Quantitative Measurement of Superoxide Generation and Oxygen Consumption from Leukocytes Using Electron Paramagnetic Resonance Spectroscopy

Roubaud, Valérie; Sankarapandi, Sornampillai; Kuppusamy, Periannan; Tordo, Paul; Zweíer, Jay L.

doi:10.1006/abio.1997.2542

Cited by 69 publications

(69 citation statements)

References 44 publications

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“…After COR reaction at 30°C in the dark for 10 min, the mixture was placed in quartz EPR flat cell, and the spectrum was recorded at ambient temperature. The EPR-settings were as follows: modulation amplitude 0.32 G, time constant 0.16 s, scan time 60 s, modulation frequency 100 kHz, microwave power 20 milliwatts, and microwave frequency 9.76 GHz (32). A standard superoxide-generating mixture of xanthine (100 M) and xanthine oxidase (0.004 units) in 0.1 M potassium phosphate (pH 7.4) buffer (33) under the gas phase of 20% O 2 and 80% N 2 (DO, 205 Ϯ 11 M) was included as a positive control.…”

Section: Superoxide Determinationmentioning

confidence: 99%

Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Kim

Lee

et al. 2008

Journal of Biological Chemistry

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Chlorophyllide a reductase of Rhodobacter sphaeroides, which were reconstituted with the purified subunits of BchX, BchY, and BchZ, reduced ring B of chlorophyllide a using NADH under anaerobic conditions. Interestingly, suppressor mutations rescuing the inability of R. sphaeroides Fe-SOD mutant to grow in succinate-based minimal medium were predominantly mapped to BchZ subunit of chlorophyllide a reductase. The enzyme is labile in the presence of O 2 . However, it generates superoxide at low O 2 . The enzymes reconstituted with BchX, BchY, and the mutein subunit of BchZ from suppressor mutants showed less activity not only for chlorophyllide a reduction but also for superoxide generation compared with the enzyme reconstituted with the wild-type subunits. BchX, which contains FMN, and BchY are iron-sulfur proteins, whereas BchZ is a hemoprotein containing b-type heme. Neither chlorophyllide a reduction nor superoxide generation was observed with the enzyme reconstituted with the wild-type subunits of BchX and BchY, and the apo-subunit of BchZ that had been refolded without heme, in which FMN of BchX was fully reduced. Thus, superoxide is generated not from FMN of BchX but from heme of BchZ. Consistently, the heme of BchZ muteins was half-reduced in its redox state compared with that of wild-type BchZ.Rhodobacter sphaeroides, a facultative photosynthetic bacterium, contains two SODs 3 ; CuZn-SOD is detected only under the conditions where photosynthetic complexes are formed (1), whereas Fe-SOD is constitutively expressed, although its activity of the aerobically grown cell nearly doubled as compared with that of the anaerobically grown cell. The role of CuZn-SOD in protecting the photoheterotrophic cells from periplasmic superoxide upon exposure to O 2 was proposed (1). A cambialistic SOD using manganese or iron is the only SOD found in Rhodobacter capsulatus and is essential for cell viability (2, 3). Oxidative stress defense in R. sphaeroides and R. capsulatus is also mediated by catalases as well as by glutathione-glutaredoxin and thioredoxin systems that act as thiol-disulfide redox buffer to reduce the protein thiols that were oxidized (4, 5). Mutations in glutathione-glutaredoxin and thioredoxin systems lowered the formation of the photosynthetic complex (4, 5).The formation of photosynthetic complexes of R. sphaeroides is redox-dependent. Lowering oxygen tension induces the formation of intracytoplasmic membrane housing the photosynthetic complexes of R. sphaeroides. Light captured by B800 -850 and B875 LH complexes is transferred to reaction center complex, where the redox reactions are initiated to convert light energy into ATP and reducing power. The oxygenregulated expression of apoproteins of LH and reaction center complexes, which are encoded by puc, puf, and puh operons, was elucidated (for reviews see Refs. 6 and 7).The expression of several enzymes for Bchl a synthesis is also subject to anaerobic induction (8 -10). Protoporphyrin IX, which is synthesized from 5-aminolevulinic acid, is a common in...

