Measurement of OH radical CT for inactivating Cryptosporidium parvum using photo/ferrioxalate and photo/TiO2 systems

Aim: This study investigates the inactivation of Cryptosporidium parvum using the OH radical and reports the OH radical CT (OH radical concentration × contact time) values for C. parvum inactivation. Methods and Results: Although a wealth of information has demonstrated the efficacy of the microbial inactivation activity of the OH radical, no studies have performed a quantitative estimation of the OH radical for C. parvum inactivation. The CT value of the OH radical required for 2 log C. parvum inactivation was measured with two OH radical‐generating systems, photo/ferrioxalate and photo/TiO₂. The OH radical was approx. 10⁴–10⁷‐fold more effective for microbial inactivation than other popular chemical disinfectants such as ozone, chlorine dioxide and free chlorine. Conclusions: The OH radical appears to be suitable for microbial inactivation with a calculated CT value required for 2 log C. parvum inactivation of 9·3 × 10^?5 mg min l^?1. Significance and Impact of the Study: This study is the first report of an investigation on the role of the OH radical in the photo/ferrioxalate and photo/TiO₂ systems and on the OH radical CT required for C. parvum inactivation.     

NADH-stimulated, cyanide-resistant superoxide production in maize coleoptiles analyzed with a tetrazolium-based assay

 Using the tetrazolium salt XTT (Na,3′-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) as a sensitive and physiologically compatible probe for the determination of superoxide (O₂ ^·−) production in vivo, we have shown that maize (Zea mays L.) coleoptiles possess the capacity of generating O₂ ^·− in the apoplastic space. Our results are in agreement with the notion that this activity is localized at the plasma membrane and can be attributed to an O₂ ^·−-synthesizing enzyme with catalytic and kinetic properties similar to that of the NADPH oxidase of mammalian phagocytes, with the important exception that it utilizes NADH instead of NADPH as electron donor. When applied to the apoplastic space, NADH strongly increased the O₂ ^·−-producing activity of coleoptiles. The maize NADH-dependent O₂ ^·−-synthase activity could clearly be differentiated from peroxidase-mediated O₂ ^·−-synthesizing activity by its insensitivity to cyanide and azide, as well as by its much higher affinity to O₂. Formation of O₂ ^·−, and concomitantly appearing H₂O₂, was preferentially localized in the outer epidermis of the coleoptile. The physiological significance of O₂ ^·− and H₂O₂ production in relation to the growth-controlling function of the epidermal cell wall is discussed. Received: 14 January 2000 / Accepted: 2 May 2000     

Cholic acid changes defense response to oxidative stress in soybean induced by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The oxidative stress and antioxidant systems in soybean leaves and roots infected with plant pathogen Aspergillus niger were studied following treatment with different concentrations of cholic acid. Several oxidative stress parameters were analyzed: production of superoxide (O₂ ^·−) and hydroxyl radicals (^·OH), lipid peroxidation (LP), and superoxide dismutase (SOD; EC 1.15.1.1) activity, as well as the content of reduced glutathione (GSH). Results showed that inoculation with A. niger led to the increase of O₂ ^·− production and GSH quantities in leaves and ^·OH in roots. The highest activity of SOD occured in infected plants treated with cholic acid in concentrations of 40 and 60 mg L⁻¹ which ultimately led to a decrease in O₂ ^·− production. Inoculation with Aspergillus in combination with elevated cholic acid concentrations also increased ^·OH production which is correlated with increased LP. These results may support the idea of using cholic acid as an elicitor to trigger hypersensitive response in plant cells. Use of cholic acid may also actively contribute to soybean plants defense response against pathogen attack.     

