Departamento de BioquímicaInstituto de Química Universidade de São Paulo, Brasil

Paolo Di Mascio

How to Measure ROS and RNS in BiologySome generality and a singlet molecular oxygen case

Sunrise Free Radical School

Society for Free Radical Biology and Medicine

Washington, DC November 2007

Reactive Species measurement methods must be:

-Very, very sensitive-Highly selective-Fast-In situ

Reactive species have , in generally, a very short half-life, are produced in low amounts. Multiple methods of measurement are available today, each with their own benefits and limits.

How to Measure Reactive Species?DirectElectron paramagnetic resonance, EPR (free radical detection)+Trapping (Spin-traps, “chemical”)

Indirect (but specific)Mass spectrometry, MS/MS (ESI, MALDI)+ScavengersProbes (spectroscopic investigation)Luminescence (direct and indirect)

Combination of Different Techniques !Free Radicals in Biology and MedicineB. Halliwell ans J.M.C. Gutteridge, 2007.

Direct detectione.g.: semiquinones, nitroxides, thiyl, ROO•…

Indirect detectionSpin-trapping

Species: superoxide, hydroxyl, alkyl, NOSpin-traps: DMPO, PBN, DEPMPO, Fe-DTCs

Intact tissues, organs … can be measured.

We can use EPR to measure free radicals from biological systems (in vivo or ex vivo)

Electron Paramagnetic Resonance (EPR)

We can detect & measure free radicals and paramagnetic species

• “High” sensitivity (nanomolar concentrations)

Other Trapping methods

Hydroxyl radical OH▪ [Trapping methods (without EPR)]-Reaction with aromatic compounds (Phenylalanine ‘Tyrosine’, Salicylate, …-Attack of OH▪ on 2-deoxyribose produces a range of products…malondialdehyde

Superoxide O2▪-

- [Fluorescent probes] Dihydroethidium (DHE) conversion to 2-hydroxyethidium and lucigenin =- Ability to reduce cytochrome c or nitroblue tetrazolium- Superoxide electrodes- Histochemical detection: Conversion of diaminobenzidine (DAB) to an insoluble product

Peroxynitrite and other nitrogen species ….Nitric oxide NO ▪

-NO ▪ : -Light emission in the presence of O3 (excited NO2▪)

-NO electrodes (porphyrinic sensors)-Haemoglobin trapping (ΔA is measured)-Spin trapping (Haemoglobin, other haem proteins, …)-4,5-Diaminofluorescein diacetate (DAF-2-diacetate), fluorescent productDiaminoanthroquinone, red fluorescent product-“indirect methods”: NO2

- measurement, use NOS inhibitors, …

Nitration assaysONOO- : ONOO- nitrates many aromatic compounds: tyrosine (3-nitrotyrosine), tryptophan, phenylalanine

Reactive halogen speciesHOCl / HOBr- [taurine assay] Conversion of taurine to a chloramine-bromo- and chlorotyrosine ?

Hydrogen peroxide H2O2

- [fluorescent products] Amplex red and DCF-DA- [non fluorescent products] Fluorescent compound scopoletin-Inactivation of catalase by aminotriazole….in cells…

Singlet oxygen 1O2……..O2 (1Δg) in biological system-Monomol and dimol Light emission-Use of scavengers (azide), traps (histidine, anthracene derivatives)and D2O effect

Studies using low-level / ultraweak / dark chemiluminescence ….

