The uremic solute indoxyl sulfate acts as an antioxidant against superoxide anion radicals under normal-physiological conditions

The effect of the uremic solute indoxyl sulfate (IS) on scavenging superoxide anion radicals () generated from both the xanthine/xanthine oxidase (X/XO) system and activated neutrophils was investigated by electron paramagnetic resonance spectroscopy, combined with 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). The findings show that the presence of normal-physiological serum concentrations of IS (0.1-10 μM) resulted in decreased formation of EMPO-superoxide adduct without affecting XO activity. Furthermore, IS showed scavenging activity against cell-derived generated from activated neutrophils. In addition, IS also eliminated hydroxyl radicals. These findings suggest that IS acts as a novel endogenous antioxidant under normal-physiological conditions.     

Dismutation of superoxide radicals by ceruloplasmin--details of the mechanism

Like superoxide dismutase (SOD), human ceruloplasmin (Cp) scavenges superoxide anion radicals injected into the solution with the aid a high-voltage generator, hydrogen peroxide being the product of reaction. The O2-/H2O2 ratio is close to 2:1. The dismutase activity of Cp is about 1500 times lower than that of Cu, Zn-SOD isolated from human erythrocytes. The dismutation of O2- accomplished by SOD, "free" copper ions, native Cp or partly copper-depleted Cp, is inhibited with equal efficiency by cyanide. All the copper ions of the multicopper catalytic center of Cp are not essentially required for the dismutation of O2-, since the enzyme depleted of all type 2 Cu2+ and partly of type 1 Cu2+ lost none of its dismutase activity. Type 1 copper ions of Cp seem to play the leading role in the one-electron transfer occurring upon dismutation of O2-.     

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.     

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.

     

Role of superoxide anion radicals in the bacterial corrosion of metals

It was found that seven strains of bacteria can cause corrosion damage to aluminum, its alloys, and zinc. With respect to the studied metals, the most active bacteria were Proteus vulgaris 1212 and Pseudomonas aeruginosa 969. Superoxide anion radicals were demonstrated to play a role in the initiation of corrosive damage to aluminum and zinc, while bacterial exometabolites participate in the later stages of this process.     

Role of superoxide anion radicals in the bacterial corrosion of metals

It was found that seven strains of bacteria can cause corrosion damage to aluminum, its alloys, and zinc. With respect to the studied metals, the most active bacteria were Proteus vulgaris 1212 and Pseudomonas aeruginosa 969. Superoxide anion radicals were demonstrated to play a role in the initiation of corrosive damage to aluminum and zinc, while bacterial exometabolites participate in the later stages of this process.     

The mechanism of superoxide anion generation by the interaction of phenylhydrazine with hemoglobin.

The mechanism by which superoxide anion is generated by the interaction of phenylhydrazine with either oxy- or methemoglobin was investigated. Rather than superoxide anion generation resulting from an accelerated autooxidation of oxyhemoglobin, it was found that both oxy- and methemoglobin function as peroxidases toward phenylhydrazine with the resultant oxidation of this compound to phenyldiazine. Generation of phenyldiazine from the oxidation of phenylhydrazine by hemoglobin or by the hydrolysis and subsequent decarboxylation of methyl phenylazoformate (C6H5N=NCOOCH3) resulted in the production of superoxide anion. It is suggested that under certain conditions hemoglobin may function as a drug-metabolizing peroxidase.     

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%.     

Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion

The temperature and pH dependence of the reaction of the superoxide radical anion with ferricytochrome c have been measured using the pulse-radiolysis technique. The temperature dependence of the reaction at low ionic strength yields an activation energy of 31 +/- 5 kJ/mol as compared to 14 +/- 3 kJ/mol for the reaction of CO2.(-) under the same conditions. The pH dependence fits the single pK'a of ferricytochrome c of 9.1. The bimolecular rate constant for the reaction of the superoxide anion with ferricytochrome c at pH 7.8, 21 +/- 2 degrees C, in the presence of 50 mM phosphate and 0.1 mM EDTA is (2.6 +/- 0.1) X 10(5) M-1 s-1. Using this value, 1 unit of superoxide dismutase activity (McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055) is calculated to be 3.6 +/- 0.3 pmol of enzyme if the assay is performed in a total volume of 3.0 ml. Copper ions reduce the yield of the reaction of ferricytochrome c with CO2.(-). The reactivities of native and singly modified 4-carboxy-2,4-dinitrophenyllysine cytochromes c towards the superoxide anion radical are in the order native greater than 4-carboxy-2,4-dinitrophenyllysine 60 greater than lysine 13 greater than lysine 87 greater than lysine 27 greater than lysine 86 greater than lysine 72, indicating that electron transfer takes place at or close to the solvent accessible heme edge. The mechanism of the reaction is discussed in terms of the approach of superoxide anion radicals to the heme edge and the available molecular orbitals of both heme and free radicals.     

