pm 1.1$ | 16 | | $10 \mu \mathrm{~g} \cdot \mathrm{~mL}^{-1}$ | ND | - | $13.5 \pm 0.2$ | 43 | | $50 \mu \mathrm{~g} \cdot \mathrm{~mL}^{-1}$ | $0.04 \pm 0.02$ | 96 | ND | - | | Desferrioxamine | | | | | | $10 \mu \mathrm{~m}$ | $0.82 \pm 0.05$ | 25 | $20.6 \pm 0.3$ | 13 | | $100 \mu \mathrm{M}$ | $0.53 \pm 0.08$ | 52 | $17.4 \pm 0.6$ | 27 | | 1 mm | $0.38 \pm 0.04$ | 66 | $13.0 \pm 0.3$ | 46 | | $\mathrm{NaN}_{3}$ | | | | | | $1 \mu \mathrm{M}$ | $0.96 \pm 0.03$ | 13 | $23.0 \pm 0.8$ | 4 | | $100 \mu \mathrm{M}$ | $0.46 \pm 0.03$ | 58 | $16.8 \pm 0.5$ | 29 | | $500 \mu \mathrm{~m}$ | $0.14 \pm 0.03$ | 88 | $11.6 \pm 0.1$ | 51 | | KCN | | | | | | $10 \mu \mathrm{~m}$ | $0.95 \pm 0.02$ | 14 | $19.6 \pm 0.3$ | 18 | | 1 mm | $0.48 \pm 0.04$ | 56 | $14.1 \pm 0.6$ | 41 | | 10 mm | $0.28 \pm 0.03$ | 75 | $11.5 \pm 1.4$ | 52 | | Diphenyleneiodonium ${ }^{\text {a }}$ | | | | | | 20 nM | $0.99 \pm 0.05$ | 10 | ND | - | | 100 nM | $0.50 \pm 0.07$ | 55 | $17.5 \pm 0.6$ | 26 | | $1 \mu \mathrm{M}$ | $0.07 \pm 0.02$ | 94 | $11.8 \pm 0.2$ | 50 |

^{a}

The concentration of DMSO in diphenyleneiodonium solutions had no effect on

^{\circ} OH

production.

^{a}

二苯基碘溶液中二甲基亚砜的浓度对

^{\circ} OH

的产生没有影响。
mechanisms are involved. However, these discrepancies may be partly explained by different degrees of competition between rate-limiting reactants, e.g. by different abilities of benzoate and deoxyribose to react with the extremely shortlived

^{\circ} OH

molecule, and thus to compete with particular scavengers, at the site of

^{\circ} OH

generation. Similar inconsistencies have been described by Gutteridge [27,37]. Irrespective of the precise explanation of these differences, the results of Table 1 provide qualitative evidence for the involvement of

\cdot OH, O_{2}^{- -}, H_{2} O_{2}

and reactive iron in the reactions mediated by horseradish peroxidase, which thus show the typical features of the Haber-Weiss reaction catalyzed by Fe-EDTA [15,20].
这些差异的部分原因可能是限速反应物之间的竞争程度不同。然而，这些差异的部分原因可能是限速反应物之间的竞争程度不同，例如苯甲酸酯和脱氧核糖与寿命极短的

^{\circ} OH

分子发生反应的能力不同，因而在

^{\circ} OH

生成部位与特定清除剂发生竞争的能力也不同。Gutteridge [27,37] 也描述了类似的不一致性。不管这些差异的确切解释是什么，表 1 的结果提供了定性证据，证明

\cdot OH, O_{2}^{- -}, H_{2} O_{2}

和活性铁参与了辣根过氧化物酶介导的反应，从而显示出 Fe-EDTA 催化的哈伯-魏斯反应的典型特征 [15,20]。

{Fe}^{2 +}

-EDTA, or

{Fe}^{3 +}

-EDTA in the presence of a

{Fe}^{2 +}

-forming reductant such as

O_{2}^{- -}

, has often been used as an effective inorganic reagent for mediating

^{\circ} OH

production from

H_{2} O_{2}

according to Eqn (1) [14]. Rowley and Halliwell [38] have shown that

{Fe}^{2 +}

-EDTA and

{Fe}^{3 +}

-EDTA are equally effective in catalyzing

^{\circ} OH

production in the presence of NADH. Comparative experiments with Fe-EDTA and horseradish peroxidase have shown, however, that at equimolar Fe concentrations up to

1 μ M

, horseradish peroxidase was

1 - 2

orders of magnitude more effective than Fe -EDTA in mediating

^{\circ} OH

production in the presence of

200 μ m

NADH and

100 μ m H_{2} O_{2}

(Fig. 5). The 'OH production catalyzed by horseradish peroxidase can be

{Fe}^{2 +}

-EDTA 或

{Fe}^{3 +}

-EDTA 在

{Fe}^{2 +}

形成还原剂（如

O_{2}^{- -}

）的存在下，经常被用作一种有效的无机试剂，用于根据公式 (1) 从

H_{2} O_{2}

催化

^{\circ} OH

的产生 [14]。Rowley 和 Halliwell [38] 的研究表明，

{Fe}^{2 +}

-EDTA 和

{Fe}^{3 +}

-EDTA 在 NADH 存在下催化

^{\circ} OH

生成的效果相同。然而，用铁-EDTA 和辣根过氧化物酶进行的比较实验表明，在等摩尔铁浓度达到

1 μ M

时，辣根过氧化物酶在

200 μ m

NADH 和

100 μ m H_{2} O_{2}

存在的情况下介导

^{\circ} OH

生成的效果比铁-EDTA 高

1 - 2

个数量级（图 5）。辣根过氧化物酶催化的 "OH "生成可以是

Table 2. Activity of ferri-peroxidase, Compound I, Compound II and Compound III in

O H

production. Horseradish peroxidase (

1 μ m

) was preincubated for 5 min with various reagents. Then benzoate ( 2 mm ) was added for determination of the

^{\circ} OH

production rate during the following 10 min . The presence of the indicated enzyme forms after the pretreatment was checked by measuring the absorption spectra.
表 2.铁过氧化物酶、化合物 I、化合物 II 和化合物 III 在