Many methods are available to identify Reactive Species in cultured cells1- Cell culture process itself causes oxidative stress2-Trypsinization increases ROS3- Artifacts are produced

-Dichlorofluorescein diacetate (DCF-DA) is deacetylated by esterases to dichlorofluorescein (DCFH) which can be visualized by fluorescence at 525 nmMore specific for H2O2?-Dihydrorhodamine 123 (DHR) is used to detect several reactive speciesConversion to rhodamine 123, highly fluorescent 536 nm-Luminol and lucigenin

“Biomarkers”Of oxidative DNA damageOf lipid peroxidationOf protein damage

by reactive species

DNA base oxidation: oxidation of guanine by

OH•

O

HN

N N

N

H2N

R

HN

N N

N•

H2N

O

R

OH

H

HN

N N

N•

H2N

O

R

OH

H

HN

N NH

HN

H2N

O

R

O

Guan(os)ine

FAPy-Guanine

HN

N N

N

H2N

O

R

OH

8-oxo-Guanine

OxidationRing opening

ReductionG8OH•

OH•

• Single and double strand breaks (Comet assay)• Oxidative modification of DNA-bases (e.g. 8-oxo-dGuo… LC/MS/MS)• Formation of DNA adducts with oxidized lipids or proteins (e.g. dGuo-MDA,

dT-Tyr….LC/MS/MS).

“Biomarkers”Of oxidative DNA damage

Arachidonic acid oxidation products:

Isoprostanes: Relatively stable endproducts that are currently viewed as most reliable marker of lipid oxidation in vivo

Isoprostanes comprise four regioisomers with eight stereoisomers. in vivo (Lawson et al., J. Biol. Chem. 1999: 274, 2444-24444).

“Biomarkers”

Of lipid peroxidation

(Roberts LJ, II, et. al. J. Biol. Chem :271:20617, 1996)

Metabolic Fate of 15-F2t-IsoP (8-Iso-PGF2α) in Humans

A GC/MS assay for F2-IsoP-M has been developed

COOH

OHOH

OH

OHOH

OHCOOH

15-F2t-IsoP

2,3-Dinor-5,6-Dihydro-15-F2t-IsoP(F2-IsoP-M)

β-oxidation Δ5-reduction

Major urinary metabolite

“Biomarkers”

Of lipid peroxidation

o,o’-dityrosine

+

NH3+

COO–

O•

NH3+

COO–

OH

NH3

COO–

OH

NH3+

COO–

OH

HOCl, Cl2 NO2•

Tyr•

Ox

Ox

Tyrosine Tyrosylradical

NH3+

COO–

OH

Cl

3-Cl-tyrosine(RHS)

NH3+

COO–

OH

NO2

3-NO2-tyrosine(RNS)

NH3+

COO–

OH

OH

3,4,-diOH-Phe(DOPA)

Stable oxidation markers: Tyrosine oxidation, chlorination, and nitration

“Biomarkers”

Of protein damage

DNA / Protein Adducts

HN

N N

N

H2N

O

dR

deoxy-Guanosine

N

N N

N

O

dR

NH

N

N N

N

O

dR

NH

HO

OH

R’

-R’Acr-dG3 -HCro-dG -CH3HNE-dG -CH(OH)-CH2-CH2-CH2-CH2-CH3

Acr-dGuo 1&2

AcroleinCrotonaldehyde

Hydroxynonenal (HNE)

R O

H

H

H

R O

N

N

HN

O

R O

S

HN

O

R O

HN

HN

O

Histidine Cysteine Lysine

α,β-unsaturated carbonyls(e.g. acrolein, HNE, cyclopentenoneprostaglandins)

Half-life of some reactive speciesHalf-life of some reactive species

RReactive specieseactive species Half-life(s)

HalfHalf--lifelife(s)(s)

Hydroxyl radical (•OH)Alcoxyl radical (RO•)Singlet oxygen (1O2)Peroxynitrite anion (ONOO-)Peroxyl radical (ROO•)Nitric oxide (•NO)Semiquinone radicalHydrogen peroxide (H2O2)

Superoxide anion (O2•-)

Hypochlorous acid (HOCl)

Hydroxyl radical (Hydroxyl radical (••OH)OH)Alcoxyl radical (ROAlcoxyl radical (RO••))Singlet oxygen (Singlet oxygen (11OO22))Peroxynitrite anion (ONOOPeroxynitrite anion (ONOO--))Peroxyl radical (ROOPeroxyl radical (ROO••))Nitric oxide (Nitric oxide (••NO)NO)Semiquinone radicalSemiquinone radicalHydrogen peroxide (HHydrogen peroxide (H22OO22))

