Substrate reduction and oxygen activation during microsomal metabolism of quinones

2-Dimethylamino-3-chloro-1,4-naphthaquinone (DCNQ) was used to study oxygen and substrate activation in microsomal system. DCNQ was shown to be bound to microsomal cytochrome P-450 as a type I substrate; its N-demethylation was catalyzed by cytochrome P-450. Cytochrome P-450 and NADPH-cytochrome P-450 reductase are capable of DCNQ reduction to semi- and hydroquinones. The OH-radical formed in the presence of DCNQ, NADPH and reductase was detected, using a spin trap (5,5-dimethylpyrroline-N-oxide). The OH-radical formation was shown to be stimulated by the Fe-EDTA complex. Using the OH-radical scavengers (mannitol, N-butanol, alpha-naphthol) and the catalase inhibitor sodium azide, it was shown that the OH-radical participates in microsomal oxidation of DCNQ and aminopyrine. It was assumed that in the course of microsomal oxidation the reduced DCNQ is responsible for: i) stimulation of molecular oxygen reduction to H2O2; ii) reduction of Fe ions (Fe3+----Fe2+) which cause the decomposition of H2O2 in the Fenton reaction resulting in the formation of a strong oxidizing agent--a hydroxyl radical.     

Role of hydroxyl radicals in the iron-ethylenediaminetetraacetic acid mediated stimulation of microsomal oxidation of ethanol

The microsomal oxidation of ethanol or 1-butanol was increased by ferrous ammonium sulfate-ethylenediaminetetraacetic acid (1:2) (Fe-EDTA) (3.4-50 microM). The increase was blocked by hydroxyl radical scavenging agents such as dimethyl sulfoxide or mannitol. The activities of aminopyrine demethylase or aniline hydroxylase were not affected by Fe-EDTA. The accumulation of H2O2 was decreased in the presence of Fe-EDTA, consistent with an increased utilization of H2O2. Other investigators have shown that Fe-EDTA increases the formation of hydroxyl radicals in systems where superoxide radicals are generated. The stimulation by Fe-EDTA appears to represent a pathway involving hydroxyl radicals rather than catalase because (1) stimulation occurred in the presence of azide, which inhibits catalase, (2) stimulation occurred in the presence of 1-butanol, which is not an effective substrate for catalase, and (3) stimulation was blocked by hydroxyl radical scavenging agents, which do not affect catalase-mediated oxidation of ethanol. A possible role for contaminating iron in the H2O or buffers could be ruled out since similar results were obtained with or without chelex-100 treatment of these solutions. The stimulatory effect by Fe-EDTA required microsomal electron transfer with NADPH, and H2O2 could not replace the NADPH-generating system. In the absence of microsomes or catalase, Fe-EDTA also stimulated the coupled oxidation of ethanol during the oxidation of xanthine by xanthine oxidase. These results suggest that during microsomal electrom transfer, conditions may be appropriate for a Fenton type or a modified Haber-Weiss type of reaction to occur, leading to the production of hydroxyl radicals.     

Effect of thiourea on microsomal oxidation of alcohols and associated microsomal functions.

Thiourea and diethylthiourea, two compounds which react with hydroxyl radicals, inhibited NADPH-dependent microsomal oxidation of ethanol and 1-butanol. Inhibition by both compounds was more effective in the presence of the catalase inhibitor, azide. Inhibition by thiourea was noncompetitive with respect to ethanol in the absence of azide but was competitive in the presence of azide. Urea, a compound which does not react with hydroxyl radicals or H2O2, was without effect. Thiourea had no effect on NADH- and NADH-cytochrome c reductase, NADPH oxidase, and NADH- and NADPH-dependent oxygen uptake. Thiourea inhibited the activities of aniline hydroxylase and aminopyrine demethylase. Thiourea, but no other hydroxyl radical scavengers, e.g., dimethyl sulfoxide, mannitol, and benzoate, reacted directly with H202 and decreased H2O2 accumulation in the presence of azide. Therefore the actions of thiourea are complex because it can react with both hydroxyl radicals and H2O2. Differences between the actions of thiourea and those previously reported for dimethyl sulfoxide, mannitol, and benzoate, e.g., effects on drug metabolism, effectiveness of inhibition in the absence of azide, or kinetics of the inhibition, probably reflect the fact that thiourea reacts directly with H2O2 whereas the other agents do not. The current results remain consistent with the concept that microsomal oxidation of alcohols involves interactions of the alcohols with hydroxyl radicals generated from microsomal electron transfer.     