O H

生产中的活性。将辣根过氧化物酶（

1 μ m

）与各种试剂预孵 5 分钟。然后加入苯甲酸盐（2 毫米），测定随后 10 分钟内

^{\circ} OH

的生成率。通过测量吸收光谱，检查预处理后是否存在指定形式的酶。

Pretreatment 预处理

Enzyme form produced 生产的酶形式

- OH 生成率

(Δ F_{407} .10 \min^{- 1})

- OH production rate

(Δ F_{407} .10 \min^{- 1})

None 无

Ferri-peroxidase 铁过氧化物酶

1 μ M H H_{2} O_{2}

Compound I 化合物 I

10 μ M H H_{2} O_{2}

Compound II 化合物 II

1 μ m H_{2} O_{2} + 1 μ m K_{4} Fe (CN)_{6}

Compound II 化合物 II

200 μ m

dihydroxyfumarate

200 μ m

二羟基富马酸盐

Compound III 化合物 III

2.3 \pm 0.1

200 μ m

NADH

Compound III 化合物 III

0.99 \pm 0.02

200 μ m

dihydroxyfumarate

+ 200 μ m

ascorbate

200 μ m

二羟富马酸

+ 200 μ m

抗坏血酸

Ferri-peroxidase 铁过氧化物酶

0.11 \pm 0.01

200 μ m

NADH

+ 200 μ m

ascorbate

200 μ m

NADH

+ 200 μ m

抗坏血酸

Ferri-peroxidase 铁过氧化物酶

0.10 \pm 0.01

promoted by the addition of

H_{2} O_{2}

indicating that

H_{2} O_{2}

is a ratelimiting substrate of this reaction. Even in the absence of added

H_{2} O_{2}

, i.e. supported solely by the endogenously produced

H_{2} O_{2}

, horseradish peroxidase was more than 10 times as effective as Fe -EDTA acting at a 10 -fold excess of

H_{2} O_{2}

. Similar to the reaction in the absence of

H_{2} O_{2}

(Fig. 2), the

H_{2} O_{2}

-promoted

\cdot OH

production by horseradish peroxidase can be inhibited by superoxide dismutase (Fig. 6).

H_{2} O_{2}

的加入促进了该反应的进行，这表明

H_{2} O_{2}

是该反应的限速底物。即使在没有添加

H_{2} O_{2}

的情况下（即仅由内源产生的

H_{2} O_{2}

支持），辣根过氧化物酶的作用效果也是过量 10 倍

H_{2} O_{2}

的 Fe -EDTA 的 10 倍以上。与没有

H_{2} O_{2}

时的反应类似（图 2），辣根过氧化物酶产生的

H_{2} O_{2}

促进的

\cdot OH

可被超氧化物歧化酶抑制（图 6）。

Various substrates that can be oxidized by horseradish peroxidase were tested for their ability to support

^{\circ} OH

production by the enzyme. Fig. 7 shows that NADH can be replaced by NADPH and cysteine, although these reagents are less effective than NADH. Ascorbate is totally ineffective. With the nonphysiological substrate dihydroxyfumarate, activity is substantially higher than with NADH. Because of their absorption at 305 nm , both

NAD (P) H

and dihydroxyfumarate interfere with the excitation of benzoate fluorescence in a concentration-dependent manner. This effect can not be disregarded in measurements utilizing different concentrations of these substrates. The measurements in Fig. 7 were corrected for this effect using calibration curves for the attenuation of 3-hydroxybenzoate fluorescence by

NAD (P) H

and dihydroxyfumarate under the conditions of the assay.
对辣根过氧化物酶可氧化的各种底物进行了测试，以确定它们支持该酶产生

^{\circ} OH

的能力。图 7 显示，NADH 可以被 NADPH 和半胱氨酸取代，尽管这些试剂的效果不如 NADH。抗坏血酸则完全无效。在使用非生理底物二羟富马酸时，其活性大大高于使用 NADH 时。由于它们在 305 纳米波长处的吸收，

NAD (P) H

和二羟富马酸都会以浓度依赖的方式干扰苯甲酸酯荧光的激发。在使用不同浓度的这些底物进行测量时，不能忽略这种影响。图 7 中的测量结果利用了

NAD (P) H

和二羟基富马酸在检测条件下对 3- 羟基苯甲酸酯荧光衰减的校正曲线来校正这种效应。

We tested the ability of various commercially available peroxidases to catalyze OH production in the presence of NADH (Fig. 8). On the basis of equal activity units of the standard peroxidase assay with pyrogallol, the highest specific activity of ’ OH production was found in a preparation of alkaline horseradish peroxidase isoforms and in peroxidase prepared from the fungus Arthromyces ramosus. Standard horseradish peroxidase, the acidic horseradish peroxidase fraction, lactoperoxidase from bovine milk and a soybean peroxidase preparation were less active, while myeloperoxidase from human leukocytes was virtually inactive. Thus this reaction appears to be a common property of peroxidases from different sources, with the exception of myeloperoxidase; this enzyme is also exceptional because of its ability to oxidize halides to reactive products [25].
我们测试了各种市售过氧化物酶在 NADH 存在下催化 OH 生成的能力（图 8）。在用焦棓酚进行标准过氧化物酶测定的同等活性单位的基础上，发现碱性辣根过氧化物酶同工型制备物和从真菌 Arthromyces ramosus 中制备的过氧化物酶产生 OH 的特异活性最高。标准辣根过氧化物酶、酸性辣根过氧化物酶部分、牛乳中的乳过氧化物酶和大豆过氧化物酶制剂的活性较低，而人类白细胞中的髓过氧化物酶几乎没有活性。因此，这种反应似乎是不同来源过氧化物酶的共同特性，但髓过氧化物酶除外；这种酶的特殊之处还在于它能将卤化物氧化成活性产物 [25]。

The separation of Compound III from dihydroxyfumarate or NADH by chromatography on Sephadex G-25 [23] made it possible to investigate the substrate requirements for

