Ascorbate peroxidase–thioredoxin interaction

Proteomics data have suggested ascorbate peroxidase (APX) to be a potential thioredoxin-interacting protein. Using recombinant enzymes, we observed that incubation of pea cytosolic APX with reduced poplar thioredoxins h drastically inactivated the peroxidase. A similar inactivation is induced by reduced glutathione and dithiothreitol, whereas diamide and oxidized glutathione have no effect. Oxygen consumption measurements, modifications of the APX visible spectrum and protection by hydrogen peroxide scavenging enzymes suggest that APX oxidizes thiols leading to the generation of thiyl radicals. These radicals can in turn react with thiyl anions to produce the disulfide radical anions, which are responsible for oxygen reduction and subsequent hydrogen peroxide production. The APX inactivation is not due solely to hydrogen peroxide since fluorimetry indicates that the environment of the APX tryptophan residues is dramatically modified only in the presence of thiol groups. The physiological implications of this interaction are discussed.     

Hypochlorite-induced oxidation of thiols: Formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.     

Hypochlorite-induced oxidation of thiols: formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.     

Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts and Photoreduction of Its Primary Oxidation Product Monodehydroascorbate Radicals in Thylakoids

Ascorbate peroxidase, a key enzyme for the scavenging of hydrogenperoxide in chloroplasts, was found in a thylakoid-bound formin spinach chloroplasts at comparable activity to that in thestroma. The activity of peroxidase was detectable in the thylakoidsonly when prepared by an ascorbate-containing medium, and enrichedin the stroma thylakoids. The thylakoid enzyme was not releasedfrom the membranes by either 2 mM EDTA, 1 M KCl, 2 M NaBr or2 M NaSCN, but was solubilized by detergents. Enzymatic propertiesof the thylakoid-bound ascorbate peroxidase were very similarto those of the stromal ascorbate peroxidase. Thylakoid-bound ascorbate peroxidase could scavenge the hydrogenperoxide either added or photoproduced by the thylakoids. Nophotoreduction of hydrogen peroxide was observed, however, inthe thylakoids whose ascorbate peroxidase was inhibited by KCNand thiol reagents or inactivated by the treatment with ascorbate-depletion.The primary oxidation product of ascorbate in a reaction ofascorbate peroxidase, monodehydroascorbate (MDA) radical, wasphotoreduced in the thylakoids, as detected by the quenchingof chlorophyll fluorescence, disappearance of EPR signals ofthe MDA radicals and the MDA radical-induced oxygen evolution.Thus, ascorbate is photoregenerated in the thylakoids from theMDA radicals produced in a reaction of ascorbate peroxidasefor the scavenging of hydrogen peroxide. (Received March 26, 1992; Accepted April 22, 1992)     

Evidence for free radical formation during horseradish peroxidase-catalyzed N-demethylation of crystal violet.

Crystal violet (gentian violet) can undergo an oxidative metabolism, catalyzed by horseradish peroxidase, resulting in formaldehyde formation. The N-demethylation reaction was strongly inhibited by reduced glutathione. Evidence for the formation of a crystal violet radical during the horseradish peroxidase catalyzed reaction was the detection of thiyl and ascorbate radicals from glutathione and ascorbate, respectively. The concentration of radicals from both compounds was significantly increased in the presence of crystal violet. Oxygen uptake was stimulated when glutathione was present in the system and this oxygen uptake was dependent on the dye and enzyme concentration. Oxygen uptake did not occur when ascorbate, instead of glutathione, was present in the system. However, when glutathione was present, ascorbate totally inhibited the glutathione-stimulated oxygen uptake in the crystal violet/horseradish peroxidase/hydrogen peroxide system. Although a weak ESR spectrum from a crystal violet-derived free radical was detected when the dye reacted with H2O2 and horseradish peroxidase, using the fast flow technique, this spectrum could not be interpreted.     

The roles of thiol-derived radicals in the use of 2',7'-dichlorodihydrofluorescein as a probe for oxidative stress