Different Inactivation Behaviors of MS-2 Phage and Escherichia coli in TiO2 Photocatalytic Disinfection

Despite a wealth of experimental evidence concerning the efficacy of the biocidal action associated with the TiO₂ photocatalytic reaction, our understanding of the photochemical mechanism of this particular biocidal action remains largely unclear. It is generally accepted that the hydroxyl radical (·OH), which is generated on the surface of UV-illuminated TiO₂, plays the main role. However, our understanding of the exact mode of action of the hydroxyl radical in killing microorganisms is far from complete, and some studies report that other reactive oxygen species (ROS) (H₂O₂ and O₂·⁻, etc.) also play significant roles. In particular, whether hydroxyl radicals remain bound to the surface or diffuse into the solution bulk is under active debate. In order to examine the exact mode of action of ROS in inactivating the microorganism, we tested and compared the levels of photocatalytic inactivation of MS-2 phage and Escherichia coli as representative species of viruses and bacteria, respectively. To compare photocatalytic microbial inactivation with the photocatalytic chemical degradation reaction, para-chlorobenzoic acid, which rapidly reacts with a hydroxyl radical with a diffusion-limited rate, was used as a probe compound. Two different hydroxyl radical scavengers, tert-butanol and methanol, and an activator of the bulk phase hydroxyl radical generation, Fe²⁺, were used to investigate their effects on the photocatalytic mode of action of the hydroxyl radical in inactivating the microorganism. The results show that the biocidal modes of action of ROS are very different depending on the specific microorganism involved, although the reason for this is not clear. It seems that MS-2 phage is inactivated mainly by the free hydroxyl radical in the solution bulk but that E. coli is inactivated by both the free and the surface-bound hydroxyl radicals. E. coli might also be inactivated by other ROS, such as O₂·⁻ and H₂O₂, according to the present results.     

Factors influencing hydroxylamine inactivation of photosynthetic water oxidation

The kinetics of Mn release during NH₂OH inactivation of the water oxidizing reaction is largely insensitive to the S-state present during addition of NH₂OH. This appears to reflect reduction by NH₂OH of higher S-states to a common more reduced state (S₀ or S_?1) which alone is susceptible to NH₂OH inactivation. Sequences of saturating flashes with dark intervals in the range 0.2–5 s^?1 effectively prevent NH₂OH inactivation and the associated liberation of manganese. This light-induced protection disappears rapidly when the dark interval is longer than about 5 s. Under continuous illumination, protection against NH₂OH inactivation is maximally effective at intensities in the range 10³–10⁴ erg · cm^?2 · s^?1. This behavior differs from that of NH₂OH-induced Mn release, which is strongly inhibited at all intensities greater than 10³ erg · cm^?2 · s^?1. This indicates that two distinct processes are responsible for inactivation of water oxidation at high and low intensities. Higher S-states appear to be immune to the reaction by which NH₂OH liberates manganese, although the overall process of water oxidation is inactivated by NH₂OH in the presence of intense light. The light-induced protection phenomenon is abolished by 50 μM DCMU, but not by high concentrations of carbonyl cyanide m-chlorophenylhydrazone, which accelerates inactivation reactions of the water-splitting enzyme, Y (an ADRY reagent). The latter compound accelerates both inactivation of water oxidation and manganese extraction in the dark.     

Bioenergetics and RNA/DNA ratios in the common carp (Cyprinus carpio ) under hypoxia

Hypoxia caused by eutrophication occurs over large areas in aquatic systems worldwide. Common carp (Cyprinus carpio) exposed to hypoxia (1 mg · O₂ · l⁻¹ and 2 mg · O₂ · l⁻¹) for 1 week showed a significant reduction in feeding rate, respiration rate, faecal production and nitrogenous excretion compared to those maintained at normoxia (7 mg · O₂ · l⁻¹). Fish exposed to hypoxia showed negative scope for growth (SfG), but no significant difference in the specific growth rate was revealed after 1 week in both hypoxic groups. A significant reduction in RNA/DNA ratio was, however, clearly evident in the white muscle of the 1 mg · O₂ · l⁻¹ treatment group, but not in the 2 mg · O₂ · l⁻¹ treatment group. Both specific growth rate and RNA/DNA ratio were significantly reduced when fish were exposed to severe hypoxia (0.5 mg · O₂ · l⁻¹) for 4 weeks. At all levels of hypoxia, growth reduction was accompanied by a significant decrease in RNA/DNA ratio in white muscle. Covariance analysis showed no significant difference between the slope of RNA/DNA ratio and growth rate under normoxic conditions and 0.5 mg · O₂ · l⁻¹ for 4 weeks (F=1.036, P > 0.326), as well as 1.0 mg · O₂ · l⁻¹ and 2.0 mg · O₂ · l⁻¹ for 1 week (F = 0.457, P > 0.5), indicating that the RNA/DNA ratio serves as a biomarker of growth under all oxygen levels, at least under controlled experimental conditions. SfG also appears to be more sensitive than the RNA/DNA ratio in responding to hypoxia in fish. Accepted: 15 September 2000     