Bleomycin-dependent damage to the bases in DNA is a minor side reaction.

The antitumor antibiotic bleomycin degrades DNA in the presence of ferric ions and H2O2 or in the presence of ferric ions, oxygen, and ascorbic acid. When DNA degradation is measured as formation of base propenals by the thiobarbituric acid assay, it is not inhibited by superoxide dismutase and scavengers of the hydroxyl radical or by catalase (except that catalase inhibits in the bleomycin/ferric ion/H2O2 system by removing H2O2). Using the technique of gas chromatography/mass spectrometry with selected-ion monitoring, we show that DNA degradation is accompanied by formation of small amounts of modified DNA bases. The products formed are identical with those generated when hydroxyl radicals react with DNA bases. Base modification is significantly inhibited by catalase and partially inhibited by scavengers of the hydroxyl radical and by superoxide dismutase. We suggest that the bleomycin-oxo-iron ion complex that cleaves the DNA to form base propenals can decompose in a minor side reaction to generate hydroxyl radical, which accounts for the base modification in DNA. However, hydroxyl radical makes no detectable contribution to the base propenal formation.     

Iodide oxidation and iodine reduction mediated by horseradish peroxidase in the presence of ethylenediaminetetraacetic acid (EDTA): the superoxide effect

Ethylenediaminetetraacetic acid (EDTA) is an inhibitor of iodide (I-) oxidation that is catalyzed by horseradish peroxidase (HRP). HRP-mediated iodine (I2) reduction and triiodide (I3+) disappearance occur in the presence of this inhibitor. It is interesting that in the presence of EDTA, HRP produces superoxide radical, a reactive oxygen species that is required for iodine reduction. Substitution of potassium superoxide (KO2) or a biochemical superoxide generating system (xanthine/xanthine oxidase) for HRP and H2O2 in the reaction mixture also can reduce iodine to iodide. Thus, iodine reduction mediated by HRP occurs because HRP is able to mediate the formation of superoxide in the presence of EDTA and H2O2. Although superoxide is able to mediate iodine reduction directly, other competing reactions appear to be more important. For example, high concentrations (mM range) of EDTA are required for efficient iodine reduction in this system. Under such conditions, the concentration (microM range) of contaminating EDTA-Fe(III) becomes catalytically important. In the presence of superoxide, EDTA-Fe(III) is reduced to EDTA-Fe(II), which is able to reduce iodine and form triiodide rapidly. Also of importance is the fact that EDTA-Fe(II) reacts with hydrogen peroxide to form hydroxyl radical. Hydroxyl radical involvement is supported by the fact that a wide variety of hydroxyl radical (OH) scavengers can inhibit HRP dependent iodine reduction in the presence of EDTA and hydrogen peroxide.     

Effect of oxygen concentration on microsomal oxidation of ethanol and generation of oxygen radicals.

The iron-catalysed production of hydroxyl radicals, by rat liver microsomes (microsomal fractions), assessed by the oxidation of substrate scavengers and ethanol, displayed a biphasic response to the concentration of O2 (varied from 3 to 70%), reaching a maximal value with 20% O2. The decreased rates of hydroxyl-radical generation at lower O2 concentrations correlates with lower rates of production of H2O2, the precursor of hydroxyl radical, whereas the decreased rates at elevated O2 concentrations correlate with lower rates (relative to 20% O2) of activity of NADPH-cytochrome P-450 reductase, which reduces iron and is responsible for redox cycling of iron by the microsomes. The oxidation of aniline or aminopyrine and the cytochrome P-450/oxygen-radical-independent oxidation of ethanol also displayed a biphasic response to the concentration of O2, reaching a maximum at 20% O2, which correlates with the dithionite-reducible CO-binding spectra of cytochrome P-450. Microsomal lipid peroxidation increased as the concentration of O2 was raised from 3 to 7 to 20% O2, and then began to level off. This different pattern of malondialdehyde generation compared with hydroxyl-radical production probably reflects the lack of a role for hydroxyl radical in microsomal lipid peroxidation. These results point to the complex role for O2 in microsomal generation of oxygen radicals, which is due in part to the critical necessity for maintaining the redox state of autoxidizable components of the reaction system.     