^{\circ} OH

production by Compound III without interference of these putative substrates. Fig. 9A shows that Compound III alone does not hydroxylate benzoate but responds to the addition of

H_{2} O_{2}

with a transient hydroxylation reaction. At the same time

H_{2} O_{2}

induces a similarly rapid decay of Compound III (Fig. 9B). In the absence of

H_{2} O_{2}

, Compound III decays spontaneously with a much slower rate that is not affected by benzoate. However, the decay of Compound III can be strongly enhanced by agents reacting with

O_{2}^{- -}

such as superoxide dismutase

(40 μ g \cdot {mL}^{- 1})

,
通过 Sephadex G-25 色谱[23]将化合物 III 与二羟富马酸或 NADH 分离，可以在不受这些假定底物干扰的情况下研究化合物 III 生成

^{\circ} OH

所需的底物。图 9A 显示，化合物 III 本身不会羟化苯甲酸酯，但加入

H_{2} O_{2}

后会发生瞬时羟化反应。同时，

H_{2} O_{2}

也会诱导化合物 III 发生类似的快速衰变（图 9B）。在没有

H_{2} O_{2}

的情况下，化合物 III 自发衰变的速度要慢得多，而且不受苯甲酸盐的影响。但是，与

O_{2}^{- -}

发生反应的物质（如超氧化物歧化酶

(40 μ g \cdot {mL}^{- 1})

）会强烈促进化合物 III 的衰变、
ascorbate

(200 μ m), {CuCl}_{2} (10 μ m)

, Tiron

(100 μ m)

, but not by catalase

(10 μ g \cdot {mL}^{- 1})

and diphenyleneiodonium (

1 μ M

) (data not shown). These data demonstrate that although

O_{2}^{- -}

can be easily removed from the enzyme by scavengers, Compound III is sufficiently stable in their absence to react with

H_{2} O_{2}

.
抗坏血酸

(200 μ m), {CuCl}_{2} (10 μ m)

、铁龙

(100 μ m)

，但过氧化氢酶

(10 μ g \cdot {mL}^{- 1})

和二苯基碘铵 (

1 μ M

) 却不能（数据未显示）。这些数据表明，虽然

O_{2}^{- -}

很容易被清除剂从酶中清除，但在没有清除剂的情况下，化合物 III 具有足够的稳定性，可以与

H_{2} O_{2}

发生反应。

In the presence of

200 μ m

NADH, which allows the continuous reformation of Compound III from ferri-peroxidase by

O_{2}^{- -}

, the addition of

H_{2} O_{2}

induces a strong and long-lasting increase in benzoate hydroxylation (Fig. 9C), indicating that

\cdot OH

production utilizing

H_{2} O_{2}

can be maintained in a steadystate under these conditions.
在

200 μ m

NADH 的存在下，可以通过

O_{2}^{- -}

使铁过氧化物酶中的化合物 III 持续转化，加入

H_{2} O_{2}

会引起苯甲酸羟基化强烈而持久的增加（图 9C），这表明利用

H_{2} O_{2}

产生的

\cdot OH

可以在这些条件下保持稳定状态。

As a negative control, experiments were conducted to check whether other oxidation states of horseradish peroxidase, besides Compound III, can contribute to the

^{\circ} OH

-producing activity of the enzyme. Horseradish peroxidase was converted into Compound I, II, or III by established methods reported in literature and their activity tested with the benzoate hydroxylation assay. Table 2 shows that horseradish peroxidase can produce

^{OH}

only in the state of Compound III and that this reaction can be inhibited by ascorbate, functioning as a

O_{2}^{- -}

scavenger rather than a substrate for

^{\circ} OH

production by horseradish peroxidase (see Fig. 7).
作为阴性对照，我们还进行了实验，以检测除化合物 III 外，辣根过氧化物酶的其他氧化态是否也能促进该酶的

^{\circ} OH

生成活性。辣根过氧化物酶通过文献报道的既定方法转化为化合物 I、II 或 III，并用苯甲酸酯羟化测定法检测它们的活性。表 2 显示，辣根过氧化物酶只有在化合物 III 的状态下才能产生

^{OH}

，并且该反应可被抗坏血酸抑制，抗坏血酸的作用是

O_{2}^{- -}

清除剂，而不是辣根过氧化物酶产生

^{\circ} OH

的底物（见图 7）。

The phenol-hydroxylating reaction of horseradish peroxidase with dihydroxyfumarate

+ O_{2}

resembles the reaction of horseradish peroxidase with

NADH + O_{2}

with respect to the production of

O_{2}^{- -}

and

H_{2} O_{2}

and the superoxide dismutasesensitive formation of Compound III [21,22]. However, according to another report, the dihydroxyfumarate-dependent reaction differs from the NADH-dependent reaction, in so far as the former could not be inhibited by catalase. Moreover, it apparently requires dihydroxyfumarate instead of

H_{2} O_{2}

as a reaction partner in the hydroxylating activity of Compound III. Therefore, it has been concluded that dihydroxyfumaratedependent hydroxylation by horseradish peroxidase does not involve

H_{2} O_{2}

and is mechanistically different from a Haber-Weiss-type reaction [23]. However, these results were obtained at a relatively high concentration of dihydroxyfumarate ( 4 mm ), saturated

O_{2}

, and at a low temperature (

4^{\circ} C)

, conditions that presumably favour high concentrations of

H_{2} O_{2}

. Fig. 10 shows that under reaction conditions more closely resembling the experiments with NADH shown in Figs 2 and 6, the production of ’ OH by dihydroxyfumarate + horseradish peroxidase can indeed be inhibited by catalase and enhanced by addition of exogenous

H_{2} O_{2}

. Moreover, purified Compound III has been shown to be capable of producing

^{\circ} OH

by utilizing

H_{2} O_{2}

in the absence of dihydroxyfumarate (Fig. 9A). Thus, both dihydroxyfumarate and NADH support