2',7'-Dichlorodihydrofluorescein (DCFH2) is one of the most widely used probes for detecting intracellular oxidative stress, but requires a catalyst to be oxidized by hydrogen peroxide or superoxide and reacts nonspecifically with oxidizing radicals. Thiyl radicals are produced when many radicals are "repaired" by thiols, but are oxidizing agents and thus potentially capable of oxidizing DCFH2. The aim of this study was to investigate the reactivity of thiol-derived radicals toward DCFH2 and its oxidized, fluorescent form 2',7'-dichlorofluorescein (DCF). Thiyl radicals derived from oxidation of glutathione (GSH) or cysteine (CysSH) oxidized DCFH2 with rate constants at pH 7.4 of approximately 4 or approximately 2x10(7) M(-1) s(-1), respectively. Both the rates of oxidation and the yields of DCF were pH-dependent. Glutathione-derived radicals interacted with DCF, resulting in the formation of DCFH* absorbing at 390 nm and loss of fluorescence; in contrast, cysteine-derived radicals did not cause any depletion of DCF fluorescence. We postulate that the observed apparent difference in reactivity between GS* and CysS* toward DCF is related to the formation of carbon-centered, reducing radicals from base-catalyzed isomerization of GS*. DCF formation from interaction of DCFH2 with GS* was inhibited by oxygen in a concentration-dependent manner over the physiological range. These data indicate that in applying DCFH2 to measure oxidizing radicals in biological systems, we have to consider not only the initial competition between thiols and DCFH2 for the oxidizing radicals, but also subsequent reactions of thiol-derived radicals, together with variables--including pH and oxygen concentration--which control thiyl radical chemistry.     

Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase

Guaiacol peroxidase from spinach catalyzes the oxidation of p-aminophenol to produce the aminophenoxy radical as the primary product which is converted further into a stable oxidation product with an absorption peak at 470 nm. The p-aminophenol radicals oxidize ascorbate (AsA) to produce monodehydroascorbate radicals. Kinetic analysis indicates that p-aminophenol radicals also oxidize monodehydroascorbate to dehydroascorbate. Incubation of AsA peroxidase from tea leaves and hydrogen peroxide with p-aminophenol, p-cresol, hydroxyurea, or hydroxylamine results in the inactivation of the enzyme. No inactivation of the enzyme was found upon incubation of the enzyme with these compounds either in the absence of hydrogen peroxide or with the stable oxidized products of these compounds. The enzyme was protected from inactivation by the inclusion of AsA in the incubation mixture. The radicals of p-aminophenol and hydroxyurea were produced by AsA peroxidase as detected by their ESR signals. These signals disappeared upon the addition of AsA, and the signal characteristic of monodehydroascorbate was found. Thus, AsA peroxidase is inactivated by the radicals of p-aminophenol, p-cresol, hydroxyurea, and hydroxylamine which are produced by the peroxidase reaction, and it is protected from inactivation by AsA via the scavenging of the radicals. Thus, these compounds are the suicide inhibitors for AsA peroxidase. Isozyme II of AsA peroxidase, which is localized in chloroplasts, is more sensitive to these compounds than isozyme I. In contrast to AsA peroxidase, guaiacol peroxidase was not affected by these various compounds, even though each was oxidized by it and the corresponding radicals were produced.     

Specific and reversible inactivation of Phycomyces blakesleeanus isocitrate lyase by ascorbate-iron: role of two redox-active cysteines

Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe(2+) system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg(2+), isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe(3+) ions produced in a "site-specific" Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.     

Inactivation Mechanism of Ascorbate Peroxidase at Low Concentrations of Ascorbate; Hydrogen Peroxide Decomposes Compound I of Ascorbate Peroxidase

One of the characteristic properties of ascorbate peroxidase(APX), which distinguishes it from guaiacol peroxidase, Cytc peroxidase and glutathione peroxidase, is the rapid inactivationof the enzyme under conditions where an electron donor is absent.When thylakoid-bound APX (tAPX) in 100 µM ascorbate wasdiluted 500-fold with an ascorbate-depleted medium, the enzymaticactivity was lost with half time of about 15 s. The inactivationof tAPX was suppressed under anaerobic conditions and also bythe addition of catalase, but it was unaffected by the additionof superoxide dismutase. These observations suggest that hydrogenperoxide at nanomolar levels, produced by autooxidation of ascorbateat lower than micromolar levels, might participate in the inactivationof tAPX. The participation of hydrogen peroxide was confirmedby the inactivation of tAPX upon incubation with hydrogen peroxideunder anaerobic conditions. In the absence of ascorbate, theheme of the two-electron-oxidized intermediate of tAPX (designatedCompound I) is decomposed by hydrogen peroxide. Thus, the instabilityof Compound I to hydrogen peroxide is responsible for the inactivationof APX when ascorbate is not available for Compound I and theenzyme cannot turnover. (Received October 16, 1995; Accepted February 21, 1996)     

Myeloperoxidase-catalyzed redox-cycling of phenol promotes lipid peroxidation and thiol oxidation in HL-60 cells