Superoxide dismutase enhanced the formation of hydroxyl radicals in a reaction mixture containing xanthone under UVA irradiation

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 microM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O(2), H(2)O(2), and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O(2)(-.).     

Cytochrome P-450-mediated oxidation of substrates by electron-transfer; role of oxygen radicals and of 1- and 2-electron oxidation of paracetamol

The mechanism by which the hepatic cytochrome P-450 (Cyt. P-450) containing mixed-function oxidase system oxidizes the analgesic drug paracetamol (PAR) to a hepatotoxic metabolite was studied. Since previous studies excluded the possibility of oxygenation of PAR, three other mechanisms, namely direct 1-electron oxidation by a Cyt. P-450-ferrous-dioxygen complex under concomitant formation of H2O2 to N-acetyl-p-semiquinone imine (NAPSQI), direct 2-electron oxidation by a Cyt. P-450-ferric-oxene complex to N-acetyl-p-benzoquinone imine (NAPQI) and indirect oxidation by active oxygen species released from Cyt. P-450, were considered. Indirect oxidation by active oxygen species was not involved, as active oxygen scavengers such as superoxide dismutase, catalase and DMSO did not affect the oxidation of PAR in hepatic microsomes. No reaction products characteristic for a direct 1-electron oxidation of PAR by Cyt. P-450 were observed: neither NAPSQI radical formation was detectable by ESR, nor PAR-dimer formation, nor stimulation of the microsomal H2O2 production was found to occur. In fact, PAR inhibited the spontaneous microsomal H2O2 formation. Studies on the reactions of NAPSQI with glutathione (GSH) revealed that NAPSQI hardly conjugated with GSH to a 3-glutathionyl-paracetamol conjugate (PAR-GSH) conjugate. The reactions of the elusive reactive metabolite formed during microsomal oxidation of PAR in the presence of GSH closely resembled those of synthetic NAPQI: both PAR-GSH and oxidized glutathione (GSSG) formation occurred. Furthermore, in agreement with a 2-electron oxidation hypothesis, iodosobenzene-dependent oxidation of PAR by cyt. P-450 in the presence of GSH resulted in the formation of the PAR-GSH conjugate. It is concluded that bioactivation of PAR by the Cyt. P-450 containing mixed-function oxidase system consists of a direct 2-electron oxidation to NAPQI.     

Increased phospholipid methylation in the myocardium of alcoholic rats

NAD(P)H is known to be oxidized by singlet molecular oxygen, perhydroxyl radical, and hydroxyl radical. In marked contrast to these reactive oxygen species, NAD(P)H is stable in the presence of micromolar concentrations of H2O2. The experiments herein demonstrate that NADPH is rapidly oxidized by H2O2 in the presence of a heme-peptide. The oxidation product is enzymatically active NADP+. In the absence of NADPH, the heme-peptide undergoes rapid degradation via reaction with H2O2. In the presence of NADPH, the reduced nucleotide is oxidized to NADP and the heme-peptide is partially protected from oxidation. It is suggested that under certain conditions the reduced nucleotides may contribute to the protection of intracellular heme moieties from degradation engendered by endogenous or exogenous H2O2.     

Free Radical Metabolism of Alcohols by Rat Liver Microsomes

By using e.s.r. spectroscopy coupled with the spin trapping technique we have detected the formation of free radical intermediates by rat liver microsomes incubated with either ethanol, 2-propanol or 2-butanol in the presence of a NADPH regenerating system and 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) as spin trap. The e.s.r. spectra have been identified as due to the hydroxyalkyl free radical adducts of 4-POBN.

The free radical formation depends upon the activity of the microsomal monoxygenase system and is blocked by omitting NADP⁺ from the incubation mixture, by anaerobic incubation or by enzyme denaturation. The involvement of hydroxyl radicals (OH) produced through a Fenton-type reaction from endogenously formed hydrogen peroxide is suggested by the opposite effects exerted on the e.s.r. signal intensity by azide and catalase. Consistently, iron chelation by desferrioxamine inhibits the free radical formation, while the supplementation of EDTA-iron increases it by several fold. Inhibitors of cytochrome P₄₅₀-dependent monoxygenase system reduce to various extents the production of free radical intermediates suggesting that reactive oxygen species might be formed at the active site of cytochrome P₄₅₀ where they react with alkyl alcohol molecules.