^{\circ} OH

production by horseradish peroxidase by a reaction mechanism involving

H_{2} O_{2}

as a substrate.
辣根过氧化物酶与二羟富马酸

+ O_{2}

发生的酚羟化反应与辣根过氧化物酶与

NADH + O_{2}

发生的反应相似，都会生成

O_{2}^{- -}

和

H_{2} O_{2}

以及对超氧化物歧化酶敏感的化合物 III [21,22]。然而，另一份报告指出，依赖富马酸二氢酯的反应不同于依赖 NADH 的反应，因为前者不能被过氧化氢酶抑制。此外，在化合物 III 的羟化活性中，显然需要二羟富马酸盐而不是

H_{2} O_{2}

作为反应伙伴。因此，有人认为辣根过氧化物酶的羟基化反应不涉及

H_{2} O_{2}

，在机理上不同于哈伯-魏斯型反应 [23]。然而，这些结果是在相对较高浓度的富马酸二氢酯（4 毫米）、饱和

O_{2}

和低温（

4^{\circ} C)

）条件下得到的，这些条件可能有利于高浓度的

H_{2} O_{2}

。图 10 显示，在更接近于图 2 和图 6 中所示的 NADH 实验的反应条件下，二羟基富马酸盐 + 辣根过氧化物酶产生的 ' OH 确实可以被过氧化氢酶抑制，并通过添加外源

H_{2} O_{2}

而增强。此外，纯化的化合物 III 已被证明能够在没有二羟基富马酸盐的情况下利用

H_{2} O_{2}

产生

^{\circ} OH

（图 9A）。因此，二羟富马酸和 NADH 都支持辣根过氧化物酶通过以

H_{2} O_{2}

为底物的反应机制产生

^{\circ} OH

。

Fig. 3. Effect of NADH and NADH + superoxide dismutase on the absorption spectrum of horseradish peroxidase. Enzyme (

1 μ m

) was incubated for 5 min with

200 μ m

NADH without or with

20 μ g \cdot {mL}^{- 1}

superoxide dismutase before recording the spectra. HRP, horseradish peroxidase; SOD, superoxide dismutase.
图 3.NADH 和 NADH + 超氧化物歧化酶对辣根过氧化物酶吸收光谱的影响。酶（

1 μ m

）与

200 μ m

NADH（不含

20 μ g \cdot {mL}^{- 1}

超氧化物歧化酶）或与

200 μ m

NADH（含

20 μ g \cdot {mL}^{- 1}

超氧化物歧化酶）孵育 5 分钟后记录光谱。HRP：辣根过氧化物酶；SOD：超氧化物歧化酶。

DISCUSSION 讨论

Peroxidases are very versatile enzymes that can react with numerous different substrates and catalyze different types of reactions in vivo [25]. The current knowledge of the basic peroxidase-dependent reactions in the peroxidatic and oxidative cycles is summarized in Fig. 11. The classic peroxidatic cycle is initiated by the transformation of the ferri-peroxidase into Compound I by

H_{2} O_{2}

, followed by the successive generation of two phenoxy radicals (Phe.) by hydrogen abstraction from
过氧化物酶是一种用途非常广泛的酶，可与多种不同的底物发生反应，并在体内催化不同类型的反应 [25]。图 11 总结了目前对过氧化循环和氧化循环中依赖过氧化物酶的基本反应的了解。经典的过氧化循环是由铁过氧化物酶通过

H_{2} O_{2}

转化为化合物 I 而开始的，随后通过从化合物 I 中抽取氢气而连续生成两个苯氧自由基 (Phe.)。

Fig. 4. pH Dependence of the transformation of ferri-horseradish peroxidase into Compound III and

O H

production in the presence of NADH. (A) Compound-III formation from horseradish peroxidase (

1.4 μ M

) was measured as the rate of

A_{452}

increase in the presence of

200 μ m

NADH. (B) Production of

^{\circ} OH

was measured either with the benzoate hydroxylation assay or with the deoxyribose degradation assay in the presence of

200 μ M

NADH.
图 4. 在 NADH 存在下铁-辣根过氧化物酶转化为化合物 III 和

O H

生成的 pH 依赖性。 (A) 在

200 μ m

NADH 存在下，辣根过氧化物酶生成化合物 III (

1.4 μ M

) 的速率随着

A_{452}

的增加而增加。(B) 在有

200 μ M

NADH 的情况下，用苯甲酸羟化测定法或脱氧核糖降解测定法测量

^{\circ} OH

的产生。

Fig. 5. Comparison of

O H

production by horseradish peroxidase and

{Fe}^{3 +}

-EDTA in the presence of NADH. (A) Reaction rates produced by

0 -

1 μ M

native or boiled horseradish peroxidase in the absence and presence of

H_{2} O_{2}

. (B) Reaction rates produced by

0 - 20 μ M {Fe}^{3 +}

-EDTA in the absence and presence of

H_{2} O_{2}

. The corresponding curves for horseradish peroxidase from panel A are included for comparison (dashed lines). Reaction mixtures contained

200 μ M NADH

and

\pm 100 μ M H_{2} O_{2}

.
图 5.比较辣根过氧化物酶和

{Fe}^{3 +}

-EDTA 在 NADH 存在下产生

O H

的情况。 (A)

0 -

1 μ M

本机或煮沸的辣根过氧化物酶在

H_{2} O_{2}

不存在和

H_{2} O_{2}

存在的情况下产生的反应速率。 (B)

0 - 20 μ M {Fe}^{3 +}

-EDTA 在

H_{2} O_{2}

不存在和

H_{2} O_{2}

存在的情况下产生的反应速率。A 组中辣根过氧化物酶的相应曲线（虚线）可供比较。反应混合物中含有

200 μ M NADH

和

\pm 100 μ M H_{2} O_{2}

。
substrates such as coniferyl alcohol. The spontaneous polymerization of these radicals leads to complex phenolic polymers such as lignins. If, however, the phenolic substrate is replaced by NADH or related reduced compounds, the resulting radicals (NAD•) initiate a nonenzymatic oxidative cycle in which

O_{2}

can be reduced to

O_{2}

. As

O_{2}^{- -}

can react with another NADH molecule to produce

H_{2} O_{2}

and NAD •, a chain reaction starts that provides the basis for the

H_{2} O_{2}

-producing NADH -oxidase activity of peroxidases. It is under these conditions that the enzyme can be partly converted to Compound III by binding

