The mechanism of interaction of ceruloplasmin with superoxide radicals

• 1.1. The mechanism of interaction of CP with O₂ radicals in chemical and enzymatic systems of Superoxide radical generation as well as in the pulse radiolysis technique was studied.
• 2.2. It is found that CP does not exert any kinetic influence on the decomposition of Superoxide radical and, unlike SOD, cannot catalyze the reaction of disproportionation of these radicals in systems with chemical and enzymatic generation of O₂⁻.
• 3.3. The data obtained confirm the suggestion that CP interacts with precursors of ₂⁻ radicals.
• 4.4. The irradiation of CP does not change its inhibiting activity in the reaction of the formation of Superoxide radicals in systems with enzymatic O₂⁻ generation, but decreases its oxidase activity.
• 5.5. The results obtained demonstrated that the increase in the radiation dose resulted in the decrease of the inhibiting activity of SOD, whereas the activity of CP did not change.

     

Reduction of iodonitrotetrazolium violet by superoxide radicals

p-Iodonitrotetrazolium (2-(4-iodophenyl-3-(4-nitrophenyl)- 5-phenyltetrazolium; INT) was reduced to a water-soluble product with an absorbance maxima at about 505 nm (reddish pink) by superoxide anion (O2-.) generated by xanthine/xanthine oxidase. The rate of INT reduction was linearly related to the xanthine oxidase activities, and was inhibited by superoxide dismutase. The soluble product may further be converted to an insoluble product, presumably nonionic formazan, with an absorbance maxima of 490 nm (purplish), under certain conditions, and the rate of the formazan formation depended on pH and protein concentration.     

The mechanism of the enhanced antioxidant effects against superoxide anion radicals of reduced water produced by electrolysis

We reported that reduced water produced by electrolysis enhanced the antioxidant effects of proton donors such as ascorbic acid (AsA) in a previous paper. We also demonstrated that reduced water produced by electrolysis of 2 mM NaCl solutions did not show antioxidant effects by itself. We reasoned that the enhancement of antioxidant effects may be due to the increase of the ionic product of water as solvent. The ionic product of water (pKw) was estimated by measurements of pH and by a neutralization titration method. As an indicator of oxidative damage, Reactive Oxygen Species- (ROS) mediated DNA strand breaks were measured by the conversion of supercoiled phiX-174 RF I double-strand DNA to open and linear forms. Reduced water had a tendency to suppress single-strand breakage of DNA induced by reactive oxygen species produced by H2O2/Cu (II) and HQ/Cu (II) systems. The enhancement of superoxide anion radical dismutation activity can be explained by changes in the ionic product of water in the reduced water.     

Generation of superoxide radicals by ischemic heart mitochondria

Tiron (1,2-dihydroxybenzene-3,5-disulfonic acid, disodium salt) was used as a spin trap to detect superoxide radicals produced by rat heart mitochondria. It was shown that ischemia results in the enhancement of the mitochondrial superoxide-forming activity. In the presence of the oxidative phosphorylation uncoupler mesoxalonitrile (3-chlorophenyl)-hydrazone the superoxide production rate in the control mitochondria increases, that in the ischemic mitochondria remains unchanged.     

Quenching of superoxide radicals by green fluorescent protein

Green fluorescent proteins (GFP) are widely used in vivo molecular markers. These proteins are particularly resistant, and maintain function, under a variety of cellular conditions such as pH extremes and elevated temperatures. Green fluorescent proteins are also abundant in several groups of marine invertebrates including reef-forming corals. While molecular oxygen is required for the post-translational maturation of the protein, mature GFPs are found in corals where hyperoxia and reactive oxygen species (ROS) occur due to the photosynthetic activity of algal symbionts. In vitro spin trapping electron paramagnetic resonance and spectrophotometric assays of superoxide dismutase (SOD)-like enzyme activity show that wild type GFP from the hydromedusa, Aequorea victoria, quenches superoxide radicals (O2*-)) and exhibits SOD-like activity by competing with cytochrome c for reaction with O2*-. When exposed to high amounts of O2*- the SOD-like activity and protein structure of GFP are altered without significant changes to the fluorescent properties of the protein. Because of the distribution of fluorescent proteins in both the epithelial and gastrodermal cells of reef-forming corals we propose that GFP, and possibly other fluorescent proteins, can provide supplementary antioxidant protection.     