The data presented support the hypothesis that free radical species are generated during the microsomal metabolism of alcohols and suggest the possibility that ethanol-derived radicals might play a role in the pathogenesis of the liver lesions consequent upon alcoholic abuse.     

Generation of reactive oxygen species in the enzymatic reduction of PbCrO4 and related DNA damage

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO4 by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (.OH) and caused DNA damage. The .OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H2O2 scavenger, inhibited the .OH radical generation, indicating the involvement of H2O2 in the mechanism of Cr(VI)-induced .OH generation. Catalase reduced .OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating .OH radicals are involved in the damage measured. The H2O2 formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO4 impaired the generation of .OH radical. The results obtained from this study show that reduction of insoluble PbCrO4 by glutathione reductase/NADPH generates .OH radicals. The mechanism of .OH generation involves reduction of molecular oxygen to H2O2, which generates .OH radicals through a Fenton-like reaction. The .OH radicals generated by PbCrO4 caused DNA strand breakage.     

Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique.

Copper-induced oxidative damage is generally attributed to the formation of the highly reactive hydroxyl radical by a mechanism analogous to the Haber-Weiss cycle for Fe(II) and H2O2. In the present work, the reaction between the Cu(I) ion and H2O2 is studied using the EPR spin-trapping technique. The hydroxyl radical adduct was observed when Cu(I), dissolved in acetonitrile under N2, was added to pH 7.4 phosphate buffer containing 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Formation of the hydroxyl radical was dependent on the presence of O2 and subsequent formation of H2O2. The kscav/kDMPO ratios obtained were below those expected for a mechanism involving free hydroxyl radical and reflect the interference of nucleophilic addition of H2O to DMPO to form the DMPO/.OH adduct in the presence of nonchelated copper ion. Addition of ethanol or dimethyl sulfoxide to the reaction suggests that a high-valent metal intermediate, possibly Cu(III), was also formed. Spin trapping of hydroxyl radical was almost completely inhibited upon addition of Cu(I) to a solution of either nitrilotriacetate or histidine, even though the copper was fully oxidized to Cu(II) and H2O2 was formed. Bathocuproinedisulfonate, thiourea, and reduced glutathione all stabilized the Cu(I) ion toward oxidation by O2. Upon addition of H2O2, the Cu(I) in all three complexes was oxidized to varying degrees; however, only the thiourea complex was fully oxidized within 2 min of reaction and produced detectable hydroxyl radicals. No radicals were detected from the bathocuproinedisulfonate or glutathione complexes. Overall, these results suggest that the deleterious effects of copper ions in vivo are diminished by biochemical chelators, especially glutathione, which probably has a major role in moderating the toxicological effects of copper.     

The transition metal-mediated formation of the hydroxyl free radical during the reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase

The NADPH-supported enzymatic reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase was investigated. The ESR spin trapping technique was employed to identify the free radical metabolites of oxygen. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to trap and identify the oxygen-derived free radicals. [17O]Oxygen was employed to demonstrate that the oxygen-centered radicals arose from molecular oxygen. From the data, the following scheme is proposed: (Formula:see text). The formation of the free hydroxyl radical during the reduction of oxygen was demonstrated with quantitative competition experiments. The hydroxyl radical abstracted hydrogen from ethanol or formate, and the resulting scavenger-derived free radical was trapped with known rate constants. If H2O2 was added to the enzymatic reaction, a stimulation of the production of the hydroxyl radical was obtained. This stimulation was manifested in both the concentration and the rate of formation of the DMPO/hydroxyl radical adduct. Catalase was shown to inhibit formation of the hydroxyl radical adduct, further supporting the formation of hydrogen peroxide as an intermediate during the reduction of oxygen. All three components, ferredoxin, ferredoxin:NADP+ oxidoreductase, and NADPH, were required for reduction. Ferredoxin:NADP+ oxidoreductase reduces ferredoxin, which in turn is responsible for the reduction of oxygen to hydrogen peroxide and ultimately the hydroxyl radical. The effect of transition metal chelators on the DMPO/hydroxyl radical adduct concentration suggests that the reduction of chelated iron by ferredoxin is responsible for the reduction of hydrogen peroxide to the hydroxyl radical via Fenton-type chemistry.     