The role of interactions between O2, H2O2, .OH,e- and O2- in free radical damage to biological systems

The occurrence of the Haber-Weiss reaction and other interactions between free radicals has been investigated in the effects of mixtures of free radicals on the permeability of resealed erythrocyte ghosts and on the activity of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. The following mixtures were found to induce damage greater than that which could be accounted for by the independent actions of the constituent free radicals: (i) · OH + H₂O₂, and (ii) · OH + H₂O₂ + O₂^?. In contrast, the following mixtures were found to induce less damage than that predicted on the basis of independent actions of constituent free radicals: (i) H₂O₂ + O₂^?, and (ii) oxidizing radicals ( · OH, H₂O₂) + reducing radicals (e^?, H · ). These results suggest a Haber-Weiss-like interaction between H₂O₂ and O₂^?and an interaction between H₂O₂ and · OH to produce a species more potent than either in causing increased permeability. The decrease in damage observed in the simultaneous presence of oxidizing and reducing radicals suggests an antagonistic effect by which each tends to moderate damage by the other. Inactivation of glyceraldehyde-3-phosphate dehydrogenase was found to be more sensitive to radiation than permeability by an order of magnitude, while permeability was more sensitive to the enhancement of damage by oxygen. Comparison of the effectiveness of free radical scavengers in inhibiting the increase in permeability caused by free radicals showed the following order of effectiveness, expressed in terms of percentage protection: formate (90%) > nitrogen (65%) > catalase (60%) > dismutase (32%), and with respect to enzymatic inactivation, nitrogen (100%) > formate (77%) > dismutase (48%) > catalase (44%). The relative rates observed anaerobically and aerobically in the presence and absence of the above scavengers suggest that (at least in the case of radiation damage to the membranes of erythrocyte ghost cells) the “oxygen effect” is due to the interaction of oxygen with e^? and H., producing O₂^? which aggravates damage under conditions which allow consequent Haber-Weiss-like reactions. The further increase in damage when oxygen concentration is raised yet higher is due to the interaction of oxygen with the sites of initial damage.     

NADPH oxidase as the source of ROS produced under waterlogging in roots of mung bean

The objective of this study was to examine the role of NADPH oxidase on superoxide radical production under waterlogging in mung bean (Vigna radiata) cvs. T 44 (tolerant) and Pusa Baisakhi (PB) (susceptible), and wild species Vigna luteola. Two days of waterlogging caused decline in superoxide radical (O₂ ^·−) contents in all the genotypes, however, further waterlogging up to 8 d caused significant increase in O₂ ^·− contents. In control and revived plants O₂ ^·− contents were higher in PB, while under waterlogging stress T 44 and V. luteola showed greater increases in the O₂ ^·− contents. During waterlogging the increase in O₂ ^·− content was found to be due to the diphenylene iodonium chloridesensitive NADPH oxidase (NOX). This was further confirmed by the waterlogging induced increase in NOX activity, which was higher in tolerant genotypes T 44 and V. luteola compared with PB. Gene expression studies showed enhanced expression of NOX in the roots of waterlogged V. luteola and T 44, while little expression was observed in control or treated plants of PB. PCR band products were cloned and sequenced, and partial cDNAs of NOX was obtained. Results suggest that increase in O₂ ^·− content during waterlogging could be due to the induction of membrane linked NOX.     

