Role of chloride ion in hydroxyl radical production in photosystem II under heat stress: Electron paramagnetic resonance spin-trapping study

Hydroxyl radical (HO?) production in photosystem II (PSII) was studied by electron paramagnetic resonance (EPR) spin-trapping technique. It is demonstrated here that the exposure of PSII membranes to heat stress (40 °C) results in HO? formation, as monitored by the formation of EMPO-OH adduct EPR signal. The presence of different exogenous halides significantly suppressed the EMPO-OH adduct EPR signal in PSII membranes under heat stress. The addition of exogenous acetate and blocker of chloride channel suppressed the EMPO-OH adduct EPR signal, whereas the blocker of calcium channel did not affect the EMPO-OH adduct EPR signal. Heat-induced hydrogen peroxide (H?O?) production was studied by amplex red fluorescent assay. The presence of exogenous halides, acetate and chloride blocker showed the suppression of H?O? production in PSII membranes under heat stress. Based on our results, it is proposed that the formation of HO? under heat stress is linked to uncontrolled accessibility of water to the water-splitting manganese complex caused by the release of chloride ion on the electron donor side of PSII. Uncontrolled water accessibility to the water-splitting manganese complex causes the formation of H?O? due to improper water oxidation, which leads to the formation of HO? via the Fenton reaction under heat stress.     

Hydroxyl radical generation by photosystem II

The photogeneration of hydroxyl radicals (OH(*)) in photosystem II (PSII) membranes was studied using EPR spin-trapping spectroscopy. Two kinetically distinguishable phases in the formation of the spin trap-hydroxyl (POBN-OH) adduct EPR signal were observed: the first phase (t(1/2) = 7.5 min) and the second phase (t(1/2) = 30 min). The generation of OH(*) was found to be suppressed in the absence of the Mn-complex, but it was restored after readdition of an artificial electron donor (DPC). Hydroxyl radical generation was also lost in the absence of oxygen, whereas it was stimulated when the oxygen concentration was increased. The production of OH(*) during the first kinetic phase was sensitive to the presence of SOD, whereas catalase and EDTA diminished the production of OH(*) during the second kinetic phase. The POBN-OH adduct EPR signal during the first phase exhibits a similar pH-dependence as the ability to oxidize the non-heme iron, as monitored by the Fe(3+) (g = 8) EPR signal: both EPR signals gradually decreased as the pH value was lowered below pH 6.5 and were absent at pH 5. Sodium formate decreases the production of OH(*) in intact and Mn-deleted PSII membranes. Upon illumination of PSII membranes, both superoxide, as measured by EPR signal from the spin trap-superoxide (EMPO-OOH) adduct, and H(2)O(2), measured colormetrically, were generated. These results indicated that OH(*) is produced on the electron acceptor side of PSII by two different routes, (1) O(2)(*)(-), which is generated by oxygen reduction on the acceptor side of PSII, interacts with a PSII metal center, probably the non-heme iron, to form an iron-peroxide species that is further reduced to OH(*) by an electron from PSII, presumably via Q(A)(-), and (2) O(2)(*)(-) dismutates to form free H(2)O(2) that is then reduced to OH(*) via the Fenton reaction in the presence of metal ions, the most likely being Mn(2+) and Fe(2+) released from photodamaged PSII. The two different routes of OH(*) generation are discussed in the context of photoinhibition.     

Production of reactive oxygen species in chloride- and calcium-depleted photosystem II and their involvement in photoinhibition

Mixed photosystem II (PSII) samples consisting of Cl(-)-depleted and active, or Ca(2+)-depleted and active PSII enriched membrane fragments, respectively, were investigated with respect to their susceptibility to light. In the presence of Cl(-)-depleted PSII, active centers were damaged more severely, most likely caused by a higher amount of reactive oxygen species formed in the nonfunctional centers. Cl(-) depletion led to an increased H(2)O(2) production, which seemed to be responsible for the stimulation of PSII activity loss. To distinguish between direct H(2)O(2) formation by partial water oxidation and indirect H(2)O(2) formation by oxygen reduction involving the prior formation of O(2)(-?), the production of reactive oxygen species was followed by spin trapping EPR spectroscopy. All samples investigated, i.e. PSII with a functional water splitting complex, Ca(2+)- and Cl(-)-depleted PSII, produced upon illumination O(2)(-?) and OH(?) radicals on the acceptor side, while Cl(-)-depleted PSII produced additionally OH(?) radicals originating from H(2)O(2) formed on the donor side of PSII.     

