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Excited singlet molecular O₂(¹Δg) is generated enzymatically from excited carbonyls in the dark.

Mano CM, Prado FM, Massari J, Ronsein GE, Martinez GR, Miyamoto S, Cadet J, Sies H, Medeiros MH, Bechara EJ, Di Mascio P - Sci Rep (2014)

Bottom Line: This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source.This corroborates formation of O2 ((1)Δg).Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates (18)O2 ((1)Δg) by triplet-triplet energy transfer to ground state oxygen O2 ((3)Σg(-)), and supports the long formulated hypothesis of O2 ((1)Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CEP 05513-970, CP 26077, São Paulo, SP, Brazil.

ABSTRACT
In mammalian tissues, ultraweak chemiluminescence arising from biomolecule oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [O2 ((1)Δg)] and electronically excited triplet carbonyl products involving dioxetane intermediates. Herein, we describe evidence of the generation of O2 ((1)Δg) in aqueous solution via energy transfer from excited triplet acetone. This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source. Both sources of excited carbonyls showed characteristic light emission at 1,270 nm, directly indicative of the monomolecular decay of O2 ((1)Δg). Indirect analysis of O2 ((1)Δg) by electron paramagnetic resonance using the chemical trap 2,2,6,6-tetramethylpiperidine showed the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl. Using [(18)O]-labeled triplet, ground state molecular oxygen [(18)O2 ((3)Σg(-))], chemical trapping of (18)O2 ((1)Δg) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[(18)O]-labeled 9,10-endoperoxide, was detected through mass spectrometry. This corroborates formation of O2 ((1)Δg). Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates (18)O2 ((1)Δg) by triplet-triplet energy transfer to ground state oxygen O2 ((3)Σg(-)), and supports the long formulated hypothesis of O2 ((1)Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light.

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EAS chemical trapping of O2 (1Δg) generated by the HRP-catalyzed oxidation of IBAL in the presence of [18O]-labeled molecular oxygen.HPLC-ESI-MS/MS analysis of 8 mM EAS upon incubation for 24 h with 5 µM HRP and 50 mM IBAL at 37°C in deuterated phosphate buffer (pD 7.4). (A) UV chromatogram at 210 nm. Endoperoxides EAS16O16O and EAS18O18O eluted at 7.8 min. (B) SRM chromatogram of EAS18O18O (m/z 230→212). (C) SRM chromatogram of EAS16O16O (m/z 228→212). (D) Full mass spectrum obtained from peak at 7.8 min within mass range of 100–280 m/z. (E) Product ion spectrum from precursor ion at m/z 230. (F) Product ion spectrum from precursor ion at m/z 228.
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f6: EAS chemical trapping of O2 (1Δg) generated by the HRP-catalyzed oxidation of IBAL in the presence of [18O]-labeled molecular oxygen.HPLC-ESI-MS/MS analysis of 8 mM EAS upon incubation for 24 h with 5 µM HRP and 50 mM IBAL at 37°C in deuterated phosphate buffer (pD 7.4). (A) UV chromatogram at 210 nm. Endoperoxides EAS16O16O and EAS18O18O eluted at 7.8 min. (B) SRM chromatogram of EAS18O18O (m/z 230→212). (C) SRM chromatogram of EAS16O16O (m/z 228→212). (D) Full mass spectrum obtained from peak at 7.8 min within mass range of 100–280 m/z. (E) Product ion spectrum from precursor ion at m/z 230. (F) Product ion spectrum from precursor ion at m/z 228.

Mentions: Figures 5 and 6 and Supplementary Fig. 3 to 7 show the typical chromatograms for EASxOxO analysis with UV and MS/MS detection. Analysis of the products by UV absorption at 210 nm showed two peaks corresponding to the endoperoxides EAS18O18O and EAS16O16O and to EAS at the time windows 7.2 to 7.9 min and 9.2 to 12.2 min, respectively, for the TMD (Fig. 5A and Supplementary Fig. 3A) and HRP/IBAL systems (Fig. 6A and Supplementary Fig. 4A and 5A). The tandem mass spectrometry detection of EAS18O18O (m/z 230→212) and EAS16O16O (m/z 228→212) was performed by the Selected Reaction Monitoring (SRM) mode. SRM detection based on the fragmentation of precursor ions at m/z 230 (Fig. 5B and 6B) and 228 (Fig. 5C and 6C), which generated the product ion at m/z 212, shows the presence of EAS18O18O and EAS16O16O, respectively. The identity of the precursor ions was confirmed based on an analysis of the mass spectra of product ions derived from each of the endoperoxides (Fig. 5E and 5F,and Fig. 6E and 6F).


