Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase.

Accompanying the autoxidation of hydroxylamine at pH 10.2, nitroblue tetrazolium was reduced and nitrite was produced in the presence of EDTA. The rate of autoxidation was negligible below pH 8.0, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, indicating the participation of superoxide anion radical in the autoxidation. Hydrogen peroxide stimulated the autoxidation and superoxide dismutase inhibited the hydrogen peroxide-induced oxidation, results which suggest the participation of hydrogen peroxide in autoxidation and in the generation of superoxide radical. An assay for superoxide dismutase using autoxidation of hydroxylamine is described.     

Evidence against superoxide involvement in tyrosine hydroxylation by mushroom tyrosinase

The possible involvement of superoxide anions in the hydroxylation of tyrosine by mushroom tyrosinase was studied. Superoxide dismutase and scavengers of superoxide ions of smaller MW than superoxide dismutase, such as nitroblue tetrazolium and copper salicylate, had no direct effect on the monohydroxyphenolase activity of mushroom tyrosinase. The kinetics of tyrosine hydroxylation, but not of DOPA oxidation, by mushroom tyrosinase was atrected by the addition of a xanthine-xanthine oxidase system. In the presence of the xanthine-xanthine oxidase system, the lag period of tyrosine hydroxylation was shortened compared to the lag period in the absence of the xanthine-xanthine oxidase system. The xanthine- xanthine oxidase system alone (without mushroom tyrosinase) had no effect on tyrosine conversion to dopachrome. Superoxide dismutase, catalase and hydroxyl radical scavengers counteracted to some extent the shortening of the lag period of tyrosine hydroxylation by mushroom tyrosinase caused by the xanthin e-xanthine oxidase system. It is suggested that the shortening of the lag period is due mainly to hydroxyl radicals generated by the xanthine-xanthine oxidase system via interaction of O₂^?. and hydrogen paroxide (a Haber-Weiss type reaction). The data do not support the direct participation of superoxide anions in tyrosine hydroxylation by mushroom tyrosinase.     

Negative air ions as a source of superoxide

The physico-chemical characteristics and possible formation mechanisms of negative air ions are considered. It was found that the products of oxygen and nitrogen negative ionization reduce ferricytochromec and nitroblue tetrazolium, and that these reactions were inhibited by superoxide dismutase. The interaction of negatively ionized oxygen with water led to hydrogen peroxide accumulation, which was inhibited by tetranitromethane or catalase. Nitrogen ionization under these conditions caused the formation of the hydrated electron e _aq ^— and the superoxide anion O ₂ ^— . The data obtained indicate that the biological activity of negative air ions may be dependent on superoxide. The generation of reactive oxygen ions in the gas phase and also at a gas/water interface is described. A scheme for superoxide production under oxygen and nitrogen ionization is proposed.     

Superoxide dismutase and glutathione peroxidase activities in erythrocytes as indices of oxygen loading in disease: a survey of one hundred cases

It has been established that the pyrogallol autoxidation method for the estimation of the activity of superoxide dismutase (SOD) (EC 1.15.1.1) is superior in precision and sensitivity to a superoxide-generating method (NADH/phenazine methosulfate linked to nitroblue tetrazolium reduction). Reference intervals were established in an urban population in the Far East for SOD activity in erythrocytes using the pyrogallol method, and for glutathione peroxidase (GSH-Px) (EC 1.11.1.9) activity in erythrocytes using a standard glutathione reductase-linked method. On this basis, erythrocyte SOD activities were significantly (P less than 0.05) depressed in cases of visceral cancer, acute myocardial infarct, congestive heart failure, respiratory failure, chronic renal failure, and diabetes mellitus, but within the reference interval in cases of lung cancer and asthma. Erythrocyte GSH-Px activity was significantly (P less than 0.05) depressed in cases of diabetes mellitus and chronic renal failure but elevated in respiratory failure and asthma. GSH-Px and SOD activities were well correlated in patients but not in the reference population.     

Reactivity of antiinflammatory and superoxide dismutase active Cu(II)-salicylates.