Dark production of reactive oxygen species in photosystem II membrane particles at elevated temperature: EPR spin-trapping study

In our study, EPR spin-trapping technique was employed to study dark production of two reactive oxygen species, hydroxyl radicals (OH) and singlet oxygen (¹O₂), in spinach photosystem II (PSII) membrane particles exposed to elevated temperature (47 °C). Production of OH, evaluated as EMPO-OH adduct EPR signal, was suppressed by the enzymatic removal of hydrogen peroxide and by the addition of iron chelator desferal, whereas externally added hydrogen peroxide enhanced OH production. These observations reveal that OH is presumably produced by metal-mediated reduction of hydrogen peroxide in a Fenton-type reaction. Increase in pH above physiological values significantly stimulated the formation of OH, whereas the presence of chloride and calcium ions had the opposite effect. Based on our results it is proposed that the formation of OH is linked to the thermal disassembly of water-splitting manganese complex on PSII donor side. Singlet oxygen production, followed as the formation of nitroxyl radical TEMPO, was not affected by OH scavengers. This finding indicates that the production of these two species was independent and that the production of ¹O₂ is not closely linked to PSII donor side.     

Production of reactive oxygen species by photosystem II

Photosysthetic cleavage of water molecules to molecular oxygen is a crucial process for all aerobic life on the Earth. Light-driven oxidation of water occurs in photosystem II (PSII) — a pigment-protein complex embedded in the thylakoid membrane of plants, algae and cyanobacteria. Electron transport across the thylakoid membrane terminated by NADPH and ATP formation is inadvertently coupled with the formation of reactive oxygen species (ROS). Reactive oxygen species are mainly produced by photosystem I; however, under certain circumstances, PSII contributes to the overall formation of ROS in the thylakoid membrane. Under limitation of electron transport reaction between both photosystems, photoreduction of molecular oxygen by the reducing side of PSII generates a superoxide anion radical, its dismutation to hydrogen peroxide and the subsequent formation of a hydroxyl radical terminates the overall process of ROS formation on the PSII electron acceptor side. On the PSII electron donor side, partial or complete inhibition of enzymatic activity of the water-splitting manganese complex is coupled with incomplete oxidation of water to hydrogen peroxide. The review points out the mechanistic aspects in the production of ROS on both the electron acceptor and electron donor side of PSII.     

叶绿体光合代谢中活性氧的产生与清除

有氧代谢不可避免产生活性氧(ROS),叶绿体的PSI和PSII反应中心均是ROS产生的主要位点。叶绿体产生的ROS主要有超氧阴离子(O2-)、过氧化氢(H2O2)、羟自由基(.OH)和单线氧(1O2),其中在PSI产生的O2-将进一步产生H2O2和.OH,而1O2产生在PSII。正常生理代谢条件下,叶绿体内抗氧化系统和光能吸收利用的调节保持活性氧产生和消灭的平衡,不会影响植物的正常生理功能。     

Evidence on the Formation of Singlet Oxygen in the Donor Side Photoinhibition of Photosystem II: EPR Spin-Trapping Study

When photosystem II (PSII) is exposed to excess light, singlet oxygen (¹O₂) formed by the interaction of molecular oxygen with triplet chlorophyll. Triplet chlorophyll is formed by the charge recombination of triplet radical pair ³[P680^•+Pheo^•−] in the acceptor-side photoinhibition of PSII. Here, we provide evidence on the formation of ¹O₂ in the donor side photoinhibition of PSII. Light-induced ¹O₂ production in Tris-treated PSII membranes was studied by electron paramagnetic resonance (EPR) spin-trapping spectroscopy, as monitored by TEMPONE EPR signal. Light-induced formation of carbon-centered radicals (R^•) was observed by POBN-R adduct EPR signal. Increased oxidation of organic molecules at high pH enhanced the formation of TEMPONE and POBN-R adduct EPR signals in Tris-treated PSII membranes. Interestingly, the scavenging of R^• by propyl gallate significantly suppressed ¹O₂. Based on our results, it is concluded that ¹O₂ formation correlates with R^• formation on the donor side of PSII due to oxidation of organic molecules (lipids and proteins) by long-lived P680^•+/TyrZ^•. It is proposed here that the Russell mechanism for the recombination of two peroxyl radicals formed by the interaction of R^• with molecular oxygen is a plausible mechanism for ¹O₂ formation in the donor side photoinhibition of PSII.     