Various types of cancer occur in peroxidase-rich target tissues of animals exposed to aryl alcohols and amines. Unlike biotransformation by cytochrome P450 enzymes, peroxidases activate most substrates by one-electron oxidation via radical intermediates. This work analyzed the peroxidase-dependent formation of phenoxyl radicals in HL-60 cells and its contribution to cytotoxicity and genotoxicity. The results showed that myeloperoxidase-catalyzed redox cycling of phenol in HL-60 cells led to intracellular formation of glutathionyl radicals detected as GS-DMPO nitrone. Formation of thiyl radicals was accompanied by rapid oxidation of glutathione and protein-thiols. Analysis of protein sulfhydryls by SDS-PAGE revealed a significant oxidation of protein SH-groups in HL-60 cells incubated in the presence of phenol/H2O2 that was inhibited by cyanide and azide. Additionally, cyanide- and azide-sensitive generation of EPR-detectable ascorbate radicals was observed during incubation of HL-60 cell homogenates in the presence of ascorbate and H2O2. Oxidation of thiols required addition of H2O2 and was inhibited by pretreatment of cells with the inhibitor of heme synthesis, succinylacetone. Radical-driven oxidation of thiols was accompanied by a trend toward increased content of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the DNA of HL-60 cells. Membrane phospholipids were also sensitive to radical-driven oxidation as evidenced by a sensitive fluorescence HPLC-assay based on metabolic labeling of phospholipids with oxidation-sensitive cis-parinaric acid. Phenol enhanced H2O2-dependent oxidation of all classes of phospholipids including cardiolipin, but did not oxidize parinaric acid-labeled lipids without addition of H2O2. Induction of a significant hypodiploid cell population, an indication of apoptosis, was detected after exposure to H2O2 and was slightly but consistently and significantly higher after exposure to H2O2/phenol. The clonogenicity of HL-60 cells decreased to the same extent after exposure to H2O2 or H2O2/phenol. Treatment of HL-60 cells with either H2O2 or H2O2/phenol at concentrations adequate for lipid peroxidation did not cause a detectable increase in chromosomal breaks. Detection of thiyl radicals as well as rapid oxidation of thiols and phospholipids in viable HL-60 cells provide strong evidence for redox cycling of phenol in this bone marrow-derived cell line.     

4-Mercaptoimidazoles derived from the naturally occurring antioxidant ovothiols 2. Computational and experimental approach of the radical scavenging mechanism

The radical-scavenging mechanism of fourteen 4-mercaptoimidazoles, derived from the natural family of ovothiols, was studied via a QSAR approach, cyclic voltammetry, ESR and NMR spectroscopy. A significant correlation was found between the DPPH scavenging abilities of test compounds and thermodynamic parameters like overall ease of disulphide formation. The production of a disulphide compound via thiyl radical formation is proposed. Upon DPPH scavenging, hydrogen abstraction from thiols yields transient short-lived thiyl radicals, which were characterised by ESR and rapidly dimerise to form a disulphide compound. Cyclic voltammetry showed that the best DPPH scavengers exhibit low oxidation potentials for their oxidation to disulphides.     

4-Mercaptoimidazoles derived from the naturally occurring antioxidant ovothiols 2. Computational and experimental approach of the radical scavenging mechanism

The radical-scavenging mechanism of fourteen 4-mercaptoimidazoles, derived from the natural family of ovothiols, was studied via a QSAR approach, cyclic voltammetry, ESR and NMR spectroscopy. A significant correlation was found between the DPPH scavenging abilities of test compounds and thermodynamic parameters like overall ease of disulphide formation. The production of a disulphide compound via thiyl radical formation is proposed. Upon DPPH scavenging, hydrogen abstraction from thiols yields transient short-lived thiyl radicals, which were characterised by ESR and rapidly dimerise to form a disulphide compound. Cyclic voltammetry showed that the best DPPH scavengers exhibit low oxidation potentials for their oxidation to disulphides.     

Glutathione oxidation during peroxidase catalysed drug metabolism

The peroxidase catalyzed oxidation of certain drugs in the presence of glutathione (GSH) resulted in extensive oxidation to oxidized glutathione (GSSG). Extensive oxygen uptake ensued and thiyl radicals could be trapped. Only catalytic amounts of drugs were required indicating a redox cycling mechanism. Active drugs included phenothiazines, aminopyrine, p-phenetidine, acetaminophen and 4-N,N-(CH3)2-aminophenol. Other drugs, including dopamine and alpha-methyl dopa, did not catalyse oxygen uptake, nor were GSSG or thiyl radicals formed. Instead, GSH was depleted by GSH conjugate formation. Drugs of the former group, e.g. acetaminophen, aminopyrine or N,N-(CH3)2-aniline have also been found by other investigators to form GSSG and hydrogen peroxide when added to hepatocytes or when perfused through an isolated liver. Although cytochrome P-450 normally catalyses a two-electron oxidation of drugs, serious consideration should be given for some one-electron oxidation resulting in radical formation, oxygen activation and GSSG formation.     