O_{2}^{- -}

to the haem iron. As Compound III does not support NADH oxidation, it is generally considered as an inactive form of peroxidase, at least as far as physiologically-relevant reactions are concerned [25].
底物，如针叶醇。这些自由基的自发聚合会产生复杂的酚类聚合物，如木质素。但是，如果酚类底物被 NADH 或相关的还原化合物取代，所产生的自由基（NAD-）就会启动一个非酶促性氧化循环，其中

O_{2}

可以还原成

O_{2}

。由于

O_{2}^{- -}

可以与另一个 NADH 分子反应生成

H_{2} O_{2}

和 NAD-，因此连锁反应开始，为过氧化物酶的

H_{2} O_{2}

生成 NADH - 氧化酶活性提供了基础。正是在这些条件下，酶可以通过与血红素铁结合

O_{2}^{- -}

部分转化为化合物 III。由于化合物 III 不支持 NADH 氧化，因此一般认为它是过氧化物酶的非活性形式，至少就生理相关反应而言是如此 [25]。

The results presented in this paper add a new facet to this picture. We have shown that horseradish peroxidase, in a manner similar to chelated Fe ions, is capable of reducing

H_{2} O_{2}

^{\circ} OH

in the presence of a suitable reductant. Various lines of evidence indicate that this reaction is catalyzed by horseradish peroxidase in the form of Compound III. Firstly, it has been shown that reagents such as superoxide dismutase or

{Cu}^{2 +}

which promote the conversion of

O_{2}^{- -}

H_{2} O_{2}

, thereby preventing the formation of Compound III, strongly inhibit

^{\circ} OH

production in the presence of NADH (Fig. 2, Table 1). As superoxide dismutase has little inhibitory effect on NADH oxidation by Compounds I and II [39], it can be concluded that sufficient

H_{2} O_{2}

is available under these conditions to maintain high levels of Compound I and II which are, however, unable to catalyze reactions leading to

^{\circ} OH

production. Moreover, the finding that superoxide dismutase inhibits

^{\circ} OH

production even in the
本文介绍的结果为这幅图景增添了一个新的侧面。我们已经证明，辣根过氧化物酶能够以类似于螯合铁离子的方式，在适当还原剂的存在下将

H_{2} O_{2}

还原成

^{\circ} OH

。各种证据表明，辣根过氧化物酶以化合物 III 的形式催化了这一反应。首先，研究表明，超氧化物歧化酶或

{Cu}^{2 +}

等试剂会促进

O_{2}^{- -}

向

H_{2} O_{2}

的转化，从而阻止化合物 III 的形成，但它们会在 NADH 的存在下强烈抑制

^{\circ} OH

的生成（图 2，表 1）。由于超氧化物歧化酶对化合物 I 和 II 的 NADH 氧化几乎没有抑制作用 [39]，因此可以得出结论：在这些条件下，有足够的

H_{2} O_{2}

维持高水平的化合物 I 和 II，但它们无法催化导致

^{\circ} OH

生成的反应。此外，超氧化物歧化酶抑制

^{\circ} OH

生成的发现甚至在

Fig. 6. Effect of superoxide dismutase on OH production by horseradish peroxidase in the presence of NADH and

H_{2} O_{2}

, determined by benzoate hydroxylation. Benzoate ( 2 mm ), NADH (

200 μ m

), horseradish peroxidase

(1.4 μ M), H_{2} O_{2} (100 μ M)

and superoxide dismutase

(50 μ g \cdot {mL}^{- 1})

were added as indicated. The two lower curves show controls omitting either BA or NADH from the initial reaction mixture. BA, benzoate, HRP, horseradish peroxidase; SOD, superoxide dismutase.
图 6.超氧化物歧化酶对辣根过氧化物酶在 NADH 和

H_{2} O_{2}

存在下产生 OH 的影响，通过苯甲酸酯羟化测定。苯甲酸盐（2 毫米）、NADH（

200 μ m

）、辣根过氧化物酶

(1.4 μ M), H_{2} O_{2} (100 μ M)

和超氧化物歧化酶

(50 μ g \cdot {mL}^{- 1})

按所示添加。下面两条曲线是初始反应混合物中没有加入 BA 或 NADH 的对照组。BA，苯甲酸；HRP，辣根过氧化物酶；SOD，超氧化物歧化酶。
presence of high levels of added

H_{2} O_{2}

(Fig. 6) confirms that Compound III, rather than Compounds I and II, is essential in this reaction. This conclusion is supported by the finding that neither Compound I nor Compound II can produce

^{\circ} OH

from

H_{2} O_{2}

(Table 2). On the other hand, purified Compound III, containing no spectroscopically detectable amounts of Compounds I and II, readily converts

H_{2} O_{2}

^{\circ} OH

and simultaneously reverts to the ferric form of the enzyme (Fig. 9A,C). We conclude therefore that Compound III catalyzes a hydroxylic cycle (Fig. 11) by which

^{OH}

can be continuously produced utilizing

H_{2} O_{2}

and

O_{2}^{\cdot -}

as formally described by the ironcatalyzed Haber-Weiss reaction (Eqn 3). According to the scheme shown in Fig. 11, peroxidase can act in two different catalytic modes. In the presence of

H_{2} O_{2}

and phenolic substrates, the enzyme operates in the peroxidatic cycle and is engaged in the synthesis of lignins and other phenolic polymers. Alternatively, upon addition of

O_{2}^{- -}

the enzyme can be switched to Compound III and now catalyzes the reduction of

H_{2} O_{2}

^{\circ} OH

in a Haber-Weiss-type reaction cycle. This changeover in catalytic function can be brought about by NADH or a similar substrate that supports an oxidative cycle producing

O_{2}^{- -}

. Although Compound III can also be produced by a large excess of

H_{2} O_{2}

[19], this may not be of biological relevance. The operation of a NADH-dependent oxidative cycle is of course not an essential requirement for the OH -producing mode of peroxidase action as the availability of