Heme and apoprotein modification of cytochrome P450 2B4 during its oxidative inactivation in monooxygenase reconstituted system

The mechanism of the cytochrome P450 2B4 modification by hydrogen peroxide (H2O2) formed as a result of partial coupling of NADPH-dependent monooxygenase reactions has been studied in the monooxygenase system reconstituted from the highly purified microsomal proteins: cytochrome P450 2B4 (P450) and NADPH-cytochrome P450 reductase in the presence of detergent Emulgen 913. It was found, that H2O2-mediated P450 self-inactivation during benzphetamine oxidation is accompanied by heme degradation and apoenzyme modification. The P450 heme modification involves the heme release from the enzyme under the action of H2O2 formed within P450s active center via the peroxycomplex decay. Additionally, the heme lost is destroyed by H2O2 localized outside of enzyme's active center. The modification of P450 apoenzyme includes protein aggregation that may be due to the change in the physico-chemical properties of the inactivated enzyme. The modified P450 changes the surface charge that is confirmed by the increasing retention time on the DEAE column. Oxidation of amino acid residues (at least cysteine) may lead to the alteration into the protein hydrophobicity. The appearance of the additional ionic and hydrophobic attractions may lead to the increase of the protein aggregation. Hydrogen peroxide can initiate formation of crosslinked P450 dimers, trimers, and even polymers, but the main role in this process plays nonspecific radical reactions. Evidence for the involvement of hydroxyl radical into the P450 crosslinking is carbonyl groups formation.     

Copper-catalyzed protein oxidation and its modulation by carbon dioxide: enhancement of protein radicals in cells

It is well known that hydrogen peroxide (H2O2)-induced copper-catalyzed fragmentation of proteins follows a site-specific oxidative mechanism mediated by hydroxyl radical-like species (i.e. Cu(I)O, Cu(II)/*OH or Cu(III)) that ends in increased carbonyl formation and protein fragmentation. We have found that the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) prevented such processes by trapping human serum albumin (HSA)-centered radicals, in situ and in real time, before they reacted with oxygen. When (bi)carbonate (CO2, H2CO3, HCO3- and CO3(-2)) was added to the reaction mixture, it blocked fragmentation mediated by hydroxyl radical-like species but enhanced DMPO-trappable radical sites in HSA. In the past, this effect would have been explained by oxidation of (bi)carbonate to a carbonate radical anion (CO3*) by a bound hydroxyl radical-like species. We now propose that the CO3* radical is formed by the reduction of HOOCO2- (a complex of H2O2 with CO2) by the protein-Cu(I) complex. CO3* diffuses and produces more DMPO-trappable radical sites but does not fragment HSA. We were also able, for the first time, to detect discrete but highly specific H2O2-induced copper-catalyzed CO3*-mediated induction of DMPO-trappable protein radicals in functioning RAW 264.7 macrophages. We conclude that carbon dioxide modulates H2O2-induced copper-catalyzed oxidative damage to proteins by preventing site-specific fragmentation and enhancing DMPO-trappable protein radicals in functioning cells. The pathophysiological significance of our findings is discussed.     

Oxygen metabolism and the microbicidal activity of macrophages.

Like neutrophils, phagocytizing macrophages undergo a "respiratory burst" in which significant quantities of oxygen are drawn into the cell. The consumed oxygen is not used in oxidative phosphorylation but, rather, in the formation of superoxide anion (O2) and H2O2. These oxygen metabolites and the products of their interaction, in particular hydroxyl radical (OH), have been implicated in the killing of ingested bacteria by neutrophils. Their role in macrophage microbicidal activity has not been fully defined. However, activated macrophages, which mediate increased resistance to infection in vivo, have a markedly increased capacity to generate O2 and H2O2 in vitro when stimulated by phagocytosis or surface perturbation. The enhanced capacity of activated macrophages to generate highly reactive oxygen metabolites during phagocytosis could contribute to the improved microbicidal and tumoricidal activity of these cells.     

