Application Techniques of Electron Spin Resonance

Ronald P. Mason and JinJie Jiang

National Institute of Environmental Health Sciences, NIH

Research Triangle Park, NC 27709

DIVISION OF INTRAMURAL RESEARCH Laboratory of Pharmacology and Chemistry

Methods

Direct ESR

Spin-Trapping

Techniques

Freeze Quench

Snap Freeze

Flat Cells

AquaX

Steady-State

Fast-Flow

Stopped-Flow

Rapid Sampling

Folch Extraction

Bile Cannulation

Other Techniques

Applications

In Vivo

In Vitro

In Situ

Direct ESR

“Freeze” the reaction

1) freeze quench (in vitro)

2) snap freeze (in vitro, ex vivo)

Steady-State

1) Rapid sampling (in vitro )

2) Fast-flow (in vitro)

Freeze Quench: O-17 Hyperfine Splitting in Electron Paramagnetic Resonance Spectrum of Enzymically Generated Superoxide

The electron paramagnetic

resonance spectrum of 17O in O2

.- generated during steady-state oxidation of xanthine catalyzed by xanthine oxidase. Both the 11-line

spectrum from 17O17O.- and the six-

line spectrum from 17O16O.- were

detected. The results provide final confirmation that one-electron reduction of oxygen can occur in biological systems

Bray, R.C., Pick, F.M. and Samuel, D., Eur J. Biochem, 15 352-355, 1970

Snap Freeze: Detection of Nitrosyl Hemoglobin in Venous Blood in the Treatment of Sickle Cell Anemia with Hydroxyurea

The nitrosyl hemoglobin complex could be detected as early as 30 min after administration of hydroxyurea and persisted up to 4 h. Our observations support the hypothesis that the ability of hydroxyurea to ease the vaso-occlusive phenomena may, in part, be attributed to vasodilation and/or decreased platelet activation induced by nitric oxide.

Glover RE, Ivy ED, Orringer EP, Maeda H, Mason RP, Mol. Pharm., 55 1006-1010, 1999

Steady-State Condition Is When the Rate of Formation Is Equal to the Rate of Decay

XR

.

R-RR.+ R

.

2 Ms-1

8 X 105 M -1s-1

0.1

1

10

0 200 400 600 800 1000 1200Time (S)

R. (

M)

1

10

100

0 200 400 600 800 1000 1200

Time (S)

X (

mM

)

Mendes, P., GEPASI: A software package for modeling the dynamics, steady states and control of biochemical and other systems. Comput. Applic. Biosci. 9, 563-571, 1993

Mendes, P. 0.2

0.6

0.8

1.0

1.2

0 200 400 600 800 1000 1200

Time (S)

R-R

(m

M)

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Detection of Nitrobenzene Anion Radical in An Anaerobic Microsomal Incubation

• NADP+

• Glucose-6-phosphate• Glucose-6-phosphate dehydrogenase• KCl-Tris-MgCl2 buffer: 150 mM KCl, 20

mM Tris (pH7.4), and 5 mM MgCl2

• Nitrobenzene

Equipment and reagents

• Fresh rat liver microsomes (40 mg protein/ml)

• Rubber stopped serum bottle • Nitrogen tank (oxygen-free)• ESR spectrometer

A. Preparation of incubation mixture1. Mix nitrobenzene (2 mM) and an NADPH-generating system consisting of

NADP+ (0.8 mM), glucose-6-phosphate (11 mM), and 4 units of glucose-6-phosphate dehydrogenase in 3 ml of KCl-Tris-MgCl2 buffer.

2. Add to rubber-stopped serum bottle.3. Bubble nitrogen gas into solutions for 5 min with the only exit being

through the aqueous flat cell.4. Add 12 mg of rat hepatic microsomal protein through the rubber stopper

with a syringe.5. Continue bubbling with nitrogen gas for 20 sec.

Protocol 1.