The development of an isokinetic squat device: reliability and relationship to functional performance

The aims of the present study were: (1) to assess aerobic metabolism in paraplegic (P) athletes (spinal lesion level, T4–L3) by means of peak oxygen uptake (V˙O_2peak) and ventilatory threshold (VT), and (2) to determine the nature of exercise limitation in these athletes by means of cardioventilatory responses at peak exercise. Eight P athletes underwent conventional spirographic measurements and then performed an incremental wheelchair exercise on an adapted treadmill. Ventilatory data were collected every minute using an automated metabolic system: ventilation (l · min⁻¹), oxygen uptake (V˙O₂, l · min⁻¹, ml · min⁻¹ · kg⁻¹), carbon dioxide production (V˙CO₂, ml · min⁻¹), respiratory exchange ratio, breathing frequency and tidal volume. Heart rate (HR, beats · min⁻¹) was collected with the aid of a standard electrocardiogram. V˙O_2peak was determined using conventional criteria. VT was determined by the breakpoint in the V˙CO₂−V˙O₂ relationship, and is expressed as the absolute VT (V˙O₂, ml · min⁻¹ · kg⁻¹) and relative VT (percentage of V˙O_2peak). Spirometric values and cardioventilatory responses at rest and at peak exercise allowed the measurement of ventilatory reserve (VR), heart rate reserve (HRr), heart rate response (HRR), and O₂ pulse (O₂ P). Results showed a V˙O_2peak value of 40.6 (2.5) ml · min⁻¹ · kg⁻¹, an absolute VT detected at 23.1 (1.5) ml · min⁻¹ · kg⁻¹ V˙O₂ and a relative VT at 56.4 (2.2)% V˙O_2peak. HRr [15.8 (3.2) beats · min⁻¹], HRR [48.6 (4.3) beat · l⁻¹], and O₂ P [0.23 (0.02) ml · kg⁻¹ · beat⁻¹] were normal, whereas VR at peak exercise [42.7 (2.4)%] was increased. As wheelchair exercise excluded the use of an able-bodied (AB) control group, we compared our V˙O_2peak and VT results with those for other P subjects and AB controls reported in the literature, and we compared our cardioventilatory responses with those for respiratory and cardiac patients. The low V˙O_2peak values obtained compared with subject values obtained during an arm-crank exercise may be due to a reduced active muscle mass. Absolute VT was somewhat comparable to that of AB subjects, mainly due to the similar muscle mass involved in wheelchair and arm-crank exercise by P and AB subjects, respectively. The increased VR, as reported in patients with chronic heart failure, suggested that P athletes exhibited cardiac limitation at peak exercise, and this contributed to the lower V˙O_2peak measured in these subjects. Accepted: 22 April 1997     

Nitric oxide retards xanthine oxidase-mediated superoxide anion generation in Phalaenopsis flower: an implication of NO in the senescence and oxidative stress regulation

Senescence is a developmentally regulated and highly ordered sequence of events. Senescence leads to abscission of plant organs and eventually leads to death of a plant or part of it. Present study revealed that Phalaenopsis flower undergo senescence due to over activation of O₂ ^·−generating xanthine oxidase (XO), which consequently increases the concentrations of O₂ ^·− leading to enhanced oxidative damage and disturbed cellular redox environment as indicated by increased lipid peroxidation and DHA/AsA + DHA ratio, respectively. While activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and non-specific peroxidase (POD) were enhanced in sepals and petals of old flower, activities of catalase (CAT) and glutathione reductase (GR) were decreased. Exogenous application of nitric oxide (NO) retarded H₂O₂-induced senescence of Phalaenopsis flower by downregulating activity of XO and concentrations of O₂ ^·−, H₂O₂ and malondialdehyde (MDA, an index of lipid peroxidation). Exogenous application of NO also downregulated SOD activity and upregulated antioxidant enzymes involved in the detoxification of H₂O₂ (CAT and APX), and in the regulation of redox couples viz, monodehydroascorbate reductase (MDHAR) and GR, together with the modulation in non-protein thiol status and DHA/AsA + DHA ratio.     