Superoxide anion (OSuperoxide anion (O22••--))

HypochloHypochlorous acidrous acid (HOCl)(HOCl)

10-9

10-6

10-5

0.05 – 1.07

1 - 10minutes/hours

spontan. hours/days(accelerated by enzymes)spontan. hours/days

(by SOD accel. to 10-6)dep. on substrate

10-9

10-6

10-5

0.05 – 1.07

1 - 10minutes/hours

spontan. hours/days(accelerated by enzymes)spontan. hours/days

(by SOD accel. to 10-6)dep. on substrate

Physiolconc. (mol/l)PhysiolPhysiolconc.conc. ((mol/lmol/l))

1010--99

1010--99 -- 1010--77

1010--12 12 -- 1010--1111

Free Radical Reaction Rate

Lethality Mutagenesis Carcinogenesis Aging

Photosensitization

Ionizing RadiationUV Laser Pulses Oxidative Metabolism

XenobioticsEnzymeMyeloperoxidase

1O2 - e- •OH H2O2 O2•-

ROOH

DNA LipidsProteins

Diseases

ROS generated by physical or chemical processes can damage biomolecules. -difficulties and how to measure ROS with the singlet oxygen case

ROO.ROOHX = HOCl, ONOOH, ….

1O2 ?O2

•¯H2O2

HO•?

?What’s the Problem?

Singlet Molecular Oxygen in biological systems as an example

DirectLuminescence (Light emission) +Quenchers (N3

-), Solvent effect (Deuterated solvent, D2O)

Indirect but specificIsotopically labeled oxygenChemical trapping and HPLC-mass spectrometry(18O-labeled compounds)EPR

Synthesis / Characterization (LAOOH, PCOOH)18O-Labeled CompoundsChemical source of 18[1O2]

-Development of a pure source of isotopically labeled singlet oxygen, 18[1O2]

Tool for mechanistic studies !

Singlet Molecular Oxygen in biological systems as an example

Photosensitized generation of singlet O2

3sens *

1sens *

sens + hv

sens

sens + 1O2

3O2

SO2

[S + 3sens* ]S

ISC

3O2

SType II

S* + sensSO2

S + sens

S- + sens+

Type I

3O2

Energy transfer

Eletrontransfer

Hydrogen transfer

3sens *

1sens *

sens + hv

sens

sens + 1O2

3O23O2

SO2

[S + 3sens* ]S

ISC

3O2

SType II

S* + sensSO2SO2

S + sens

S- + sens+

Type I

3O23O2

Energy transfer

Eletrontransfer

Hydrogen transfer

Porphyria Patient

Porphyrin Nucleus

XNH

N

HN

R4

R3

R7

R8

R1 R2

N

R5R6

Porphyria is a diseases in which pigments called porphyrins accumulate in the skin, bones and teeth.

Angew-Chem.Int. Ed., 21, 343 ,1982

Activated leukocytes

Steinbeck et al. (1992) J. Biol. Chem., 267, 13425 Wentworth et al. (2002) Science, 298, 2195.Babior et al. (2003) Proc. Natl. Acad. Sci. USA, 100, 3031.

Bacterial killing: Respiratory burst

Fagocytosis

1O2

Lipid peroxidation

X

XH

X

XH

O2O2 LH

L

LH

LOO•

OO

H

X = OH, RO , ROO , CO3−

LH L LOO LOOH

ONOO-,

?

Russell Mechanism?Is singlet oxygen generated from lipid hydroperoxides?

Generation of Singlet Oxygen by the “Russell Mechanism”

1957Russell proposed that

termination reaction of 2 peroxyl radicals involves a

tetraoxide intermediary state.Russel J. Am. Chem. Soc., 79, 3871, 1957

1968Howard & Ingold showed the formation

of singlet oxygen in the reaction of sec-butylhydroperoxide with Ce4+.