The activity of chelated Cu(II) with four different aspirin-like drugs in various superoxide dismutase assays was examined. Prior to these studies the oxidation state of the involved copper was measured by x-ray photoelectron spectrometry and was found to be +II throughout. All copper complexes were able to suppress the xanthine-xanthine oxidase mediated reduction of both cytochrome c and nitroblue tetrazolium as well as the formazan formation by KO2 in a specific manner. The hydroxylation of benzo-[alpha]-pyrene as well as the demethylation of 7-ethoxycoumarin using induced hepatic rat microsomes could be successfully inhibited by the employed Cu(II) chelates. Cu(II)-acetylsalicylate was the most active copper complex. Our findings support the proposal that Cu(II) chelates are the active forms of aspirin-like antiinflammatory agents.     

A comparative study of superoxide dismutase activity assays in Crocus sativus L. corms

Superoxide dismutase catalyzes the breakdown of superoxide radical anion and provides the first line of defense against oxygen toxicity. Its vital importance has made it the subject of numerous investigations. Several assays have been proposed for the detection and quantitation of superoxide dismutase activity, but their use has remained controversial and no comparative studies have been reported. In this investigation, three commonly used methods for the measurement of superoxide dismutase activity were compared to assay the enzyme in Crocus sativus L. corm extract. The methods, based on the competition between the enzyme itself and a superoxide scavenger, involved cytochrome c reduction, nitro blue tetrazolium reduction, and pyrogallol autooxidation, respectively. Because of its accuracy, reproducibility, simplicity, and cost benefit, the latter method was preferred. The text was submitted by the authors in English.     

Reduction of mercuric ion in vitro by superoxide anion

The reduction rate of mercuric ion to metallic mercury by a superoxide anion produced by a xanthine-xanthine oxidase system increased with an increased concentration of xanthine oxidase in the presence of enough xanthine. The reduction rate of mercuric ion by a superoxide anion in the presence of nitroblue tetrazolium (NBT) was proportional to the concentration of NBT. The result suggests that NBT was reduced to diformazan by a superoxide anion produced by a xanthine-xanthine oxidase system and that mercuric ion will be reduced to metallic mercury by diformazan. The reduction rate of mercuric ion was also indicative that a superoxide anion produced by an NADH-phenazine methosulfate (PMS) system increased with an increased concentration of PMS.     

Superoxide dismutase-like activities of copper(II) complexes tested in serum

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.     

鸦肝超氧化物歧化酶的纯化

本文用低浓度氯化钠与肝脏一起匀浆,75℃加热,硫酸铵分级沉淀,在Cu~(2+)存在下透析,sephadex G-75柱层析等方法,从寒鸦肝脏中纯化出铜锌超氧化物歧化酶。对其理化性质鉴定表明,用此法纯化的SOD为均一性纯酶,比活性为4734U/mg pr,分子量32.6kD,紫外吸收峰在258.6nm。理化性质与文献报道的不同来源的同类酶基本相同。     

Comparative study of superoxide dismutase activity assays in Crocus sativus L. corms

Superoxide dismutase catalyzes the breakdown of the superoxide radical anion and provides the first line of defense against oxygen toxicity. Its vital importance has made it the subject of numerous investigations. Several assays have been proposed for the detection and quantitation of superoxide dismutase activity, but their use has remained controversial and no comparative studies have been reported. In this investigation, three commonly used methods were compared for the measurement of superoxide dismutase activity in Crocus sativus L. corm extract. The methods, based on a competition between the enzyme itself and another superoxide scavenger, involved respectively cytochrome c reduction, nitro blue tetrazolium reduction, and pyrogallol autoxidation. Because of its accuracy, reproducibility, simplicity and cost benefit, the latter method was the most appropriate.     

