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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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Chemiluminescence studies of TMD in organic solvents.TMD (10 mM) was incubated in air-equilibrated organic solvents at 70°C. (A) Time course of total UV-visible light emission of TMD in acetonitrile (line a) and in CCl4 (line b); (B) The chemiluminescence spectrum matches the phosphorescence spectrum of TMD-generated triplet excited acetone20 in CCl4; (C) NIR light emission of O2 (1Δg) at 1,270 nm during the thermolysis of TMD in CCl4 (line b) and in acetonitrile (line a); (D) O2 (1Δg) spectrum, corresponding to the monomol light emission recorded during incubation of TMD in CCl4; and (E and F) thermodissociation of 10 mM DMNO2 in methanol, as a control, which generated O2 (1Δg) monomol light emission at 1,270 nm and the NIR spectrum of released O2 (1Δg), respectively.
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f1: Chemiluminescence studies of TMD in organic solvents.TMD (10 mM) was incubated in air-equilibrated organic solvents at 70°C. (A) Time course of total UV-visible light emission of TMD in acetonitrile (line a) and in CCl4 (line b); (B) The chemiluminescence spectrum matches the phosphorescence spectrum of TMD-generated triplet excited acetone20 in CCl4; (C) NIR light emission of O2 (1Δg) at 1,270 nm during the thermolysis of TMD in CCl4 (line b) and in acetonitrile (line a); (D) O2 (1Δg) spectrum, corresponding to the monomol light emission recorded during incubation of TMD in CCl4; and (E and F) thermodissociation of 10 mM DMNO2 in methanol, as a control, which generated O2 (1Δg) monomol light emission at 1,270 nm and the NIR spectrum of released O2 (1Δg), respectively.

Mentions: The CL arising from the thermal decomposition of 10 mM TMD at 70°C in air-equilibrated CCl4 or acetonitrile (Fig. 1A(b) and 1A(a), respectively) was recorded in the UV-visible region. The CL spectrum of 10 mM TMD in CCl4 shows a peak at 430 nm (Fig. 1B), which was assigned to the triplet excited acetone14. Fig. 1C and 1D depict the time course of monomol light emission of O2 (1Δg) at λ = 1,270 nm and the NIR spectrum of O2 (1Δg), respectively. Since the lifetime of O2 (1Δg) in acetonitrile is much lower than in CCl4 (5.0–8.0 × 10−5 s and 0.02–0.08 s, respectively), the TMD/O2 (3Σg−) NIR light emission in acetonitrile was very low under similar experimental conditions6162. For comparison, the time course and spectrum of NIR light emission were recorded during the thermolysis of 1,4-dimethylnaphthalene-1,4-endoperoxide (DMNO2)60 in methanol (Fig. 1E and 1F).


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)

Chemiluminescence studies of TMD in organic solvents.TMD (10 mM) was incubated in air-equilibrated organic solvents at 70°C. (A) Time course of total UV-visible light emission of TMD in acetonitrile (line a) and in CCl4 (line b); (B) The chemiluminescence spectrum matches the phosphorescence spectrum of TMD-generated triplet excited acetone20 in CCl4; (C) NIR light emission of O2 (1Δg) at 1,270 nm during the thermolysis of TMD in CCl4 (line b) and in acetonitrile (line a); (D) O2 (1Δg) spectrum, corresponding to the monomol light emission recorded during incubation of TMD in CCl4; and (E and F) thermodissociation of 10 mM DMNO2 in methanol, as a control, which generated O2 (1Δg) monomol light emission at 1,270 nm and the NIR spectrum of released O2 (1Δg), respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Chemiluminescence studies of TMD in organic solvents.TMD (10 mM) was incubated in air-equilibrated organic solvents at 70°C. (A) Time course of total UV-visible light emission of TMD in acetonitrile (line a) and in CCl4 (line b); (B) The chemiluminescence spectrum matches the phosphorescence spectrum of TMD-generated triplet excited acetone20 in CCl4; (C) NIR light emission of O2 (1Δg) at 1,270 nm during the thermolysis of TMD in CCl4 (line b) and in acetonitrile (line a); (D) O2 (1Δg) spectrum, corresponding to the monomol light emission recorded during incubation of TMD in CCl4; and (E and F) thermodissociation of 10 mM DMNO2 in methanol, as a control, which generated O2 (1Δg) monomol light emission at 1,270 nm and the NIR spectrum of released O2 (1Δg), respectively.
Mentions: The CL arising from the thermal decomposition of 10 mM TMD at 70°C in air-equilibrated CCl4 or acetonitrile (Fig. 1A(b) and 1A(a), respectively) was recorded in the UV-visible region. The CL spectrum of 10 mM TMD in CCl4 shows a peak at 430 nm (Fig. 1B), which was assigned to the triplet excited acetone14. Fig. 1C and 1D depict the time course of monomol light emission of O2 (1Δg) at λ = 1,270 nm and the NIR spectrum of O2 (1Δg), respectively. Since the lifetime of O2 (1Δg) in acetonitrile is much lower than in CCl4 (5.0–8.0 × 10−5 s and 0.02–0.08 s, respectively), the TMD/O2 (3Σg−) NIR light emission in acetonitrile was very low under similar experimental conditions6162. For comparison, the time course and spectrum of NIR light emission were recorded during the thermolysis of 1,4-dimethylnaphthalene-1,4-endoperoxide (DMNO2)60 in methanol (Fig. 1E and 1F).

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