Dismutation of dihydrofolate by dihydrofolate reductase

Degradation of 7,8-dihydrofolate (H2folate) in the presence of dihydrofolate reductase (DHFR) has been shown due not to an oxygenase activity of the reductase as previously reported but to dismutation of H2folate to folate and 5,6,7,8-tetrahydrofolate (H4folate). The reaction can be followed spectrophotometrically or by analysis of the reaction mixture by high-performance liquid chromatography (HPLC). The products have also been isolated and characterized. Oxygen uptake during the reaction is much less than stoichiometric with H2folate disappearance and is attributed to autoxidation of the H4folate formed. The dismutation activity is a property of highly purified Streptococcus faecium DHFR isoenzyme 2 (but not isoenzyme 1) and of Lactobacillus casei DHFR, but not of bovine liver DHFR. The activity is dependent on tightly bound NADP+ and/or NADPH. Removal of the nucleotide results in loss of dismutation activity, which is restored by adding NADP+ or NADPH. Maximum activity is obtained when approximately 1 mol equiv of nucleotide is added per mol of DHFR. It is proposed that in the dismutation reaction bound NADP(H) is alternately reduced and oxidized by incoming molecules of H2folate with release of folate and H4folate, respectively. The relatively slow rate of folate formation presumably limits the rate of the overall reaction. The equilibrium constant for the dismutation reaction is 19.4 +/- 7.4 at 22 degrees C and pH 7.0. Calculation of standard oxidation-reduction potentials at pH 7 gave values of -0.230 V for the H2folate/H4 folate pair and -0.268 V for the folate/H2folate pair. The mechanism by which NADP+ is retained by the enzyme from some sources during purification procedures is unclear.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of superoxide radicals produced by human activated neutrophils by accumulation of stable nitroxide radicals detected by MR spectroscopy

An important index of neutrophil function is the production of superoxide radicals (O2-) upon activation. Thus a development of a new adequate assay of O2- generation measurement is of great interest for phagocyte researchers. The present article considers the quantitative determination of O2- generation based on the interaction of O2- with 1-oxy-2,2,6,6-tetramethyl-4-oxypiperidine producing 4-oxo-2,2,6,6-piperidine-1-oxyl, detected by ESR. The kinetic curve of nitroxyl radical (NR) formation has a linear character. The NR formation rate after a short induction period (appr. 2 min.) approaches 3.3 X 10(-3) M/s, where cell concentration was 4 X 10(5) per ml. Hydroxylamine (3.8 mM) auto-oxidation rate is negligible as compared with activated neutrophils and is equal to 2 X 10(-9) M/s. Sensitivity NR to the presence of superoxide dismutase (SOD) came as evidence that NR formation is due O2- radicals. SOD (10(-7) M) inhibits NR formation by 90%. Hydroxylamine oxidation by O2- is an irreversible reaction--20-min incubation of activated neutrophils with NR do not influence NR concentration. The NR generation rate dependence upon the neutrophil concentration is linear in the cell concentration range from 4 X 10(5 up to 6 X 10(6) per ml. In this range a quantitative measurement of O2- production is suitable. The sensitivity of hydroxylamine assay is close to the sensitivity of chemiluminescent method, but specificity is higher, as SOD inhibits chemiluminescence only by 50%.     

Detection of superoxide radicals in intact heart mitochondria by spin trapping

Tiron (4,5-dihydroxybenzene-1,3-disulfonic acid, disodium salt) has been used as a spin trap to detect superoxide radicals produced by the rat heart mitochondria. In the presence of the oxidative phosphorylation uncoupler carbonyl cyanide (3-chlorophenyl)-hydrazone the superoxide production rate increases.     