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Section: Superoxide Determinationmentioning

confidence: 99%

Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Kim

Lee

et al. 2008

Journal of Biological Chemistry

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“…The half-life for decomposition of the DEPMPO-OOH adduct in aqueous media is ∼16 min, much longer than the ∼45 s half life of DMPO-OOH [37][38][39]. EPR experiments were carried out under conditions identical to those in Fig.…”

Section: Detection Of By Epr Spin Trappingmentioning

confidence: 99%

Characterization of superoxide production from aldehyde oxidase: An important source of oxidants in biological tissues

Kundu

Hille

Murugesan

et al. 2007

Archives of Biochemistry and Biophysics

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Aldehyde oxidase, a molybdoflavoenzyme that plays an important role in aldehyde biotransformation, requires oxygen as substrate and produces reduced oxygen species. However, little information is available regarding its importance in cellular redox stress. Therefore, studies were undertaken to characterize its superoxide and hydrogen peroxide production. Aldehyde oxidase was purified to >98% purity and exhibited a single band at ∼290 kDa on native polyacrylamide gradient gel electrophoresis. Superoxide generation was measured and quantitated by cytochrome c reduction and EPR spin trapping with p-dimethyl aminocinnamaldehyde as reducing substrate. Prominent superoxide generation was observed with an initial rate of 295 nmol/min/mg. Electrochemical measurements of oxygen consumption and hydrogen peroxide formation yielded values of 650 nmol/min/mg and 355 nmol/min/mg. In view of the ubiquitous distribution of aldehydes in tissues, aldehyde oxidase can be an important basal source of superoxide that would be enhanced in disease settings where cellular aldehyde levels are increased. KeywordsAldehyde oxidase; Xanthine oxidase; Superoxide; Hydrogen peroxide; Electron paramagnetic resonance; Spin trapping; Cytochrome c reduction; Reactive oxygen species; Free radicals; Oxygen consumption Aldehyde oxidase (AO; EC 1.2.3.1) 1 is a prominent member of the molybdenum hydroxylase family of enzymes, which also includes xanthine oxidoreductase (XOR). XOR has two interconvertible forms, xanthine dehydrogenase (XDH; EC 1.1.1.204) and xanthine oxidase (XO; 1. 1.3.22). Both forms of XOR are involved in the catabolism of purines, * To whom correspondence should be addressed: Davis Heart and Lung Research Institute, 473 W. 12th Ave, Room 110G, The Ohio State University, Columbus, OH 43210. Phone: (614) . E-Mail: Jay.Zweier@osumc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Both XOR and AO are homodimeric consisting of two identical subunits with an approximate molecular mass of 145 kDa each. Each subunit consists of four discrete regions -two N-terminal domains contain distinct [2Fe-2S] centers. A linker peptide connects it to a 40 kDa FAD binding domain that positions the flavin ring in close proximity and a second linker peptide connects the FAD domain with the 85 kDa C-terminal portion of the protein that contains the molybdenum center and the substrate binding pocket [3,4]. The structure of XOR is well conserved among human, chicken, mouse and rat enzymes [5] and the amino acid sequences reveal that the molybdenum binding site is the most conserved region with 94% homo...

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“…19,20 Neutrophils (1ϫ10 6 ) were incubated with 5 mmol/L CPH and stimulated by the addition of 200 nmol/L of phorbol 12-myristate 13-acetate (PMA). All ESR samples were prepared using chelated phosphate-buffered saline, pH 7.4.…”

Section: Superoxide Measurementmentioning

confidence: 99%

C242T CYBA Polymorphism of the NADPH Oxidase Is Associated With Reduced Respiratory Burst in Human Neutrophils

et al. 2004

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Abstract-Oxidative stress contributes to the pathogenesis of atherosclerosis. p22phox -based NAD(P)H oxidases exist in the vessel wall, acting as important superoxide-generating systems in the vasculature. Some studies have identified reduced atherosclerosis in the presence of the C242T CYBA polymorphism, whereas others have not. Because vascular p22 phox is identical to neutrophil p22 phox , we studied the association between the C242T, A640G, and Ϫ930 A/G CYBA polymorphisms and the quantity of superoxide produced from neutrophils isolated from healthy adults to determine if these polymorphisms had any functional impact on NADPH oxidase function. Neutrophils were isolated from 90 subjects by Percoll density gradient centrifugation. Genotypes were determined by polymerase chain reaction (PCR) and restriction mapping, as well as real-time PCR. The oxidative burst was stimulated with phorbol 12-myristate 13-acetate. Superoxide was quantified using the superoxide dismutase inhibitable oxidation of the spin probe hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine, detected by electron paramagnetic resonance. Superoxide production was significantly affected by the C242T polymorphism, being 8.7Ϯ0.7, 7.9Ϯ0.6, and 5.9Ϯ1.2 mol/L per minute per 10 6 neutrophils for the C242T CC, CT, and TT genotypes, respectively (PϽ0.05). In contrast, the A640G and the Ϫ930

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Quantitative Measurement of Superoxide Generation and Oxygen Consumption from Leukocytes Using Electron Paramagnetic Resonance Spectroscopy

Cited by 69 publications

References 44 publications

Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Characterization of superoxide production from aldehyde oxidase: An important source of oxidants in biological tissues

C242T CYBA Polymorphism of the NADPH Oxidase Is Associated With Reduced Respiratory Burst in Human Neutrophils

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