Photocatalytic inactivation rate of phage MS2 in titanium dioxide suspensions containing various ionic species

In the TiO₂ photoreaction system, the coexistence of NO₃ ^–, SO₄ ^2–, PO₄ ^3–, K⁺ or Ca²⁺ each at 10–100 mM decreased the rate constant for phage MS2 inactivation, but Cl^–, Br^– or Na⁺ did not. The inhibitory effects of the ions could be elucidated by the proportional relation found between the rate constants and quantities of the phage on TiO₂ irrespective of the kinds of existing ions.     

DNA strand scission by enzymically generated oxygen radicals

Col E1 DNA suffers strand scission when exposed to xanthine oxidase acting aerobically on xanthine. Strand scission was prevented by low levels of superoxide dismutase or of catalase. Mannitol, benzoate, or histidine, which scavenge OH · but which react with neither O₂^? nor H₂O₂, also prevented strand scission. Replacement of 0.1 mm ethylenediaminetetraacetate by 0.1 mm diethylenetriaminepentaacetate prevented strand scission. Three mechanisms for the production of OH ·, or of a comparably powerful oxidant, by metal-catalyzed interaction of O₂^? with H₂O₂, are proposed.     

Pulse-radiolytic investigations of catechols and catecholamines II. Reactions of Tiron with oxygen radical species

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (°OH), superoxide anions (O₂^?) or a combination of both oxygen radicals. The rate constant with °OH radicals was determined at 1.0·10⁹ M^?1·s^?1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11–16), the rate constant with O₂^? of 1.0·10⁷ M^?1·s^?1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with °OH and O₂^? of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of °OH radicals, leading to the same products with catechol compounds.     

Titanium dioxide induced cell damage: A proposed role of the carboxyl radical

Titanium dioxide (TiO₂) nanoparticles have been shown to be genotoxic to cells exposed to ultraviolet A (UVA) radiation. Using the technique of electron spin resonance (ESR) spin trapping, we have confirmed that the primary damaging species produced on irradiation of TiO₂ nanoparticles is the hydroxyl (OH) radical. We have applied this technique to TiO₂-treated fish and mammalian cells under in vitro conditions and observed the additional formation of carboxyl radical anions (CO₂^?) and superoxide radical anions (O₂^?). This novel finding suggests a hitherto unreported pathway for damage, involving primary generation of OH radicals in the cytoplasm, which react to give CO₂^? radicals. The latter may then react with cellular oxygen to form O₂^? and genotoxic hydrogen peroxide (H₂O₂).     

Reduced performance of male and female athletes at 580 m altitude

This study examined the effect of mild hypobaria (MH) on the peak oxygen consumption (O_2peak) and performance of ten trained male athletes [ (SEM); O_2peak = 72.4 (2.2) ml · kg⁻¹ · min⁻¹] and ten trained female athletes [O_2peak = 60.8 (2.1) ml · kg⁻¹ · min⁻¹]. Subjects performed 5-min maximal work tests on a cycle ergometer within a hypobaric chamber at both normobaria (N, 99.33 kPa) and at MH (92.66 kPa), using a counter-balanced design. MH was equivalent to 580 m altitude. O_2peak at MH decreased significantly compared with N in both men [− 5.9 (0.9)%] and women [− 3.7 (1.0)%]. Performance (total kJ) at MH was also reduced significantly in men [− 3.6 (0.8)%] and women [− 3.8 (1.2)%]. Arterial oxyhaemoglobin saturation (S_aO₂) at O_2peak was significantly lower at MH compared with N in both men [90.1 (0.6)% versus 92.0 (0.6)%] and women [89.7 (3.1)% versus 92.1 (3.0)%]. While S_aO₂ at O_2peak was not different between men and women, it was concluded that relative, rather than absolute, O_2peak may be a more appropriate predictor of exercise-induced hypoxaemia. For men and women, it was calculated that 67–76% of the decrease in O_2peak could be accounted for by a decrease in O₂ delivery, which indicates that reduced O₂ tension at mild altitude (580 m) leads to impairment of exercise performance in a maximal work bout lasting ≈ 5 min. Accepted: 30 July 1996     