Photoproduction of catalase-insensitive peroxides on the donor side of manganese-depleted photosystem II: evidence with a specific fluorescent probe

The photoproduction of organic peroxides (ROOH) in photosystem II (PSII) membranes was studied using the fluorescent probe Spy-HP. Two types of peroxide, highly lipophilic ones and relatively hydrophilic ones, were distinguished by the rate of reaction with Spy-HP; the former oxidized Spy-HP to the higher fluorescent form Spy-HPOx within 5 min, while the latter did so very slowly (the reaction was still not completed after 180 min). The level of photoproduction of these peroxides was significantly larger in the alkaline-treated, Mn-depleted PSII membranes than that in the untreated membranes, and it was suppressed by an artificial electron donor (diphenylcarbazide or ferrocyanide) and by the electron transport inhibitor diuron. Postillumination addition of Fe(2+) ions, which degrade peroxides by the Fenton mechanism, abolished the accumulation of Spy-HPOx, but catalase did not change the peroxide level, indicating that the detected species were organic peroxides, excluding H(2)O(2). These results agreed with our previous observation of an electron transport-dependent O(2) consumption on the PSII donor side and indicated that ROOH accumulated via a radical chain reaction that started with the formation of organic radicals on the donor side. Illumination (λ > 600 nm; 1500 μmol of photons m(-2) s(-1)) of the Mn-depleted PSII membranes for 3 min resulted in the formation of nearly 200 molecules of hydrophilic ROOH per reaction center, but only four molecules of highly lipophilic ROOH. The limited formation of the latter was due to the limited supply of its precursor to the reaction, suggesting that it represented structurally fixed peroxides, i.e., either protein peroxides or peroxides of the lipids tightly bound to the core complex. These ROOH forms, likely including several species derived from lipid peroxides, may mediate the donor side-induced photoinhibition of PSII via protein modification.     

Competitive binding of acetate and chloride in photosystem II.

The binding of chloride and acetate to photosystem II (PSII) was examined to elucidate the mechanism of acetate inhibition. The mode of inhibition was studied, and individual binding sites were assigned by steady-state O2 evolution measurements in correlation with electron paramagnetic resonance (EPR) results. Two binding sites were found for acetate, one chloride-sensitive on the electron donor side and one chloride-insensitive on the electron acceptor side. The respective binding constants were as follows: KCl = 0.5 +/- 0.2 mM (chloride binding to the donor side), KI = 16 +/- 5 mM (acetate binding to the donor side), and KI' = 130 +/- 40 mM (acetate binding to the acceptor side). When acetate was bound to the acceptor side of PSII, 200 K illumination induced a narrowed form of the QA-FeII EPR signal, the yield of which was independent of the chloride concentration. When acetate was bound to the donor side, room-temperature illumination produced the S2YZ* state. EPR measurements showed that both the yield and formation rate of this state increased with acetate concentration. Increasing chloride concentrations slowed the rate of formation of the S2YZ* state, but did not affect the steady-state yield of the S2YZ* state. These findings indicate that the light-induced reactions in acetate-inhibited PSII are modulated by both donor side and acceptor side binding of acetate, while the steady-state yield of the S2YZ* state at the high PSII concentrations used for EPR measurements depends primarily on acceptor side turnover. Our data further support a close proximity of chloride to YZ*, indicating a possible role for chloride in the electron-transfer mechanism at the O2-evolving complex.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr(Z)) and side-path electron donors (Car/Chl(Z)/Cyt(b)(559)) can be oxidized by P(680)(+) at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S(1)Tyr(Z) EPR signal were independent of the treatment of K(3)Fe(CN)(6), whereas formation and decay of the Car(+)/Chl(Z)(+) EPR signal correlated with the reduction and recovery of the Fe(3+) EPR signal of the non-heme iron in K(3)Fe(CN)(6) pre-treated PSII, respectively. Based on the observed correlation between Car/Chl(Z) oxidation and Fe(3+) reduction, the oxidation of non-heme iron by K(3)Fe(CN)(6) at 0 degrees C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe(3+) EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr(Z) oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr(Z) oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K(3)Fe(CN)(6) takes place only in inactive PSII, which implies that the Fe(3+) state is probably not the intermediate species for the turnover of quinone reduction.     