Howard & Ingold J. Am. Chem. Soc., 90, 1057, 1968

2

LOO• + LOO•

O

H

O•

CO

H

R1

R2

CO

H

OO

OC

H

CO

H

OO

OC

H

LOOOOL L=O L-OH O2

C O* 3O2+ +CH OH

or

(100 kcal/mol)

C O CH OHC 1O2+ +(22 kcal/mol)

(75-80 kcal/mol)

!

Experimental Strategy Incubation: LA18O18OH + Metal ion (Cerium, Iron, peroxynitrite) → LA18O18O•

I- Synthesis of LA18O18OH

R OxOx

R

R

R

+

R: –C6H5

X : DPA DPA

R OxOx

R

R

R

+

R: –X : 18O or 16O atoms DPAxOxO

xO2(1Δg)

R OxOx

R

R

R

R

R

+

R: –C6H5

X : DPA DPA

R OxOx

R

R

R

R

R

+

R: –X : 18O or 16O atoms DPAxOxO

xO2(1Δg)

LA18O18O• [ LA18O18O 18O18OLA ]

18O

+

-

LA18O18O•

18O 18[1O2]

Chemical Trapping of [ xOxO(1Δg)] with DPA

Chemiluminescence

18O 18O •

II

III

IV- EPR

Synthesis of 18O-Labelled Linoleic AcidHydroperoxide (LA18O18OH)

Part I

18O2Methylene BlueIrradiation

Linoleic Acid (LA)OH

O

O18O18 H

O

OH

O18

OH

OO18 H

O18OH

O

O18 H

O18OH

O

O18 H

9101213

9-LA18O18OH

13-LA18O18OH

10-LA18O18OH

12-LA18O18OH

Structures of the 4 LAOOH isomers generatedfrom linoleic acid photooxidation under 18O2 atmosphere

1O2

Phosphatidylcholine(PC)

Phosphatidylcholine Hydroperoxides(PCOOH)

Synthesis of Synthesis of PhosphatidylcholinePhosphatidylcholine HydroperoxidesHydroperoxides by by PhotooxidationPhotooxidation using using MethyleneMethylene Blue as a Blue as a PhotosensitizerPhotosensitizer

OOH

Electrospray ionization mass spectra of LAOOH and LA18O18OH obtained in the negative ion mode.

LA16O16OH(M = 312)

LA18O18OH(M = 316)

+4

311

293

311

293

311

293

311

293

311

293

000000000000

311

m/z

Rel

ativ

e ab

unda

nce

(%)

315

295

00000060 80 100 120 140 160 180 200 220 240 260 280 300 320 340 36060 80 100 120 140 160 180 200 220 240 260 280 300 320 340 36060 80 100 120 140 160 180 200 220 240 260 280 300 320 340 36060 80 100 120 140 160 180 200 220 240 260 280 300 320 340 36060 80 100 120 140 160 180 200 220 240 260 280 300 320 340 36060 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360

OOH

O

OH

O18O18 H

O

OH

[M-H]– = 311

[M-H]– = 315

[M-H2O-H]– = 293

[M-H218O-H]– = 295

[M-H]–

[M-H2O-H]–

[M-H]–

[M-H218O-H]–

100

100

A

B

Generation of [18(1O2)] from LA18O18O•

18

R1

R2

LA18O18O•

•

LA18O18O 18O18OLA 1[18O18O]

H

C18

O

18O

18OOH

R2

R1

C

2 18 (1Δg)O

O

18

LA18O18OH / Metal ion (Cerium, Iron) or (peroxynitrite) → LA18O18O•

?