O_{2}^{- -}

and

H_{2} O_{2}

from other sources would have a similar effect. Pyridine nucleotides
大量添加的

H_{2} O_{2}

的存在（图 6）证实了化合物 III 而不是化合物 I 和 II 在该反应中是必不可少的。化合物 I 和化合物 II 都不能从

H_{2} O_{2}

生成

^{\circ} OH

（表 2），这一发现也支持了上述结论。另一方面，纯化的化合物 III 不含光谱上可检测到的化合物 I 和化合物 II 的量，能轻易地将

H_{2} O_{2}

转化为

^{\circ} OH

，并同时还原为铁形式的酶（图 9A、C）。因此，我们得出结论，化合物 III 催化了一个羟基循环（图 11），通过该循环，可以利用

H_{2} O_{2}

和

O_{2}^{\cdot -}

不断产生

^{OH}

，正如铁催化的哈伯-魏斯反应（公式 3）所正式描述的那样。根据图 11 所示的方案，过氧化物酶可以以两种不同的催化模式发挥作用。在

H_{2} O_{2}

和酚类底物存在的情况下，酶在过氧化循环中运行，参与木质素和其他酚类聚合物的合成。或者，在加入

O_{2}^{- -}

后，该酶可切换到化合物 III，并在哈伯-魏斯型反应循环中催化

H_{2} O_{2}

还原成

^{\circ} OH

。这种催化功能的转换可以通过 NADH 或类似的底物来实现，这些底物支持产生

O_{2}^{- -}

的氧化循环。虽然化合物 III 也可以由大量过量的

H_{2} O_{2}

生成 [19]，但这可能与生物学无关。依赖 NADH 的氧化循环的运行当然不是过氧化物酶产生 OH 作用模式的必要条件，因为从其他来源获得

O_{2}^{- -}

和

H_{2} O_{2}

也会产生类似的效果。吡啶核苷酸

Fig. 7. Comparison of

\cdot OH

production by horseradish peroxidase supported by various putative substrates. Reaction mixtures contained

1.4 μ M

horseradish peroxidase. The data were corrected for the effects of substrates (dihydroxyfumarate, NADH, NADPH) on fluorescence emisson. Note that the curve for DHF is reduced by a factor of 10. Asc, ascorbate; DHF, dihydroxyfumarate.
图 7.各种假定底物支持的辣根过氧化物酶产生

\cdot OH

的比较。反应混合物中含有

1.4 μ M

辣根过氧化物酶。根据底物（二羟基富马酸、NADH、NADPH）对荧光发射的影响对数据进行了校正。请注意，DHF 的曲线缩小了 10 倍。Asc，抗坏血酸；DHF，二羟富马酸。
can be replaced by cysteine, which is however much less effective in supporting

^{\circ} OH

production by horseradish peroxidase, presumably because it also acts as an antioxidant. It has previously been shown that the oxidation of cysteine by horseradish peroxidase results in

H_{2} O_{2}

production, unless the pro-oxidant function is superimposed by the antioxidant function at higher concentrations [40]. The pronounced antioxidant

(O_{2}^{- -}

-scavenging) function of ascorbate [41] may be responsible for the inhibitory effect of this peroxidase substrate on Compound-III formation and its inactivity in the

\cdot OH

-producing reaction of horseradish peroxidase [21,23].
然而，半胱氨酸在支持辣根过氧化物酶产生

^{\circ} OH

方面的作用要小得多，这可能是因为半胱氨酸还具有抗氧化作用。以前的研究表明，辣根过氧化物酶对半胱氨酸的氧化会导致

H_{2} O_{2}

的产生，除非在较高浓度下，促氧化功能被抗氧化功能所叠加 [40]。抗坏血酸具有明显的抗氧化

(O_{2}^{- -}

- 清除）功能 [41]，这可能是这种过氧化物酶底物对化合物-III 的形成具有抑制作用以及在辣根过氧化物酶的

\cdot OH

- 生成反应中不起作用的原因 [21,23]。

In plants, peroxidases are primarily localized in the apoplastic space where they are bound ionically or covalently to cell-wall polymers [25]. No clear functions have been attributed to cellwall peroxidases except in lignin synthesis and related oxidative cross-linking reactions [25]. In the light of the present results these enzymes may have a much wider array of physiological functions that are related to their

^{\circ} OH

-producing property. This mode of peroxidase action may be fundamentally important in physiological processes involving the destructive effects of oxygen. The prediction can be made that

^{\circ} OH

is likely to be produced whenever peroxidase comes into contact with suitable concentrations of

O_{2}^{-}

and

H_{2} O_{2}

, originating either from the oxidative cycle of peroxidase or from other sources. This situation prevails, for instance, when

O_{2}^{- -}

and

H_{2} O_{2}

levels are increased in plants in response to pathogen attack (oxidative burst) and is often followed by a hypersensitive reaction that results in host cell death [42]. However, there is accumulating evidence that plant cells can release significant amounts of

O_{2}^{-}

and

H_{2} O_{2}

in the absence of pathogenic stimuli [43-45]. The production of

^{\circ} OH

by peroxidases bound to plant cell walls could also be relevant for the controlled breakdown of structural
在植物中，过氧化物酶主要定位于细胞质空间，它们以离子方式或共价方式与细胞壁聚合物结合 [25]。除了木质素合成和相关的氧化交联反应之外，细胞壁过氧化物酶还没有明确的功能[25]。从目前的研究结果来看，这些酶可能具有与

^{\circ} OH

产生特性相关的更广泛的生理功能。这种过氧化物酶的作用模式在涉及氧的破坏作用的生理过程中可能具有根本性的重要意义。可以预测，只要过氧化物酶与适当浓度的

O_{2}^{-}

和

H_{2} O_{2}

接触，就可能产生

^{\circ} OH

，而这些

O_{2}^{-}

和

H_{2} O_{2}

要么来自过氧化物酶的氧化循环，要么来自其他来源。例如，当植物体内的

O_{2}^{- -}

和

H_{2} O_{2}

含量增加以应对病原体侵袭（氧化猝灭）时，就会出现这种情况，随后往往会出现超敏反应，导致宿主细胞死亡 [42]。然而，越来越多的证据表明，在没有病原体刺激的情况下，植物细胞也能释放大量的