Role of superoxide anion radicals in microvascular permeability and leukocyte behaviour

Inflammation is associated with the accumulation and activation of phagocytic cells, such as polymorphonuclear leukocytes, and with the subsequent release and generation of a group of activated oxygen species, some of which are free radicals. These studies were carried out to assess the influence of enzymatically generated free radicals on both microvascular permeability and leukocyte adhesion. The extravasation of fluorescein-labelled dextran mean molecular weight (MW) 150 000 was used to assess microvascular permeability and methodology was developed to measure in vivo leukocyte endothelial interactions. Enzymatically generated superoxide anion radical (O-.2) was associated with an increase in macromolecular extravasation (seen primarily from postcapillary venules) and an increase in leukocyte adhesion. Macromolecular extravasation was found to be dependent on the generation of a hydroxyl radical related interaction while leukocyte adhesion was dependent on the presence of O-.2. It is suggested that the permeability alterations and increased polymorphonuclear leukocyte adhesion seen during inflammation may be partially related to the release of free radicals from inflammatory cells.     

Reactivity of an indolinonic aminoxyl with superoxide anion and hydroxyl radicals

The increasing knowledge on the participation of free radicals in many diverse clinical and pathological conditions, has consequently expanded the search for new and versatile antioxidants aimed at combating oxidative stress. Our interest in this field concerns aromatic indolinonic aminoxyls (nitroxides) which efficiently react with alkoxyl, peroxyl, aminyl, arylthiyl and alkyl radicals to give non-paramagnetic species. This prompted us to test their antioxidant activity on different biological systems exposed to free radical-induced oxidative stress and the results obtained so far have been very promising. However little is known about their behaviour towards superoxide and hydroxyl radicals.

Here, we report on the reactivity of an indolinonic aminoxyl, with the two above mentioned radicals using hypoxanthine/xanthine oxidase and potassium superoxide for generating the former and the Fenton reagent for the latter. Besides performing the deoxyribose assay for studying the reaction of the aminoxyl with hydroxyl radical and monitoring spectral changes of the aminoxyl in the presence of superoxide radical, macroscale reactions were performed in both cases and the products of the reactions isolated and identified. The EPR technique was used in this study to help elucidate the data obtained. The results show that this compound efficiently reacts with both hydroxyl and superoxide radicals and furthermore, it is capable of maintaining iron ions in its oxidized form. The results thus contribute to increasing the knowledge on the reactivity of indolinonic aminoxyls towards free radical species and as a consequence, these compounds and/or other aminoxyl derivatives, may be considered as complementary, and sometimes alternative sources for combating oxidative damage.     

Ceruloplasmin,extracellular-superoxide dismutase,and scavenging of superoxide anion radicals

Ceruloplasmin and extracellular-superoxide dismutase are similar in physical properties. Both are found in extracellular fluids and both are scavengers of the superoxide radical. The relationship between the two proteins was further explored in the present investigation. Ceruloplasmin preparations were found to be commonly contaminated with extracellular-superoxide dismutase. In one preparation, 80% of the superoxide dismutase activity was due to extracellular-superoxide dismutase. Ceruloplasmin, freed from contaminating superoxide dismutase, was found to catalytically dismute the superoxide anion radical with a rate constant of about 1.0 × 10⁴ M⁻ s⁻¹ per copper atom. Under physiological conditions with a low rate of superoxide production, ceruloplasmin preferentially reacts stoichiometrically with the superoxide radical with a rate constant of about 2 × 10⁵ M⁻¹ s⁻¹ per copper atom. Under such conditions, the reaction does not result in hydrogen peroxide formation. From the kinetic data obtained it was calculated that in normal human plasma, extracellular-superoxide dismutase will scavenge about twice as much superoxide as ceruloplasmin. Using immobilized antibodies toward extracellular superoxide dismutase and ceruloplasmin, no antigenic cross-reactivity between the two proteins could be detected.     