Part II

Detection and quantification of 1O2 bychemical trapping with

9,10-diphenylanthrancene (DPA)

Mass spectrometryHPLC/MS-MS

(18O-labeled compounds)

H

O

OH

O

O

O

O

1O2

1O2

1O2

“ene” type reaction

Cycloaddition [2+2]

Cycloaddition [4+2]

A + 1O2 A O2

Cycloaddition of Singlet Oxygen

Singlet Molecular Oxygen QuenchingSinglet Molecular Oxygen Quenching

Chemical QuenchingChemical Quenching

Q +Q + OO22((11ΔΔgg)) QQOO22

Physical QuenchingPhysical Quenching

Q +Q + OO22((11ΔΔgg)) Q + OQ + O22((33ΣΣ −−gg) +) + heatheat

kr

kq

Lycopene

Chemical Trapping of [ xOxO(1Δg)] with DPA

R Ox

Ox

R

R

R

+

R: –C6H5

X : DPA DPA

R Ox

Ox

R

R

R

+

R: –X : 18O or 16O atoms DPAxOxO

xO2(1Δg)

R Ox

Ox

R

R

R

R

R

+

R: –C6H5

X : DPA DPA

R Ox

Ox

R

R

R

R

R

+

R: –X : 18O or 16O atoms DPAxOxO

xO2(1Δg)

LA18O18O• [ LA18O18O 18O18OLA ]

18O

+

-

LA18O18O•

18O 18[1O2]

Chemical Trapping of [ xOxO(1Δg)] with DPA

..

LAOOH

+

?Russell

1O2

Ce4+, Fe2+

ONOO-HOCl

PC-OOH

ROS / RNS “Conversion” !ROOH

LA16O16OH + HOCl

LA18O18OH+ HOCl

Chemical Trapping of [ xOxO(1Δg)] by EAS andDetectionDetection ofof EASEASxxOOxxOO byby HPLCHPLC--ESIESI--MSMS

230

222 224 226 228 230 232 234 236 2380

100

% 228229

100

0

%

0

%

228

222 224 226 228 230 232 234 236 238

EAS16O16O

m/z

EAS16O18O

EAS16O16O

EAS18O18O

OSO3-

OSO3-

OSO3-

OSO3-

OO+ xOXO (1Δg)

xx

EAS EASxO2x= 16 or 18

m/z

Probe

Part III LuminescenceLight Emission (spectroscopic investigation)

Dimol emissionO2(1Δg) + O2(1Δg) → 2O2(3Σ−

g) + hν (λ= 634 and 703 nm)

Monomol emissionO2(1Δg) → O2(3Σ −g) + hν (λ= 1274 nm)

LA18O18O• [ LA18O18O 18O18OLA ]

18O

+

-

LA18O18O•

18O 18[1O2]

Chemical Trapping of [ xOxO(1Δg)] with DPA

Chemiluminescence

Photomultiplier

Ge-DiodeSample ChamberMonochromator 1O2

Detection and Characterization of 1O2

O2(1Δg)

O2(3Σg−)

hν

Singlet Oxygen Monomol light EmissionO2(1Δg) → O2(3Σ −g) + hν (λ= 1270 nm)

Improvement / “new” systems

Dimol emissionO2(1Δg) + O2(1Δg) → 2 O2(3Σg) + hν

(λ= 634 and 703 nm)

Khan and Kasha, 1963

Emission spectrum of thehydrogen peroxide-hypochloritereaction

H2O2 + OCl- → HOCl + HO2-

HOCl + HO2- → HOOCl + HO-

HOOCl + HO- → H2O + ClOO-

ClOO- → 1O2 + Cl-

-------------------------------------------H2O2 + OCl- → 1O2 + Cl- + H2O

1200

.00

1213

.70

1227

.00

1241

.00

1255

.00

1268

.30

1282

.00

1295

.60

1309

.00

1322

.90

1337

.00

0 5 10 15 20 25 30 35 40 45 50 55 60 65

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

Light Emission (cps)

Wavelength (nm)Time (μs)

Monomol emissionO2(1Δg) → O2(3Σ −g) + hν (λ= 1274 nm)