Generation of superoxide anion radicals by the marine phytoplankton organism, Chattonella antiqua

The generation of superoxide anion radical (O₂^–, believedto be a causative factor in the killing of fish by the phytoplanktonChattonella antiqua, has been examined using several methods:electrochemical technique, reduction of ferricytochrome c andfluorescent laser microscope. Evidence is presented to suggestthat these organisms release superoxide continuously while theyare living, even in the resting state. Additional generationof O₂^– accompanies the discharge of mucocysts, and istriggered when they are exposed to mucus from the gill lamellaeof fish. Such instantaneous generation of O₂^– is alsoinduced when the organisms are in contact with an electrodepoised at a potential of +0.1 V versus Ag/AgCI, which is positiveenough to oxidize O₂^– to O₂.     

Salicylate inhibits LDL oxidation initiated by superoxide/nitric oxide radicals

Simultaneously produced superoxide/nitric oxide radicals (O2*-/NO*) could form peroxynitrite (OONO-) which has been found to cause atherogenic, i.e. oxidative modification of LDL. Aromatic hydroxylation and nitration of the aspirin metabolite salicylate by OONO- has been reported. Therefore we tested if salicylate may be able to protect LDL from oxidation by O2*-/NO* by scavenging the OONO reactive decomposition products. When LDL was exposed to simultaneously produced O2*-/NO* using the sydnonimine SIN-1, salicylate exerted an inhibitory effect on LDL oxidation as measured by TBARS and lipid hydroperoxide formation and alteration in electrophoretic mobility of LDL. The cytotoxic effect of SIN-1 pre-oxidised LDL to endothelial cells was also diminished when salicylate was present during SIN-1 treatment of LDL. Spectrophotometric analysis revealed that salicylate was converted to dihydroxybenzoic acid (DHBA) derivatives in the presence of SIN-1. 2,3- and 2,5-DHBA were even more effective to protect LDL from oxidation by O2*-/NO*. Because O2*-/NO* can occur in vivo, the results may indicate that salicylate could act as an efficacious inhibitor of O2*-/NO* initiated atherogenic LDL modification, thus further supporting the rationale of aspirin medication regarding cardiovascular diseases.     

Formation of superoxide radicals in the mitochondria of skeletal muscle

The superoxide-dependent oxidation of adrenaline by skeletal muscle mitochondria (maximal inhibition by superoxide dismutase from 50 to 80%) is described. The oxidation reaction is initiated by antimycin A but not by rotenone. It was assumed that the main source of superoxide radicals in skeletal muscle mitochondria is the site of the respiratory chain between the rotenone and antimycin block. It was found that skeletal muscle mitochondria are characterized by a higher rate of superoxide anion formation and by a lower activity of superoxide dismutase as compared to heart mitochondria.     

Oxygen free radicals mediate the induction of manganese superoxide dismutase gene expression by TNF-alpha

In this study, the hypothesis that oxygen free radicals act as intracellular messengers is examined. Treatment of human oral carcinoma SCC-25 cells with 200 ng/ml human TNF-alpha for 6 h greatly increased manganese superoxide dismutase (MnSOD) gene expression as detected by western blotting, RT-PCR, and nuclear run-on experiments. In the presence of the oxygen free radical spin trapping reagent, 5,5-dimethyl pyrroline-N-oxide (DMPO), the induction of MnSOD gene expression by TNF-alpha was significantly reduced. Electron paramagnetic resonance experiments showed that the production of oxygen free radicals was enhanced in TNF-alpha treated cells. Taken together, these observations suggest that the induction of MnSOD expression by TNF-alpha is at least partially mediated by intracellular formation of oxygen free radicals, and that superoxide is most likely the initiating species involved in the mediation of MnSOD gene expression by TNF-alpha.     

Inactivation of Escherichia coli by superoxide radicals and their dismutation products.

Escherichia coli cells are inactivated by the products of the reaction between dialuric acid and oxygen, of which the primary product is Superoxide. The rate of inactivation is decreased by Superoxide dismutase, by catalase, and by EDTA, whereas it is increased by addition of cupric ions or hydrogen peroxide. It is concluded that a toxic product is formed in a reaction involving Superoxide, hydrogen peroxide, and metal ions, which might be the Haber-Weiss reaction, O₂^? + H₂O₂ → OH + OH^? + O₂. In radiation chemical experiments it is shown that this reaction does not occur in the absence of metal ions.     