Inactivation of Ascorbate Peroxidase in Spinach Chloroplasts on Dark Addition of Hydrogen Peroxide: Its Protection by Ascorbate

Dark addition of hydrogen peroxide to intact spinach chloroplastsresulted in the inactivation of ascorbate peroxidase accompaniedby a decrease in ascorbate contents. This was also the casein reconstituted chloroplasts containing ascorbate, NADP⁺, NAD⁺and ferredoxin. The addition of hydrogen peroxide during light,however, showed little effect on ascorbate contents and ascorbateperoxidase activity in either the intact or reconstituted chloroplasts.In contrast to ascorbate peroxidase, the enzymes participatingin the regeneration of ascorbate in chloroplasts (monodehydroascorbatereductase, dehydroascorbate reductase and glutathione reductase)were not affected by the dark addition of hydrogen peroxide.Ascorbate contents increased again by illumination of the chloroplastsafter the dark addition of hydrogen peroxide. These resultsshow that the inactivation of the hydrogen peroxide scavengingsystem on dark addition of hydrogen peroxide [Anderson et al.(1983) Biochim. Biophys. Acta 724: 69, Asada and Badger (1984)Plant & Cell Physiol. 25: 1169] is caused by the loss ofascorbate peroxidase activity. Ascorbate peroxidase activitywas rapidly lost in ascorbate-depleted medium, and protectedby its electron donors, ascorbate, isoascorbate, guaiacol andpyrogallol, but not by GSH, NAD(P)H and ferredoxin. (Received June 14, 1984; Accepted August 15, 1984)     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

Peroxidase-catalyzed oxidation of eugenol: formation of a cytotoxic metabolite(s)

The oxidation of eugenol (4-allyl-2-methoxyphenol) by horseradish peroxidase was studied. Following the initiation of the reaction with hydrogen peroxide, eugenol was oxidized via a one-electron pathway to a phenoxyl radical which subsequently formed a transient, yellow-colored intermediate which was identified as a quinone methide. The eugenol phenoxyl radical was detected using fast-flow electron spin resonance. The radicals and/or quinone methide further reacted to form an insoluble complex polymeric material. The stoichiometry of the disappearance of eugenol versus hydrogen peroxide was approximately 2:1. The addition of glutathione or ascorbate prevented the appearance of the quinone methide and also prevented the disappearance of the parent compound. In the presence of glutathione, a thiyl radical was detected, and increases in oxygen consumption and in the formation of oxidized glutathione were also observed. These results suggested that glutathione reacted with the eugenol phenoxyl radical and reduced it back to the parent compound. Glutathione also reacted directly with the quinone methide resulting in the formation of a eugenol-glutathione conjugate(s). Using 3H-labeled eugenol, extensive covalent binding to protein was observed. Finally, the oxidation products of eugenol/peroxidase were observed to be highly cytotoxic using isolated rat hepatocytes as target cells.     

Ascorbate peroxidase – a hydrogen peroxide-scavenging enzyme in plants

Ascorbate peroxidase is a hydrogen peroxide-scavenging enzyme that is specific to plants and algae and is indispensable to protect chloroplasts and other cell constituents from damage by hydrogen peroxide and hydroxyl radicals produced from it. In this review, first, the participation of ascorbate peroxidase in the scavenging of hydrogen peroxide in chloroplasts is briefly described. Subsequently, the phylogenic distribution of ascorbate peroxidase in relation to other hydrogen peroxide-scavenging peroxidases using glutathione, NADH and cytochrome c is summarized. Chloroplastic and cytosolic isozymes of ascorbate peroxidase have been found, and show some differences in enzymatic properties. The basic properties of ascorbate peroxidases, however, are very different from those of the guaiacol peroxidases so far isolated from plant tissues. Amino acid sequence and other molecular properties indicate that ascorbate peroxidase resembles cytochrome c peroxidase from fungi rather than guaiacol peroxidase from plants, and it is proposed that the plant and yeast hydrogen peroxide-scavenging peroxidases have the same ancestor.     