Effect of CuSO4 and Cu(II)(Gly)2 on some indirect assays for superoxide dismutase activity

The effect of CuSO₄ and Cu(II)(Gly)₂ has been compared with that of superoxide dismutase on the ferricytochrome c reduction and on the nitroblue tetrazolium reduction by an enzymic or chemical flux of superoxide anion radicals as well as on o-dianisidine photooxidation. Both CuSO₄ and Cu(II)(Gly)₂ have been found to inhibit ferricytochrome c reduction as well as the aerobic and anaerobic nitroblue tetrazolium reduction with approximately equal efficiency. Unlike superoxide dismutase they proved capable of inhibiting o-dianisdine photooxidation. The effect of copper either as CuSO₄ or as Cu(II)(Cly)₂ has been established as being due to its interference with the indirect assays for superoxide dismutase activity used. The reasons for this interference have been examined and it is concluded that copper can react with a component of the indirect assay system and depending on the method used it either mimics SOD or acts contrary to the enzyme.     

Enhanced superoxide dismutase activity of pulsed cytochrome oxidase

The superoxide dismutase (SOD) activity of beef heart cytochrome oxidase, both in the resting (as isolated) and pulsed (reduced and reoxidized) states, has been investigated using their ability to inhibit the autoxidation rate of pyrogallol and epinephrine. Resting oxidase showed variable SOD activity, while in the pulsed state the SOD activity of cytochrome oxidase (CcO) increased by an order of magnitude. These results are discussed in terms of a physiological role for the pulsed oxidase.     

Superoxide-induced bleaching of streptocyanine dyes: Application to assay the enzymatic activity of superoxide dismutases

With the aim of developing a novel superoxide dismutase (SOD) activity assay, a series of polymethinium salts (streptocyanines) were prepared and studied for their ability to be reduced by superoxide radical anion generated either from the pyrogallol autoxidation or by the xanthine oxidase-catalyzed oxidation of xanthine. The nonacarbon chain streptocyanine 9Cl(NEt₂)₂ was found to be relatively stable in neutral buffered aqueous solutions, to be reduced at a significant rate by superoxide, and addition of iron-dependent superoxide dismutase (Fe-SOD) prevented its bleaching, thus constituting a good candidate as a possible superoxide indicator in a spectrophotometric SOD assay. The values found to be optimal for a SOD assay were defined as pH 7.4, wavelength 728 nm, xanthine and xanthine oxidase as superoxide source, and a reaction time of 5 min. Based on the color change caused by the superoxide-induced bleaching of the streptocyanine, a qualitative colorimetric method for the SOD activity detection is proposed, enabling visual detection within a short time without any instrument.     

Pretreatment with oxygen species increases the resistance of mammalian cells to hydrogen peroxide and gamma-rays

Pretreatment of Chinese hamster ovary (CHO) or H4 (rat hepatoma) cells with low non-toxic doses of H2O2 or xanthine-xanthine oxidase renders the cells more resistant to the toxic effect of H2O2 and gamma-rays. This increased resistance is observed both in exponentially growing and in plateau-phase cells. Cells pretreated with xanthine-xanthine oxidase are less mutated than control cultures when challenged with ionizing radiation. The number of DNA single-strand breaks (measured by nucleoid sedimentation) induced by a high dose of gamma-rays or H2O2 is lower in cells pretreated with xanthine-xanthine oxidase compared to control cultures. However, the pretreatment does not modify the rate of DNA single-strand breaks rejoining in cells challenged with H2O2 or gamma-rays. The catalase activity is not modified in pretreated cells, but the superoxide dismutase activity is increased about 2-fold.     

Comparison of four indirect methods for fluid superoxide dismutase activities

Relatively small sample dilutions could render fluid extracellular (EC) superoxide dismutase (SOD) activity assays more subject to interfering compounds than tissue SOD assays. Highly variable relative SOD activity were obtained when comparing four indirect assays for several fluid samples (human plasma, human synovial fluid, and plasma from healthy or inflamed rats). Analysis of rat plasma fractionated with Sephadex G-150 showed that each assay (three xanthine oxidase based assays plus a modified pyrogallol assay) detected apparent SOD almost entirely at the same molecular weight as rat lung EC SOD. However, unfractionated fluid samples caused interferences with the xanthine oxidase based SOD assays, though not with the pyrogallol method. Examples of interference were stimulation of xanthine oxidase activity, color formation without xanthine oxidase, color formation despite excess Cu-Zn SOD addition, and absorbance changes with cyanide inhibition of EC SOD that were above or below blank values. In summary, relative fluid SOD values depended on the assay used, and a modified pyrogallol assay was not subject to several interferences found for three xanthine oxidase based assays of fluid SOD activity.     