The mechanism of superoxide production by the antimycin-inhibited mitochondrial Q-cycle

Superoxide production from antimycin-inhibited complex III in isolated mitochondria first increased to a maximum then decreased as substrate supply was modulated in three different ways. In each case, superoxide production had a similar bell-shaped relationship to the reduction state of cytochrome b(566), suggesting that superoxide production peaks at intermediate Q-reduction state because it comes from a semiquinone in the outer quinone-binding site in complex III (Q(o)). Imposition of a membrane potential changed the relationships between superoxide production and b(566) reduction and between b(562) and b(566) redox states, suggesting that b(562) reduction also affects semiquinone concentration and superoxide production. To assess whether this behavior was consistent with the Q-cycle mechanism of complex III, we generated a kinetic model of the antimycin-inhibited Q(o) site. Using published rate constants (determined without antimycin), with unknown rate constants allowed to vary, the model failed to fit the data. However, when we allowed the rate constant for quinol oxidation to decrease 1000-fold and the rate constant for semiquinone oxidation by b(566) to depend on the b(562) redox state, the model fit the energized and de-energized data well. In such fits, quinol oxidation was much slower than literature values and slowed further when b(566) was reduced, and reduction of b(562) stabilized the semiquinone when b(566) was oxidized. Thus, superoxide production at Q(o) depends on the reduction states of b(566) and b(562) and fits the Q-cycle only if particular rate constants are altered when b oxidation is prevented by antimycin. These mechanisms limit superoxide production and short circuiting of the Q-cycle when electron transfer slows.     

Involvement of superoxide radicals in the mouse two-cell block.

The effect of oxygen toxicity on the development of mammalian embryos was assessed by the use of superoxide dismutase (SOD), a potent scavenger of superoxide radicals. Mouse pronuclear embryos recovered 17 h after human chorionic gonadotropin (hCG) were cultured in medium BWW at 37 degrees C under an atmosphere of 5% CO2 in air. Culture of mouse pronuclear embryos in the presence of Cu.Zn-SOD (500 micrograms/ml) significantly increased the blastulation rate (44.6%) when compared with the control culture system (4.2%). Essentially the same effects were observed in SOD containing either Mn or Fe in the catalytic center. Heat treatment of the SOD preparation, and the addition of anti-SOD antibodies to the culture medium, significantly reduced the attenuation of the two-cell block by SOD, indicating that this effect is SOD dependent. SOD activity was detected in rabbit oviduct fluid (3.675 +/- 3.084 mIU/mg protein) by electron spin resonance. These results suggest that active oxygen is involved in the two-cell block phenomenon in mouse embryos exposed to air and that SOD in the oviduct may play an important role in the protection of embryos from superoxide radicals.     

Oxidation of manganous pyrophosphate by superoxide radicals and illuminated spinach chloroplasts.

The oxidation of Mn²⁺-pyrophosphate to Mn³⁺ by superoxide (O₂^?) was quantitative as evidenced from the formation of Mn³⁺-pyrophosphate and hydrogen peroxide and from the inhibition by superoxide dismutase. Using the competitive relation between Mn²⁺-pyrophosphate and superoxide dismutase for the O₂^?, the rate constant of Mn²⁺ oxidation was estimated to be about 6 × 10⁶m^?1 s^?1. The oxidation of Mn²⁺-pyrophosphate by illuminated chloroplasts was also indicated to be stoichiometrically induced by O₂^?. In the presence of saturating amounts of the Mn²⁺, a double enhancement of hydrogen peroxide production and triple uptake of oxygen were found, as expected from the oxidation of Mn²⁺-pyrophosphate by O₂^?. Anaerobiosis or superoxide dismutase annuled these increments. We propose that the O₂^? generated as the sole initial step of the Mehler reaction oxidized Mn²⁺-pyrophosphate, and we discuss the role of free manganese in chloroplasts.     

The generation of hydroxyl radicals following superoxide production by neutrophil NADPH oxidase

Stimulated neutrophils generate superoxide and hydroxyl radicals. A membrane-bound NADPH oxidase, inactive in the resting state, is responsible for superoxide production. The production of hydroxyl radicals is through a secondary reaction. A Fenton-catalysed Haber—Weiss reaction is proposed. Transferrin was used as the catalyst in this investigation.