Reactions of captopril and epicaptopril with transition metal ions and hydroxyl radicals: An EPR spectroscopy study

In the present study, using the technique of EPR spin trapping with DMPO a spin trap, we demonstrated formation of thiyl radicals from thiol-containing angiotensin converting enzyme (ACE) inhibitor captopril (CAP) and from its stereoisomer epicaptopril (EPICAP), a non-ACE inhibitor, in the process of ^.OH radical scavenging. Splitting constants of DMPO/thiyl radical adducts were identical for both thiols and were a_N = 15.3 G, and a_H = 16.2 G. Bimolecular rate constants for the reaction of CAP and EPICAP with ^.OH radicals were close to a diffusion-controlled rate (≈ 2 × 10¹⁰ M⁻¹s⁻¹). Our data also show that both CAP and EPICAP reduce Fe(III) ions and that their respective thiyl radicals are formed in this reaction. In the presence of Fe(III), H₂O₂, and CAP, or EPICAP, ^.OH radicals were produced by a thiol-driven Fenton mechanism. Copper(II) ions were also reduced by these thiols, but no thiyl radicals could be detected in these reactions, and no ^.OH or other Fenton oxidants were observed in the presence of H₂O₂. Our data show direct evidence that thiol groups of CAP and EPICAP are involved in scavenging of ^.OH radicals. The direct ^.OH radical scavenging, together with the reductive “repair” of other sites of ^.OH radical attack, may contribute to the known protective effect of CAP against ischemia/reperfusion-induced arrhythmias. The formation of reactive thiyl radicals in the reactions of the studied compounds with ^.OH radicals and with Fe(III) ions may play a role in some of the known adverse effects of CAP.     

Purification and Molecular Properties of the Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts

The hydrogen peroxide that is photoproduced in thylakoids isscavenged by the thylakoid-bound ascorbate peroxidase (tAPX)[Miyake and Asada (1992) Plant Cell Physiol. 33: 541]. tAPXwas purified from spinach thylakoids to homogeneity as judgedby SDS-polyacrylamide gel electrophoresis, and its molecularproperties were studied. Spinach tAPX was a monomer with a molecularweight of 40,000, which is about 10,000 higher than that ofthe stromal ascorbate peroxidase (sAPX) from spinach chloroplasts.tAPX cross-reacted with the antibody raised against sAPX fromtea leaves, as determined by Western blotting, which also providedevidence for the higher molecular weight of tAPX from spinachthylakoids than that of tea sAPX. The amino acid sequence ofthe amino-terminal region of tAPX showed a low degree of homologyto those of cytosolic APXs from spinach, pea and Arabidopsisthaliana, but a high degree of homology to that of stromal APXfrom tea. Thus, the amino-terminal region of tAPX seems notto be a domain required for binding of the enzyme to the thylakoidmembranes. tAPX contained protoheme IX, as identified by itspyridine hemochromogen, and gave a Soret peak at 403 nm and433 nm with an a band at 555 nm in its oxidized and reducedforms, respectively. Resembling sAPX but differing from cytosolicAPX, tAPX showed high specificity for ascorbate as the electrondonor. tAPX was inhibited by cyanide, thiol-modifying reagents,thiols and several suicide inhibitors, such as hydroxyurea andp-aminophenol. ¹Present address: Beijing Vegetable Research Centre, PO Box2443, Beijing, China.     

An ESR investigation of the reactions of glutathione, cysteine and penicillamine thiyl radicals: competitive formation of RSO., R., RSSR-., and RSS(.)

The reactions of the cysteine, glutathione and penicillamine thiyl radicals with oxygen and their parent thiols in frozen aqueous solutions have been elucidated through electron spin resonance spectroscopy. The major sulfur radicals observed are: (1) thiyl radicals, RS.; (2) disulfide radical anions. RSSR-.; (3) perthiyl radicals, RSS. and upon introduction of oxygen; (4) sulfinyl radicals, RSO., where R represents the remainder of the cysteine, glutathione or penicillamine moiety. The radical product observed depends on the pH, concentration of thiol, and presence or absence of molecular oxygen. We find that the sulfinyl radical is a ubiquitous intermediate in the free radical chemistry of these important biological compounds, and also show that peroxyl radical attack on thiols may lead to sulfinyl radicals. We elaborate the observed reaction sequences that lead to sulfinyl radicals, and, using 17O isotopic substitution studies, demonstrate that the oxygen atom in sulfinyl radicals originates from dissolved molecular oxygen. In addition, the glutathione thiyl radical is found to abstract hydrogen from the alpha-carbon position on the cysteine residue of glutathione to form a carbon-centered radical.