Cyanide catalyzes the oxidation of alpha-hydroxyaldehydes and related compounds: monitored as the reduction of dioxygen, cytochrome c, and nitroblue tetrazolium

Cyanide catalyzed the oxidation of α-hydroxycarbonyls and of related compounds. In the cases of glyceraldehyde 3-phosphate and of dihydroxyacetone phosphate the tautomeric enediol was the obligatory intermediate which reacted with cyanide yielding the active reductant. Cytochrome c, nitroblue tetrazolium, and dioxygen were all reduced by this reductant. In the case of dioxygen the product was the superoxide radical which could then secondarily reduce cytochrome c or nitroblue tetrazolium. In air-equilibrated reaction mixtures, at 25 °C, approximately 35% of cytochrome c reduction and 95% of nitroblue tetrazolium reduction was mediated by superoxide, as judged from susceptibilities to inhibition by superoxide dismutase. Since the oxidations observed were univalent, carbon-centered radicals appear to be necessary intermediates, and their secondary reactions generated a multiplicity of products, seen as smears on thin-layer chromatograms. Free cyanide must be regenerated during these secondary reactions, since cyanide functioned catalytically in the overall process. A partial mechanism has been proposed in explanation of these observations.     

Important Roles of Minor Peroxide Components in the Autoxidation of Methyl Linoleate

Superoxide dismutase (SOD)-like compounds and activators of SOD were screened for in the extracts of fruits, vegetables, and mushrooms by measuring their effects on pyrogallol autoxidation, which is catalyzed by superoxide anion. SOD-like activity was high in aqueous extract of nameko, garlic, broccoli, and oriental lettuce. Ethanolic extracts of onion and watermelon could enhance human SOD activity more than 40%.     

An assay for superoxide dismutase activity in mammalian tissue homogenates

During the course of measuring superoxide dismutase (SOD) activity in rat breast tissue, interferences in the nitroblue tetrazolium (NBT) and cytochrome c assay systems were noted. These interferences inhibit accurate measurement of SOD activity in breast tissues, necessitating the development of a new NBT-based assay that includes compounds capable of inhibiting tissue specific interferences. The most effective compounds were metal chelators that were also electron transport chain inhibitors. Bathocuproine sulfonate (BCS) was the most effective of these compounds. The inclusion of BCS in the NBT assay system was shown to make the accurate measurement of SOD activity in tissues with interferences possible.     

An assay for the detection of superoxide dismutase in individual Escherichia coli colonies

A method for detecting superoxide dismutase activity in individual colonies of Escherichia coli was developed. The assay involves the lysis of individual cells in colonies on filter papers by a series of lysozyme, chloroform, and freeze-thaw treatments. Filters are placed on agar plates to allow diffusion of cellular enzymes into a solid matrix. A nitroblue tetrazolium overlay is applied to detect superoxide dismutase activity. Colonies possessing activity produce achromatic zones against a dark Formazan background. The assay can detect the presence of superoxide dismutase and the relative amount of enzyme as well. This assay provides a method for screening a population of cells for mutants deficient in or overproducing superoxide dismutase.     

Reaction of superoxide anions with melanins: electron spin resonance and spin trapping studies

Scavenging of superoxide radicals by melanin is a possible factor in the photoprotection afforded by melanin pigments. The reaction between superoxide anions and melanins has been studied by electron spin resonance and spin trapping methods. It was found that superoxide anions react to produce melanin free radicals in a reaction inhibited by superoxide dismutase but not by catalase. The rate of radical formation depends on the concentration of melanin and superoxide, the pH of the medium and the presence of diamagnetic metal ions. The melanin pigment competes with the enzyme superoxide dismutase for removal of superoxide radicals. It was found that the xanthine-xanthine oxidase system is not suitable for studying the reaction of superoxide with melanin, as the enzymatic activity of xanthine oxidase is considerably inhibited by melanin.