Near-IR

0 20 40 60 80 100 120

0

2

4

6

8

10

12

14

16

18

Ge-

Dio

de S

igna

l (10

4co

unts

)

Time (seconds)

NaN3injection

injection stop

Ce4+

injection

LAOOH + Ce4+ + NaN3 (B)

LAOOH + Ce4+ (A)

0 20 40 60 80 100 120

0

2

4

6

8

10

12

14

16

18

Ge-

Dio

de S

igna

l (10

4co

unts

)

Time (seconds)

NaN3injection

injection stop

Ce4+

injection

LAOOH + Ce4+ + NaN3 (B)

LAOOH + Ce4+ (A)

Ligh

t Em

issi

on (1

0-2

mV)

λ>57

0 nm

0

2

4

6

8

10

12

14

0 10 20 30 40 50 65Time (seconds)

H2O2 + OCl- (B)

LAOOH + Ce4+ (A)

injection

Characterization of singlet oxygen generated in the reactionof LAOOH and Ceric ion by chemiluminescence

Dimol light emission at λ > 570 nm

Monomol light emission at λ=1270 nm

O2(1Δg) + O2(1Δg) → 2O2(3Σ−g) + hν (λ= 634 and 703 nm)

O2(1Δg) → O2(3Σ −g) + hν (λ= 1270 nm)

ROOH / HOClE

mis

são

de lu

z em

127

0 nm

(102

coun

ts)

Tempo (s)

A

B

C

D

H2O2

LAOOH

t-BuOOH

CuOOH

HOClHOCl

HOCl HOCl

0 30 60 90 120 150

1

2

3

4

5

6

0 30 60 90 120 1500

1

2

3

4

5

60 30 60 90 120 150

0

1

2

3

4

5

6

0 30 60 90 120 1500

20406080

100120140160

0

CH3C CH3

OOH

CH3C CH3

OOH

C OOHH3C

H3CH3C C OOH

H3C

H3CH3C

O2 (1Δg) O2 (3Σg-) + hν (λ=1270 nm)

OOH

OHO

100 %

14 %

The presence of a hydrogen-α at the carbon to which the hydroperoxide is attached is essential for the generation of O2 (1Δg).

3400 3410 3420 3430 3440

B

A

g = 2.014

Magnetic Field (G)

EPR spectrum of linoleate peroxyl radicals, LAOO•.Reaction of LAOOH with HOCl

Direct detection

Part IV

2 LA18O18O•

[ LA18O18O 18O18OLA ] 18[1O2]

DHPN18O2 Near-IR(0,0) 1Δg →3Σg

-

Ligh

tEm

issi

on(1

03cp

s)

λ (nm)1200 1250 1300 1350

0.6

0.9

1.2

1.5

1.8

2.1

2.41270

HPLC-MS/MS

C6H5

O18

O18

C6H5

[M+H]+ = 367

[M - 18O18O]+

300 320 340 360 380 400 420 4400

100

0

100 330

367

m/z

[M+H]+%

18

(1Δg)O

O18+

OO

O

NH

O

NH

OH

OH

OH

OH

18

18

3400 3410 3420 3430 3440

B

A

g = 2.014

Magnetic Field (G)

EPR

Clean source

trapping

and Visible

Conclusion: Combination of Different Techniques !Take-Home Message

SourceFor example, lipids?

I- Synthesis of 18O-labeled sourceII- Chemiluminescence

III- Mass Spectrometry

IV- EPR

V- Clean chemical source of 18O-labeled reacive species, [1O2]

[1O2]

R

R

18O

O

R

R18

+18

Chemical source of [18O] isotopically labeled singlet oxygen 18[1O2]

? ..to elucidate mechanism of 1O2 reaction towards biological target

Excellent tool (+MS) for mechanistic studies of the reaction of singlet molecular oxygen in biological media.