Generation of oxygen free radicals in thyroid cells and inhibition of thyroid peroxidase

We examined whether superoxide (O(2)(-)) is produced as a precursor of hydrogen peroxide (H(2)O(2)) in cultured thyroid cells using the cytochrome c method and the electron paramagnetic resonance (EPR) method. No O(2)(-) or its related radicals was detected in thyroid cells under the physiological condition. The presence of quinone, 2,3-dimethoxy-l-naphthoquinone (DMNQ), or 2-methyl-1, 4-naphthoquinone (menadione), in the medium produced O(2)(-) and hydroxyl radicals (OH*); the amount of H(2)O(2) generation was also increased. Incubation of follicles with DMNQ or menadione inhibited iodine organification (a step of thyroid hormone formation) and its catalytic enzyme, thyroid peroxidase (TPO). This inhibition should be caused by reactive oxygen species because the two quinones, particularly DMNQ, exert their effect through the generation of reactive oxygen species. It is speculated that the site-specific inactivation of TPO might have occurred at the heme-linked histidine residue of the TPO molecule, a critical amino acid for enzyme activity because OH* (vicious free radicals) can be formed at the iron-linked amino acid. TPO mRNA level and electrophoretic mobility of TPO were not inhibited by quinones. Our study suggests that thyroid H(2)O(2) is produced by divalent reduction of oxygen without O(2)(-) generation. If thyroid cells happen to be exposed to significant amount of reactive oxygen species, TPO and subsequent thyroid hormone formation are inhibited.     

Exposure of nuclear medicine patients to ionizing radiation is associated with rises in HPRT- mutant frequency in peripheral T-lymphocytes

When Chinese hamster fibroblasts were exposed to hydrogen peroxide or to a system consisting of xanthine oxidase and hypoxanthine, which generates superoxide anion plus hydrogen peroxide, sister-chromatid exchanges (SCEs) were formed in a dose-dependent manner. When the iron-complexing agent o-phenanthroline was present in the medium, however, the production of these SCEs was completely inhibited. This fact indicates that the Fenton reaction: Fe2+ + H2O2----OH0 + OH- + Fe3+ is responsible for the production of SCEs. When O2- and H2O2 were generated inside the cell by incubation with menadione, the production of SCE was prevented by co-incubation with copper diisopropylsalicylate, a superoxide dismutase mimetic agent. The most likely role of O2- is as a reducing agent of Fe3+: O2- + Fe3+----Fe2+ + O2, so that the sum of this and the Fenton reaction, i.e., the iron-catalyzed Haber-Weiss reaction, provides an explanation for the active oxygen species-induced SCE: H2O2 + O2(-)----OH- + OH0 + O2. According to this view, the OH radical thus produced is the agent which ultimately causes SCE. These results are discussed in comparison with other mechanisms previously proposed for induction of SCE by active oxygen species.     

The involvement of hydroxyl radical derived from hydrogen peroxide in lignin degradation by the white rot fungus Phanerochaete chrysosporium