铜锌超氧物歧化酶对几种羟自由基产生系统的影响

应用脱氧核糖降解法研究了ＣｕＺｎ－ＳＯＤ对几种·ＯＨ产生系统的作用机理．结果证明：ＳＯＤ对Ｆｅ（３＋）·Ｏ·Ｈ２Ｏ２系统中·ＯＨ的产生有明显的抑制作用,而失活ＳＯＤ或ＢＳＡ对它的抑制作用不大;在Ｆｅ（２＋）·Ｈ２Ｏ２和ＣＵ（２＋）·Ｈ２Ｏ２系统中,ＳＯＤ、失活ＳＯＤ和ＢＡＳ均能抑制·ＯＨ的产生;在Ｆｅ（２＋）·Ｏ系统中,ＳＯＤ对·ＯＨ产生作用不大,而失活ＳＯＤ或ＢＳＡ对它有明显的抑制作用．由此推测ＳＯＤ对·ＯＨ形成可能有三方面的影响：１．对Ｏ的清除作用,阻断Ｈａｂｅｒ－Ｗｅｉｓｓ反应;２．对金属离子的络合作用,降低·ＯＨ的产额;３．促进Ｈ２Ｏ２的积累,加快Ｆｅｎｔｏｎ反应．     

Spin-trapping studies on the reactions of Cr(III) with hydrogen peroxide in the presence of biological reductants: is Cr(III) non-toxic?

Cr(III), which is thought to be relatively non-toxic, was reduced to Cr(II) ion by biological reductants such as L-cysteine and NADH and Cr(II) thus formed could easily react with hydrogen peroxide (H2O2) to yield very reactive active oxygen species, hydroxyl radical (.OH). The formation of hydroxyl radical was detected by water-soluble spin-traps, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). This result indicates that non-toxic Cr(III) compounds have the possibility of causing dangerous effects to living organism in the presence of biological reductants.     

Activation of latent human neutrophil collagenase by reactive oxygen species and serine proteases

The ability of various reactive oxygen species and serine proteases to activate latent collagenase (matrix metalloproteinase-1) purified from human neutrophils was examined. Latent 70-75 kD human neutrophil collagenase (HNC) was efficiently activated by known non-proteolytic activators phenylmercuric chloride (an organomercurial compound) and gold thioglucose (Au(I)-salt). Corresponding degree of activation was achieved by reactive oxygen species including hypochlorous acid (HOCl), hydrogen peroxide (H2O2) and hydroxyl radical generated by hypoxanthine/xanthine oxidase (HX/XAO). The presence of trace amounts of iron and EDTA were necessary and even enhanced H2O2 induced activation of latent HNC. This activation could be abolished by an iron chelator desferrioxamine and a hydroxyl radical scavenger mannitol. HOCl induced activation of latent HNC was not affected by desferrioxamine and mannitol. Thus, these compounds do not inhibit the active/activated form of HNC. Latent HNC could also be activated by trypsin and chymotrypsin but not by plasmin and plasma kallikrein. The ability of mannitol and desferrioxamine to inhibit the H2O2-induced activation of HNC suggests the transition metal dependent Fenton reaction to be responsible for localized and/or site-specific generation of hydroxyl radical/hydroxyl radical -like oxidants to act as the activating oxygen species. Our results support the ability of myeloperoxidase derived HOCl to act as a direct oxidative activator of HNC and further suggest the existence of a new/alternative oxidative activation pathway of HNC involving hydroxyl radical.     

纤维二糖脱氢酶生成羟自由基和还原各种自由基的研究

利用电子顺磁共振（ＥＳＲ）技术和硫代巴比妥酸（ＴＢＡ）反应研究了纤维二糖脱氢酶（ＣＤＨ）生成·ＯＨ和还原各种自由基的能力．以纤维二糖为电子供体时,ＣＤＨ可以生成·ＯＨ．·ＯＨ生成量与ＣＤＨ、Ｆｅ３＋和Ｏ２的浓度有关．加入过氧化氢酶可使·ＯＨ的生成明显减少．ＣＤＨ可以还原自旋加合物［ＰＢＮ－ＯＨ］·、氮氧自由基和天然木素分子中的自由基．结果表明,ＣＤＨ具有生成·ＯＨ和还原各种自由基的能力．对该酶在木质纤维素降解中的作用进行了探讨