0

100 330

363

0

100 330

363

100

Rel

ativ

e ab

unda

nce

(%)

[M+H]+ = 363

C6H5

O16

O16

C6H5

C6H5

O16

O18

C6H5

[M+H]+ = 365

[M+H]+

[M - 16O16O]+

[M - 16O18O]+

A

0

330

365

[M+H]+B

0

330

365

[M+H]+B

160 180 200 220 240 260 280 300 320 340 360 380 400 420 4400

100 330

367

m/z

C6H5

O18

O18

C6H5

[M+H]+ = 367[M - 18O18O]+

[M+H]+C

160 180 200 220 240 260 280 300 320 340 360 380 400 420 4400

100 330

367

m/z

C6H5

O18

O18

C6H5

[M+H]+ = 367[M - 18O18O]+

[M+H]+C

Positive APCI-mass spectra in the MS/MS mode of the endoperoxidesof 9,10-diphenylanthracene (DPAxOxO, x: 16O or 18O)

DPA18O18O

DPA18O16O

DPA16O16O

25 mM LA18O18OH + 25 mM Ce4+ + 60 mM DPA/37°C, 1 hour

Probe

18

18

18

18

16

16

16

16 1Δg3Σg-

+1Δg 3Σg

-+

18

18

18

18

16

16

16

16 1Δg3Σg-

+1Δg 3Σg

-+

150 160 170 180 190 200 210 220 230 240 250m/z

0

100230

228

EAS18O2 (m/z= 230)

Rel

ativ

e Ab

unda

nce

(%)

R= O SO3-

18O18O

R

R

R= O SO3-

18O18O

R

R

150 160 170 180 190 200 210 220 230 240 250m/z

0

100230

228

EAS18O2 (m/z= 230)

Rel

ativ

e Ab

unda

nce

(%)

R= O SO3-

18O18O

R

R

R= O SO3-

18O18O

R

R

150 160 170 180 190 200 210 220 230 240 2500

100 228

230

EAS16O2 (m/z= 228)

Rel

ativ

e Ab

unda

nce

(%)

m/z

R= O SO3-

16O16O

R

R

R= O SO3-

16O16O

R

R

150 160 170 180 190 200 210 220 230 240 2500

100 228

230

EAS16O2 (m/z= 228)

Rel

ativ

e Ab

unda

nce

(%)

m/z

R= O SO3-

16O16O

R

R

R= O SO3-

16O16O

R

R

R= OSO3-

R

R

R= OSO3-

R

R

EAS

EAS

18

18

18

18

16

16

16

16 1Δg3Σg-

+1Δg 3Σg

-+

18

18

18

18

16

16

16

16 1Δg3Σg-

+1Δg 3Σg

-+

150 160 170 180 190 200 210 220 230 240 250m/z

0

100230

228

EAS18O2 (m/z= 230)

Rel

ativ

e Ab

unda

nce

(%)

R= O SO3-

18O18O

R

R

R= O SO3-

18O18O

R

R

150 160 170 180 190 200 210 220 230 240 250m/z

0

100230

228

EAS18O2 (m/z= 230)

Rel

ativ

e Ab

unda

nce

(%)

R= O SO3-

18O18O

R

R

R= O SO3-

18O18O

R

R

150 160 170 180 190 200 210 220 230 240 2500

100 228

230

EAS16O2 (m/z= 228)

Rel

ativ

e Ab

unda

nce

(%)

m/z

R= O SO3-

16O16O

R

R

R= O SO3-

16O16O

R

R

150 160 170 180 190 200 210 220 230 240 2500

100 228

230

EAS16O2 (m/z= 228)

Rel

ativ

e Ab

unda

nce

(%)

m/z

R= O SO3-

16O16O

R

R

R= O SO3-

16O16O

R

R

R= OSO3-

R

R

R= OSO3-

R

R

EAS

EAS

Energy transfer between singlet (1Δg) and triplet (3Σg-)

molecular oxygen in aqueous solution.

“propagation” !