The possible involvement of hydrogen peroxide (H2O2)-derived hydroxyl radical (.OH) in lignin degradation ([14C]lignin leads to 14CO2) by Phanerochaete chrysosporium was investigated. When P. chrysosporium was grown in low nitrogen medium (2.4 mM N), an increase in the specific activity for H2O2 production in cell extracts was observed to coincide with the appearance of ligninolytic activity and both activities appeared after the culture entered stationary phase. The production of .OH in ligninolytic cultures of P. chrysosporium was demonstrated by alpha-keto-gamma-methiolbutyric acid-dependent formation of ethylene. Hydrogen peroxide-dependent .OH formation was also shown in cell extracts of ligninolytic cultures. The radical species was demonstrated to be .OH by the .OH-dependent hydroxylation of p-hydroxybenzoic acid to form protocatechuic acid and by using 5,5-dimethyl-1-pyrroline-N-oxide and detecting the production of the nitroxide radical of 5,5-dimethyl-1-pyrroline-N-oxide by EPR. These reactions were inhibited by .OH-scavenging agents and were stimulated when azide was added to inhibit endogenous catalase. Lignin degradation by P. chrysosporium was markedly suppressed in the presence of the .OH-scavenging agents mannitol, benzoate, and the nonspecific radical scavenging agent butylated hydroxytoluene. The above results indicate that .OH derived from H2O2 is involved in lignin biodegradation by P. chrysosporium.     

Spectroscopic characterization of triplet forming states in photosystem II.

Fluorescence and electron paramagnetic resonance (EPR) measurements have been applied to characterize chlorophyll triplet formation in the reaction center of photosystem II (PSII). A highly triplet forming state was generated in PSII membranes by chemical double reduction of the primary electron acceptor QA. In triplet forming PSII centers, the steady-state yield of chlorophyll fluorescence decreased to about 70% of the maximal fluorescence yield observed in closed PSII centers in which QA is singly reduced. The results are well interpreted in the framework of a model where the charge state of QA electrostatically controls the yield of primary charge separation [Schatz, G. H., Brock, H., & Holzwarth, A. R. (1988) Biophys. J. 54, 397-405]. Thus, high triplet yield and decreased, although still quite high, fluorescence indicate a charge-neutralized state of PSII in which QA is singly or doubly reduced and protonated or absent. The EPR signal of the triplet primary chlorophyll donor, 3P680, is suppressed by illumination at 77 K concomitant with the formation of a cationic radical (g = 2.0025-2.0027, and 0.92 mT wide) that is stable in the dark. This is attributed to the oxidation of an accessory chlorophyll (Chl) in the vicinity of P680. Electrostatic repulsion between Chl+ and P680+ is likely to prevent primary charge separation, and in turn triplet formation, providing a further example of electrostatic control of primary charge separation. The triplet P680 EPR signal is also suppressed in the presence of oxygen. This effect, which is almost completely reversible by removing the oxygen, is attributed to the interaction of triplet P680 with triplet O2.     

Chemical and biochemical aspects of superoxide radicals and related species of activated oxygen

The spectrum of biological processes in which oxygen is used by living systems is quite large, and the products include some damaging species of activated oxygen, particularly the superoxide radical (O-.2) and hydrogen peroxide (H2O2). Superoxide radicals and hydrogen peroxide, in turn, can lead to the formation of other damaging species: hydroxyl radicals (.OH) and singlet oxygen (1O2). Hydroxyl radicals react with organic compounds to give secondary free radicals that, in the presence of oxygen, yield peroxy radicals, peroxides, and hydroperoxides. Formation, interconversion, and reactivity of O-.2 and related activated oxygen species, methods available for their detection, and the basis of their biological toxicity are briefly reviewed.     

Molecular mechanisms of production and scavenging of reactive oxygen species by photosystem II

Photosystem II (PSII) is a multisubunit protein complex in cyanobacteria, algae and plants that use light energy for oxidation of water and reduction of plastoquinone. The conversion of excitation energy absorbed by chlorophylls into the energy of separated charges and subsequent water-plastoquinone oxidoreductase activity are inadvertently coupled with the formation of reactive oxygen species (ROS). Singlet oxygen is generated by the excitation energy transfer from triplet chlorophyll formed by the intersystem crossing from singlet chlorophyll and the charge recombination of separated charges in the PSII antenna complex and reaction center of PSII, respectively. Apart to the energy transfer, the electron transport associated with the reduction of plastoquinone and the oxidation of water is linked to the formation of superoxide anion radical, hydrogen peroxide and hydroxyl radical. To protect PSII pigments, proteins and lipids against the oxidative damage, PSII evolved a highly efficient antioxidant defense system comprising either a non-enzymatic (prenyllipids such as carotenoids and prenylquinols) or an enzymatic (superoxide dismutase and catalase) scavengers. It is pointed out here that both the formation and the scavenging of ROS are controlled by the energy level and the redox potential of the excitation energy transfer and the electron transport carries, respectively. The review is focused on the mechanistic aspects of ROS production and scavenging by PSII. This article is part of a Special Issue entitled: Photosystem II.     

