The Globin-based Free Radical of Ferryl Hemoglobin Is Detected in Normal Human Blood

Svistunenko, Dimitri A.; Patel, Rakesh P.; Voloshchenko, Sergey V.; Wilson, Michael T.

doi:10.1074/jbc.272.11.7114

Cited by 111 publications

(96 citation statements)

References 45 publications

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“…Before NO inhalation, an increase in a broad free radical species was observed upon artery to vein transit, similar to the radical species observed during oxidation of hemoglobin by either hydrogen peroxide or nitrite (28) and previously observed in venous blood (27). Balagopalakrishna et al (29) observed the formation of a radical signal at g ϭ 2.004 after freezing of partially deoxygenated hemoglobin and assigned this signal to a superoxide radical bound within the heme pocket.…”

Section: Discussionmentioning

confidence: 71%

“…The broader signal was spectrally similar to the free radical species observed upon addition of hydrogen peroxide to met-Hb and was tentatively identified as a hemoglobin protein radical. The presence of this species has been observed previously in venous blood (27). Based on these observations, for the quantification of HbNO in whole blood, we incorporated three additional basis spectra in the regression analysis, both the broad and sharp radical signals and the copper(II) species.…”

Section: Standardization and Characterization Of Hbno Analysis-in Ordermentioning

confidence: 99%

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Electron Paramagnetic Resonance Analysis of Nitrosylhemoglobin in Humans during NO Inhalation

Piknová

Gladwin

Schechter

et al. 2005

Journal of Biological Chemistry

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The reactions of nitric oxide with hemoglobin play an important role in explaining the vascular biology of this free radical. It is perhaps surprising that the level of nitrosylhemoglobin (HbNO) in which NO is bound to the ferrous hemoglobin heme in whole human blood under basal and stimulated conditions is a matter of some controversy, with measurements ranging from <1 nM to close to 10 M. In order to examine HbNO levels in human blood by using EPR spectroscopy, we have developed a regression-based spectral analysis technique that has a detection level of about 200 nM HbNO. We have utilized this methodology to detect the level of HbNO under basal conditions and during NO inhalation. The major findings of this study are as follows. (i) HbNO can be accurately detected and quantified in whole blood with a detection limit of ϳ200 nM. The role of hemoglobin in the biochemistry, physiology, and pharmacology of nitric oxide (NO) 3 and nitrovasodilators is controversial (1-3). Understanding how hemoglobin can control NO-mediated responses is crucial in order to fully appreciate the physiological limitations of endothelial derived nitric oxide, the sequelae of nitric oxide delivery (either through nitric oxide donors, nitrovasodilators, or nitric oxide inhalation), and the vasoconstrictive propensity of hemoglobinbased blood substitutes. It is generally accepted that oxygenated ferrous hemoglobin (oxy-Hb) can convert nitric oxide to nitrate, through a reductive dioxygenation reaction, to form ferric hemoglobin (met-Hb) (4). This reaction is fast (k ϭ 5 ϫ 10 7 M Ϫ1 s Ϫ1 ) (5), irreversible, and generates an inert product. It is also universally accepted that nitric oxide can bind to unliganded ferrous hemoglobin (deoxy-Hb) to form a nitrosyl derivative (HbNO) at an almost equally rapid rate (k ϭ 2.6 ϫ 10 7 M Ϫ1 s Ϫ1 ) (6). Although HbNO is reasonably stable because of the slow ligand off rate, it can be plausibly argued that the NO has not been destroyed but has been preserved, if a viable mechanism for release and utilization can be established. Two major central areas of controversy are whether sufficient quantities of HbNO can be formed in vivo, and whether the formation of S-nitrosohemoglobin from HbNO represents a viable mechanism for NO reutilization (7-10). It has been argued that at oxygen concentrations at which hemoglobin is predominantly oxygenated, the small pool of hemes that are still in the high oxygen affinity state (R-state) but are unliganded will bind NO up to 100 times faster than has been appreciated previously. This would favor the formation of HbNO over met-Hb at oxygen saturation levels less than 99% (11, 12). This conclusion has been disputed in several laboratories, and methodological mixing problems with these experiments have been highlighted (13-16). Gladwin et al. (17) examined NO/hemoglobin reactions in vivo in humans by supplementing endogenous production with inhaled NO. These experiments indicated that the major hemoglobin-derived product formed during NO inhalation was met-Hb, together...