O_{2}^{-}

和

H_{2} O_{2}

[43-45]。与植物细胞壁结合的过氧化物酶产生的

^{\circ} OH

也可能与受控的结构分解有关。

Fig. 8. Comparison of

O H

production catalyzed by various types of peroxidase in the presence of NADH. Reaction mixtures contained

200 μ M

NADH and

0 - 20

units (standard pyrogallol assay) as indicated by the manufacturer of the following enzymes: horseradish peroxidase (unfractionated enzyme mixture), acidic horseradish peroxidase fraction, alkaline horseradish peroxidase fraction, soybean peroxidase, Arthromyces peroxidase, lactoperoxidase, myeloperoxidase. aciHRP, acidic horseradish peroxidase fraction; alkHRP, alkaline horseradish peroxidase fraction; ArtPOD, Arthromyces peroxidase; lacPOD, lactoperoxidase; myPOD, myeloperoxidase; soyPOD, soybean peroxidase .
图 8.各种过氧化物酶在 NADH 存在下催化

O H

生成的比较。反应混合物中含有 NADH

200 μ M

和下列酶的制造商标明的

0 - 20

单位（标准焦棓酚测定法）：辣根过氧化物酶（未分馏的酶混合物）、酸性辣根过氧化物酶部分、碱性辣根过氧化物酶部分、大豆过氧化物酶、节杆菌过氧化物酶、乳过氧化物酶、髓过氧化物酶。aciHRP，酸性辣根过氧化物酶部分；alkHRP，碱性辣根过氧化物酶部分；ArtPOD，节霉过氧化物酶；lacPOD，乳过氧化物酶；myPOD，髓过氧化物酶；soyPOD，大豆过氧化物酶。

Fig. 9. Production of OH from

H_{2} O_{2}

by Compound III and the simultaneous conversion of Compound III to ferri-peroxidase. (A) Reaction kinetics of benzoate hydroxylation induced by adding

3 μ m

freshly purified Compound III (arrow) to assay mixture without NADH, in the absence and presence of

100 μ M H_{2} O_{2}

. (B) Reaction kinetics of CompoundIII conversion induced by adding

100 μ M H_{2} O_{2}

to buffer containing

50 μ m

freshly prepared Compound III, measured as decrease in

A_{582}

[29]. Control: Compound III in buffer; without and with added benzoate ( 2 mm ). © Reaction kinetics of benzoate hydroxylation induced by adding

1.4 μ M

horseradish peroxidase (ferri-peroxidase, arrow) to assay mixture containing

200 μ m NADH

in the absence and presence of

100 μ M H_{2} O_{2} . H_{2} O_{2}

produced no measurable fluorescence increase in the reaction mixture without NADH.
图 9.化合物 III 从

H_{2} O_{2}

中产生 OH 并同时将化合物 III 转化为铁过氧化物酶。(A) 在没有和有

100 μ M H_{2} O_{2}

的情况下，向不含 NADH 的测定混合物中加入

3 μ m

新鲜纯化的化合物 III（箭头）诱导苯甲酸羟基化的反应动力学。 (B) 向含有

50 μ m

新鲜制备的化合物 III 的缓冲液中加入

100 μ M H_{2} O_{2}

诱导化合物 III 转化的反应动力学，测量值为

A_{582}

的下降 [29]。对照组：缓冲液中的化合物 III；不添加苯甲酸盐和添加苯甲酸盐 ( 2 mm )。将

1.4 μ M

辣根过氧化物酶（铁-过氧化物酶，箭头）加入到含有

200 μ m NADH

的检测混合物中，在没有

100 μ M H_{2} O_{2} . H_{2} O_{2}

和

100 μ M H_{2} O_{2} . H_{2} O_{2}

存在的情况下，苯甲酸羟基化的反应动力学在没有 NADH 的反应混合物中没有产生可测量的荧光增加。

Fig. 10. Effect of catalase and

H_{2} O_{2}

on the production of OH by horseradish peroxidase in the presence of dihydroxyfumarate, determined by benzoate hydroxylation. Dihydroxyfumarate (

50 μ m

), horseradish peroxidase

(1.4 μ M)

, catalase

(50 μ g \cdot {mL}^{- 1})

and

H_{2} O_{2} (100 μ M)

were added as indicated.
图 10.过氧化氢酶和

H_{2} O_{2}

对辣根过氧化物酶在二羟基富马酸盐存在下产生 OH 的影响，由苯甲酸盐羟化测定。按所示添加二羟富马酸（

50 μ m

）、辣根过氧化物酶

(1.4 μ M)

、过氧化氢酶

(50 μ g \cdot {mL}^{- 1})

和

H_{2} O_{2} (100 μ M)

。
polymers. This could occur in the form of site-specific scissions in polymers to which the peroxidase molecules are attached. As

\cdot OH

can also cause oxidative cross-linking reactions [46], the as yet unexplained covalent binding of peroxidase to cell-wall polymers could be another consequence of the

^{\circ} OH

-producing activity of this enzyme. Moreover, it is well known that enzymes can be structurally modified or damaged by

^{\circ} OH

[12]. Thus, taking into account the short life-time of

^{\circ} OH

, which restricts the effective range of action to a few nanometers from its site of generation, it seems unavoidable that the peroxidase protein itself becomes a target of

^{\circ} OH

. This suicide action provides a plausible explanation for the unique and bewildering abundance of peroxidase isoforms generally found in the cell walls of plants (e.g. 47 electrophoretically-separable forms in tobacco tissues [47]), which could be partly degraded products of a smaller number of true isoenzymes.
聚合物。这可能以过氧化物酶分子所附着的聚合物中特定位点裂解的形式发生。由于