Damage to the oxygen-evolving complex by superoxide anion, hydrogen peroxide, and hydroxyl radical in photoinhibition of photosystem II

Under strong illumination of a photosystem II (PSII) membrane, endogenous superoxide anion, hydrogen peroxide, and hydroxyl radical were successively produced. These compounds then cooperatively resulted in a release of manganese from the oxygen-evolving complex (OEC) and an inhibition of oxygen evolution activity. The OEC inactivation was initiated by an acceptor-side generated superoxide anion, and hydrogen peroxide was most probably responsible for the transportation of reactive oxygen species (ROS) across the PSII membrane from the acceptor-side to the donor-side. Besides ROS being generated in the acceptor-side induced manganese loss; there may also be a ROS-independent manganese loss in the OEC of PSII. Both superoxide anion and hydroxyl radical located inside the PSII membrane were directly identified by a spin trapping-electron spin resonance (ESR) method in combination with a lipophilic spin trap, 5-(diethoxyphosphoryl)-5-phenethyl-1-pyrroline N-oxide (DEPPEPO). The endogenous hydrogen peroxide production was examined by oxidation of thiobenzamide.     

Quantitative assessment of intrinsic carbonic anhydrase activity and the capacity for bicarbonate oxidation in photosystem II

On the basis of equilibrium isotopic distribution experiments using (18)O-labeled water, it is generally accepted that water is the sole substrate for O(2) production by photosystem II (PSII). Nevertheless, recent studies indicating a direct interaction between bicarbonate and the donor side of PSII have been used to hypothesize that bicarbonate may have been a physiologically important substrate for O(2) production during the evolution of PSII [Dismukes, G. C., Klimov, V. V., Baranov, S. V., Kozlov, Y. N., DasGupta, J., and Tyryshikin, A. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2170-2175]. To test out this hypothesis and to determine whether contemporary oxygenic organisms have the capacity to oxidize bicarbonate, we employed special rapid-mixing isotopic experiments using (18)O/(13)C-labeled bicarbonate to quantify the inherent carbonic anhydrase activity in PSII samples and the potential flux of oxygen from bicarbonate into the photosynthetically produced O(2). The measurements were made on PSII samples prepared from spinach, Thermosynechococcus elongatus, and Arthrospira maxima. For the latter organism, a strain was used that grows naturally in an alkaline, high (bi)carbonate soda lake in Africa. The results reveal that bicarbonate is not the substrate for O(2) production in these contemporary oxygenic photoautotrophs when assayed under single turnover conditions.     

Metal ions and oxygen radical reactions in human inflammatory joint disease

Activated phagocytic cells produce superoxide (O2-) and hydrogen peroxide (H2O2); their production is important in bacterial killing by neutrophils and has been implicated in tissue damage by activated phagocytes. H2O2 and O2- are poorly reactive in aqueous solution and their damaging actions may be related to formation of more reactive species from them. One such species is hydroxyl radical (OH.), formed from H2O2 in the presence of iron- or copper-ion catalysts. A major determinant of the cytotoxicity of O2- and H2O2 is thus the availability and location of metal-ion catalysts of OH. formation. Hydroxyl radical is an initiator of lipid peroxidation. Iron promoters of OH. production present in vivo include ferritin, and loosely bound iron complexes detectable by the 'bleomycin assay'. The chelating agent Desferal (desferrioxamine B methanesulphonate) prevents iron-dependent formation of OH. and protects against phagocyte-dependent tissue injury in several animal models of human disease. The use of Desferal for human treatment should be approached with caution, because preliminary results upon human rheumatoid patients have revealed side effects. It is proposed that OH. radical is a major damaging agent in the inflamed rheumatoid joint and that its formation is facilitated by the release of iron from transferrin, which can be achieved at the low pH present in the micro-environment created by adherent activated phagocytic cells. It is further proposed that one function of lactoferrin is to protect against iron-dependent radical reactions rather than to act as a catalyst of OH. production.     

Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical.

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.