Use of Fenton's Reagent for the Degradation of TCE in Aqueous Systems and Soil Slurries

Fenton's reaction is comprised of hydrogen peroxide (H₂O₂) catalyzed by iron, producing the hydroxyl radical (·OH), a strong oxidant. ·OH in turn may react with H₂O₂ and iron and is capable of destroying a wide range of organic contaminants. In this laboratory study, Fenton's reaction was observed in aqueous and soil slurry systems using trichloroethylene (TCE) as the target contaminant, with the goal of maximizing TCE degradation while minimizing H₂O₂ degradation. Fenton's reaction triggers a complex matrix of reactions involving ·OH, H₂O₂, iron, TCE, and soil organics. In soil slurries with a high fraction of organic carbon (f_OC), iron tends to sorb to soil organics and/or particles. In aqueous systems the optimal ratio of H₂O₂:Fe²⁺:TCE to degrade TCE in a timely fashion, minimize costs, and minimize H₂O₂ degradation is 300?mg/L: 25?mg/L: 60?mg/L (19:1:1 molar ratio), while soil slurries with a f_OC up to approximately 1% and a soil:water ratio of 1:5 (weight ratio) require about ten times the amount of H₂O₂, the optimal ratio being 3000?mg/L: 5?mg/L: 60?mg/L (190:0.2:1 molar ratio). TCE degradation rates were observed to decrease in soil slurries with higher f_OC because of competition by soil organic matter, which appears to act as a sink for ·OH. H₂O₂ degradation rates tended to increase in soil slurries with higher f_OC, most likely due to increased demand for ·OH by soil organics, increased available iron and other oxidation processes.     

High-light-induced superoxide anion radical formation in cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach as detected by EPR spectroscopy

The generation of superoxide anion radical (O₂ ^·−) in the cytochrome b ₆ f complex (Cyt b ₆ f) of spinach under high-light illumination was studied using electron paramagnetic resonance spectroscopy. The generation of O₂ ^·− was lost in the absence of molecular oxygen. It was also suppressed in the presence of NaN₃ and could be scavenged by extraneous antioxidants such as ascorbate, β-carotene, and glutathione. The results also indicate that O₂ ^·−, which is produced under high-light illumination of the Cyt b ₆ f from spinach, might be generated from a reaction involing ¹O₂, and the Rieske Fe-S protein could serve as the electron donor in the O₂ ^·− production. The mechanism of photoprotection of the Cyt b ₆ f complex by antioxidants is discussed.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Inactivation of the human CuZn superoxide dismutase during exposure to O-2 and H2O2

Human copper-zinc superoxide dismutase undergoes inactivation when exposed to O₂^? and H₂O₂ generated during the oxidation of acetaldehyde by xanthine oxidase at pH 7.4 and 37° C. In contrast, human manganese superoxide dismutase is not inactivated under the same conditions. Catalase and Mn-superoxide dismutase protect CuZn superoxide dismutase from inactivation. Similar protection is observed with hydroxyl radical (OH.) scavengers, such as formate and mannitol. In contrast, other OH. scavengers such as ethanol and tert-butyl alcohol, have no protective action. The latter results indicate that “free OH.” is not responsible for the inactivation. Furthermore, H₂O₂ generated during the oxidation of glucose by glucose oxidase, i.e., without production of O₂^?, does not induce CuZn superoxide dismutase inactivation. A mechanism accounting for this $\text{O}{}_2{}^{\mathrm{?}}\text{H}{}_2\text{O}{}_2$-dependent inactivation of CuZn superoxide dismutase is proposed.