\cdot OH

也能引起氧化交联反应 [46]，过氧化物酶与细胞壁聚合物的共价结合尚未得到解释，这可能是这种酶的

^{\circ} OH

生成活性的另一个结果。此外，众所周知，

^{\circ} OH

可以改变或破坏酶的结构 [12]。因此，考虑到

^{\circ} OH

的寿命很短，其有效作用范围被限制在距其产生部位几纳米的范围内，过氧化物酶蛋白本身成为

^{\circ} OH

的目标似乎是不可避免的。这种自杀性作用为植物细胞壁中通常发现的独特而又令人困惑的大量过氧化物酶同工酶（例如烟草组织中的 47 种电泳分离形式 [47]）提供了一个合理的解释，这些同工酶可能是数量较少的真正同工酶的部分降解产物。

In this context it is also interesting to note that the depolymerization of lignins by wood-rotting fungi is mediated by secreted peroxidases, designated as ligninases, in the presence of

H_{2} O_{2}

[48]. The biochemical mechanism of this ecologically extremely important peroxidase reaction is not yet resolved and it may be of interest to test the hypothesis that ligninases initiate the biodegradation of lignins via the

\cdot OH

-producing mode of peroxidase action. It has been shown that ligninase Compound III can produce

^{\circ} OH

in the presence of dihydroxyfumarate in a similar fashion as horseradish peroxidase
在这方面还值得注意的是，木腐真菌在

H_{2} O_{2}

存在的情况下通过分泌的过氧化物酶（称为木质素酶）对木质素进行解聚 [48]。这种在生态学上极其重要的过氧化物酶反应的生物化学机制尚未得到解决，因此，检验木质素酶通过

\cdot OH

产生过氧化物酶作用模式启动木质素生物降解的假设可能会很有意义。研究表明，木质素酶化合物 III 能在二羟富马酸存在下产生

^{\circ} OH

，其方式与辣根过氧化物酶类似

Fig. 11. Reaction scheme combining the peroxidatic, oxidative and hydroxylic cycles of peroxidase catalysis. The classic peroxidatic cycle mediates the oxydation by

H_{2} O_{2}

of phenolic substrates ( PheH ) to phenoxy radicals (Phe.) that can polymerize to form molecules such as lignins. The corresponding reduction of substrates such as NADH (or dihydroxyfumarate) initiates the selfsustained oxidative cycle that reduces

O_{2}

O_{2}^{- -}

and

H_{2} O_{2} \cdot O_{2}^{\cdot -}

can be utilized to produce Compound III that reduces

H_{2} O_{2}

to OH in the hydroxylic cycle.
图 11.过氧化物酶催化的过氧化循环、氧化循环和羟基循环相结合的反应方案。经典的过氧化循环通过

H_{2} O_{2}

将酚类底物（PheH）氧化成苯氧自由基（Phe.），苯氧自由基可聚合形成木质素等分子。NADH （或二羟基富马酸）等底物的相应还原启动了自持氧化循环，将

O_{2}

还原成

O_{2}^{- -}

和

H_{2} O_{2} \cdot O_{2}^{\cdot -}

可用于生成化合物 III，在羟基循环中将

H_{2} O_{2}

还原成 OH。

Compound III [32]. The observation that the ligninase-like peroxidase secreted by the fungus A. ramosus [49] displays a particularly high activity of ’ OH production (Fig. 8) provides further evidence for this idea. It can also be conceived that cationic cell-wall peroxidases possessing a similarly high

^{\circ} OH -

producing activity (Fig. 8) are involved in the depolymerization of lignins under particular physiological conditions in the living plant. Thus, depending on the level of

O_{2}^{- -}

and

H_{2} O_{2}

in the cellwall space, peroxidases could mediate either wall-stiffening by lignin synthesis [50] or wall-loosening by lignin depolymerization. These and other biologically relevant implications of our results can now be explored experimentally.
化合物 III [32]。据观察，由真菌 A. ramosus [49] 分泌的类似木质素酶的过氧化物酶具有特别高的'OH 生成活性（图 8），这为这一观点提供了进一步的证据。我们还可以设想，在活体植物的特定生理条件下，具有类似高

^{\circ} OH -

生成活性的阳离子细胞壁过氧化物酶（图 8）也参与了木质素的解聚过程。因此，根据细胞壁空间中

O_{2}^{- -}

和

H_{2} O_{2}

的水平，过氧化物酶可以通过木质素合成[50]或通过木质素解聚介导细胞壁加固。现在我们可以通过实验来探索我们的研究结果所具有的这些及其他生物学意义。

ACKNOWLEDGEMENT 致谢

This work was supported by a postdoctoral fellowship to S.C. from the Alexander-von-Humboldt foundation.
这项工作得到了亚历山大-冯-洪堡基金会（Alexander-von-Humboldt foundation）为 S.C. 提供的博士后奖学金的支持。

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Correspondence to P. Schopfer, Institut für Biologie II der Universität Freiburg, Schänzlestr. 1, D - 79104 Freiburg, Germany.
通讯作者：P. Schopfer，弗莱堡大学生物二研究所，Schänzlestr.
Fax: + 49761203 2612, Tel: + 49761203 2665,
传真：+ 49761203 2612，电话：+ 49761203 2665、
E-mail: schopfer@sun2.ruf.uni-freiburg.de.
电子邮件：schopfer@sun2.ruf.uni-freiburg.de.
Enymes: catalase (EC 1.11.1.6); horseradish peroxidase (EC.1.11.1.7); superoxide dismutase (EC 1.15.1.1)
酶：过氧化氢酶（EC 1.11.1.6）；辣根过氧化物酶（EC 1.11.1.7）；超氧化物歧化酶（EC 1.15.1.1）
(Received 24 July 1998, revised 16 December 1998, accepted 17
(1998年7月24日收到，1998年12月16日修订，17日接受
December1998) 1998 年 12 月）

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

Abstract 摘要

MATERIALS AND METHODS 材料和方法

Chemicals 化学品

Enzyme assays 酶测定

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

Statistical treatment of data数据的统计处理

RESULTS 结果

DISCUSSION 讨论

ACKNOWLEDGEMENT 致谢

REFERENCES 参考文献

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

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

Statistical treatment of data
数据的统计处理