Excited singlet molecular O₂(¹Δg) is generated enzymatically from excited carbonyls in the dark.

Mano CM, Prado FM, Massari J, Ronsein GE, Martinez GR, Miyamoto S, Cadet J, Sies H, Medeiros MH, Bechara EJ, Di Mascio P - Sci Rep (2014)

EAS chemical trapping of O2 (1Δg) generated by the HRP-catalyzed oxidation of IBAL in the presence of [18O]-labeled molecular oxygen.HPLC-ESI-MS/MS analysis of 8 mM EAS upon incubation for 24 h with 5 µM HRP and 50 mM IBAL at 37°C in deuterated phosphate buffer (pD 7.4). (A) UV chromatogram at 210 nm. Endoperoxides EAS16O16O and EAS18O18O eluted at 7.8 min. (B) SRM chromatogram of EAS18O18O (m/z 230→212). (C) SRM chromatogram of EAS16O16O (m/z 228→212). (D) Full mass spectrum obtained from peak at 7.8 min within mass range of 100–280 m/z. (E) Product ion spectrum from precursor ion at m/z 230. (F) Product ion spectrum from precursor ion at m/z 228.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4120373&req=5

f6: EAS chemical trapping of O2 (1Δg) generated by the HRP-catalyzed oxidation of IBAL in the presence of [18O]-labeled molecular oxygen.HPLC-ESI-MS/MS analysis of 8 mM EAS upon incubation for 24 h with 5 µM HRP and 50 mM IBAL at 37°C in deuterated phosphate buffer (pD 7.4). (A) UV chromatogram at 210 nm. Endoperoxides EAS16O16O and EAS18O18O eluted at 7.8 min. (B) SRM chromatogram of EAS18O18O (m/z 230→212). (C) SRM chromatogram of EAS16O16O (m/z 228→212). (D) Full mass spectrum obtained from peak at 7.8 min within mass range of 100–280 m/z. (E) Product ion spectrum from precursor ion at m/z 230. (F) Product ion spectrum from precursor ion at m/z 228.
Mentions: Figures 5 and 6 and Supplementary Fig. 3 to 7 show the typical chromatograms for EASxOxO analysis with UV and MS/MS detection. Analysis of the products by UV absorption at 210 nm showed two peaks corresponding to the endoperoxides EAS18O18O and EAS16O16O and to EAS at the time windows 7.2 to 7.9 min and 9.2 to 12.2 min, respectively, for the TMD (Fig. 5A and Supplementary Fig. 3A) and HRP/IBAL systems (Fig. 6A and Supplementary Fig. 4A and 5A). The tandem mass spectrometry detection of EAS18O18O (m/z 230→212) and EAS16O16O (m/z 228→212) was performed by the Selected Reaction Monitoring (SRM) mode. SRM detection based on the fragmentation of precursor ions at m/z 230 (Fig. 5B and 6B) and 228 (Fig. 5C and 6C), which generated the product ion at m/z 212, shows the presence of EAS18O18O and EAS16O16O, respectively. The identity of the precursor ions was confirmed based on an analysis of the mass spectra of product ions derived from each of the endoperoxides (Fig. 5E and 5F,and Fig. 6E and 6F).

Bottom Line: This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source.This corroborates formation of O2 ((1)Δg).Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates (18)O2 ((1)Δg) by triplet-triplet energy transfer to ground state oxygen O2 ((3)Σg(-)), and supports the long formulated hypothesis of O2 ((1)Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CEP 05513-970, CP 26077, São Paulo, SP, Brazil.

ABSTRACT
In mammalian tissues, ultraweak chemiluminescence arising from biomolecule oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [O2 ((1)Δg)] and electronically excited triplet carbonyl products involving dioxetane intermediates. Herein, we describe evidence of the generation of O2 ((1)Δg) in aqueous solution via energy transfer from excited triplet acetone. This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source. Both sources of excited carbonyls showed characteristic light emission at 1,270 nm, directly indicative of the monomolecular decay of O2 ((1)Δg). Indirect analysis of O2 ((1)Δg) by electron paramagnetic resonance using the chemical trap 2,2,6,6-tetramethylpiperidine showed the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl. Using [(18)O]-labeled triplet, ground state molecular oxygen [(18)O2 ((3)Σg(-))], chemical trapping of (18)O2 ((1)Δg) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[(18)O]-labeled 9,10-endoperoxide, was detected through mass spectrometry. This corroborates formation of O2 ((1)Δg). Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates (18)O2 ((1)Δg) by triplet-triplet energy transfer to ground state oxygen O2 ((3)Σg(-)), and supports the long formulated hypothesis of O2 ((1)Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light.

Show MeSH
Related in: MedlinePlus