The effect of concentration on the antioxidant effectiveness of alpha-tocopherol in lipid peroxidation induced by superoxide free radicals

Egg yolk phosphatidylcholine liposomes were rapidly oxidized in the presence of chelated iron and a superoxide-generating system. alpha-Tocopherol incorporated in the bilayer was oxidized at the same time. No lipid or alpha-tocopherol oxidation occurred in liposomes composed of dimyristoyl phosphatidylcholine. The antioxidant did not inhibit lipid peroxidation until its concentration reached a critical level, which depended on the effectiveness of the oxidative stress. Beyond this level, peroxidation was inhibited completely and, simultaneously, the rate of oxidation of tocopherol was lowered. The results suggest that the antioxidant efficiency of alpha-tocopherol depends on its ability to react mainly with the chain-initiating or chain-propagating lipid radicals. This, in turn, is closely tied to the tocopherol content of the membrane. Ascorbate inhibited the consumption of alpha-tocopherol, possibly by regenerating its reduced form.     

Selective detection of superoxide anion radicals generated from macrophages by using a novel fluorescent probe

Quantitation of superoxide radical (O (2)(-).) production at the site of radical generation remains challenging. A simple method to detect nanomolar to micromolar levels of superoxide radical in aqueous solution has been developed and optimized. This method is based on the efficient trapping of O(2)(-). using a novel fluorescent probe (2-chloro-1,3-dibenzothiazolinecyclohexene), coupled with a spectra character-signaling increase event. A high-specificity and high-sensitivity fluorescent probe was synthesized in-house and used to image O(2)(-). in living cells. Better selectivity for O(2)(-). over competing cellular reactive oxygen species and some biological compounds illustrates the advantages of our method. Under optimal conditions, the linear calibration range for superoxide anion radicals was 5.03 x 10(-9)-3.33 x 10(-6) M. The detection limit was 1.68 x 10(-9) M. Fluorescence images of probe-stained macrophages stimulated with 4beta-phorbol 12-myristate 13-acetate were obtained successfully using a confocal laser scanning microscope.     

Tiron as a spin-trap for superoxide radicals produced by the respiratory chain of submitochondrial particles

Tiron can be used as a spin-trap for O2 radicals generated by the respiratory chain of submitochondrial particles (SMP). Using this sensitive method, it was shown that the O2 (radical) production by the succinate-oxidizing SMP can be reduced by antimycin or 4-nonyl-2-hydroxyquinoline-N-oxide, the effects of both antibiotics being abolished and prevented by cyanide. It is suggested tht the O2 radicals are produced due to autooxidation of ubisemiquinone which is formed as an intermediate upon one-electron oxidation of CoQH2 by cytochrome c1. The effects of antimycin, 2-nonyl-4-hydroxyquinoline-N-oxide and cyanide on the O2 (radical) generation correlate with the effects of these inhibitors on a steady-state concentration of ubisemiquinone predicted by the Mitchell's Q-cycle hypothesis.     

Interactions of superoxide anion with enzyme radicals: kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer

The kinetics of O^·-₂ reaction with semi-oxidized tryptophan radicals in lysozyme, Trp^·(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp^·(Lyz) radicals were formed by Br^·-₂ oxidation of the 3-4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 × 10⁸M^-1s^-1; but drops to 8 × 10⁷M^-1s^-1 or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp^·(Lyz) + Trp^·(Lyz) recombination reaction is about (4-7 × 10⁶M^-1s^-1) at/or below pH 6.2 then drops to 1.3-1.6 × 10⁶M^-1s^-1 at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semioxidized Trp^· residues upon Br^·-₂ reaction. The kinetics of Trp^·(Lyz) with ascorbate, a reducing species rather larger than O^·-₂ have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals-leading to the repair of the semi-oxidized Trp^·(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp^·(Lyz)+O^·-₂ reaction, as is most of the Trp^·(Lyz)+Trp^·(Lyz) reaction. However, the kinetic behavior of Trp^·(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O^·-₂. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O^·-₂ reactivity. To our knowledge, this is the first report on the kinetics of a protein-O^·-₂ reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.