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Section: Discussionmentioning

confidence: 71%

Section: Standardization and Characterization Of Hbno Analysis-in Ordermentioning

confidence: 99%

Electron Paramagnetic Resonance Analysis of Nitrosylhemoglobin in Humans during NO Inhalation

Piknová

Gladwin

Schechter

et al. 2005

Journal of Biological Chemistry

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“…23) For HRP, 24) chloroperoxidase, 25) plant ascorbate peroxidase, 26) prostaglandin H synthase, 27) and lignin peroxidase, 23) the second oxidizing equivalent is stored as a porphyrin p-cation radical. However, for compound ESs of yeast and Pseudomonas cytochrome c peroxidase, 28,29) horse myoglobin, 30) leghemoglobin (Lb), 31) and human Hb, 7) the second oxidizing equivalent is on an amino acid side chain of the protein. Recently it has been shown that catalase compound I has a porphyrin p-cation radical and it is likely to be converted to a tyrosyl radical on protein depending on pH and temperature.…”

Section: Pseudoperoxidase Cyclementioning

confidence: 99%

“…6) Actually such globin radicals in the ferryl Hb has been detected by electron spin resonance (ESR) spectroscopy in human and animal blood. 7) Basic problems are associated with the use of Hb outside the erythrocytes. Erythrocytes provide abundant antioxidant enzymes such as catalase and superoxide dismutase (SOD), that catalyze the breakdown of H2O2 and O2 -, respectively, and the reductase systems that catalyze the reduction of ferric iron (Fe III ) back to the ferrous state, the only form that reversibly binds O2.…”

Section: -mentioning

confidence: 99%

“…Hb-Fe IV (+globin radicals)+PEAªHb-Fe IV +PEA  [7] Hb-Fe IV +PEAªHb-Fe III +PEA  [8] PEA  +O 2 ªPEA + +O 2 - [9] where PEA  and PEA + are a PEA-derived radical species and a two-electron oxidation intermediate, respectively. Then H2O2, the product of concomitant O2 -dismutation, may reproduce the globin radical associated Hb-Fe IV from Hb-Fe II and Hb-Fe III by 2-electron and 1-elecron oxidation, respectively.…”

Section: Pseudoperoxidase Cyclementioning

confidence: 99%

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Monoamine-dependent Production of Reactive Oxygen Species Catalyzed by Pseudoperoxidase Activity of Human Hemoglobin

Kawano

Pinontoan

Hosoya

et al. 2002

Bioscience, Biotechnology, and Biochemistry

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An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis

Chaplin

Chicano

Hampshire

et al. 2019

Chemistry A European J

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Dye decolouring peroxidases (DyPs) are the most recent class of heme peroxidase to be discovered. On reacting with H2O2, DyPs form a high‐valent iron(IV)‐oxo species and a porphyrin radical (Compound I) followed by stepwise oxidation of an organic substrate. In the absence of substrate, the ferryl species decays to form transient protein‐bound radicals on redox active amino acids. Identification of radical sites in DyPs has implications for their oxidative mechanism with substrate. Using a DyP from Streptomyces lividans, referred to as DtpA, which displays low reactivity towards synthetic dyes, activation with H2O2 was explored. A Compound I EPR spectrum was detected, which in the absence of substrate decays to a protein‐bound radical EPR signal. Using a newly developed version of the Tyrosyl Radical Spectra Simulation Algorithm, the radical EPR signal was shown to arise from a pristine tyrosyl radical and not a mixed Trp/Tyr radical that has been widely reported in DyP members exhibiting high activity with synthetic dyes. The radical site was identified as Tyr374, with kinetic studies inferring that although Tyr374 is not on the electron‐transfer pathway from the dye RB19, its replacement with a Phe does severely compromise activity with other organic substrates. These findings hint at the possibility that alternative electron‐transfer pathways for substrate oxidation are operative within the DyP family. In this context, a role for a highly conserved aromatic dyad motif is discussed.

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The Globin-based Free Radical of Ferryl Hemoglobin Is Detected in Normal Human Blood

Cited by 111 publications

References 45 publications

Electron Paramagnetic Resonance Analysis of Nitrosylhemoglobin in Humans during NO Inhalation

Electron Paramagnetic Resonance Analysis of Nitrosylhemoglobin in Humans during NO Inhalation

Monoamine-dependent Production of Reactive Oxygen Species Catalyzed by Pseudoperoxidase Activity of Human Hemoglobin

An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis

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