Apparatus for Filling The ESR Flat Cell under A Nitrogen Atmosphere

Mason, R.P.: Assay of in situ radicals by electron spin resonance. Meth. Enzymol. 105:416‑422, 1984

B. Sample handling1. Lower the stainless-steel needle tubing below the surface of the

solution.2. Force solution into the aqueous flat cell with pressure of the

nitrogen gas until full.3. Close ground glass cap and vent nitrogen pressure by inserting

a second needle into the rubber stopper.4. Remove needle tubing from the force-fitted septum in the bottom

of the flat cell.5. Mount the flat cell in the microwave cavity with aqueous cell

holders.6. Tune and operate ESR spectrometer to obtain spectrum of

nitrobenzene anion radical.

Protocol 1. (continue)

Mason, R.P.: In vitro and in vivo detection of free radical metabolites with electron spin resonance. In: Punchard, N.A. and Kelly, F.J. (Eds.), Free Radicals: A Practical Approach. IRL Press at Oxford University Press, New York, pp. 11-24, 1996.

Apparatus for Filling The ESR Flat Cell under A Nitrogen Atmosphere

Mason, R.P.: Assay of in situ radicals by electron spin resonance. Meth. Enzymol. 105:416‑422, 1984

Electron Spin Resonance Evidence for Nitroaromatic Free Radical Intermediates

Mason, R.P. and Holtzman, J.L., Biochemistry 14:1626‑1632, 1975.

Spectrum a is of 1.1 M p-nitrobenzoate dianion radical formed in a microsomal incubation. Spectrum b is nitrobenzene anion radical under the same conditions as spectrum a. Spectrum c is of 0.2 M nitrobenzene anion radical formed in a mitochondrial incubation.

Nearly Undetectable Radical Formation When Radical Decay Is Diffusion Limited

XR

.

R-RR.+ R

.

2 s-1

5 X 109 M -1s-1

0.01

0.1

0 200 400 600 800 1000 1200

R. (

M)

Time (S)

1

10

100

0 200 400 600 800 1000 1200

X (

mM

)

Time (S)

0 200 400 600 800 1000 1200

R-R

(m

M)

Time (S)

0.2

0.6

0.8

1.0

1.2

0.0

0.4

Steady-State Condition Is Unsustainable with Rapid Substrate Depletion

XR

.

R-RR.+ R

.

200 Ms-1

5 X 109 M-1s-1

Time (S)

0

1

2

3

4

5

0 200 400 600 800 1000 1200

R-R

(m

M)

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mM

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Time (S)

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Fast-Flow Technique for Obtaining Steady-State Condition with Rapid Substrate Depletion

ESR Spectroscopy Employing A Millisecond Time Scale Fast-Flow Method Has Revealed the Formation of a Transient Phenoxyl Radical in the Reaction of Acetaminophen with Horseradish

Peroxidase/H2O2 and Bovine Lactoperoxidase/H2O2

Fischer, V., Harman L.S., West P.R., and R.P. Mason, Chem.-Biol. Interactions, 60, 115-127, 1986

Spin-Trapping

• Selecting the spin trap (stability, adduct stability, distributions, toxicity, trapping efficiency, solubility, structure information, etc.)

• Artifacts and control experiments

• Increase the spin adduct concentration: extraction

• Identify the radicals

• Increase sensitivity: flat cells, etc.

Protocol 2. In Vivo Spin Trapping of the Trichloromethyl Radical Metabolite of Carbon Tetrachloride

Equipment and reagents

• Male, Sprague-Dawley rats: 250-300 g• Phenyl-tert-butylnitrone (PBN): 1 ml of a

140 mM solution in 20 mM phosphate buffer, pH 7.4

• Carbon tetrachloride: 1.2 ml/kg body weight

• Corn oil

• Chloroform• Methanol• Anhydrous sodium sulfate• Nitrogen tank• No plasticware (will leach nitroxides

into organic solvents)

A. Administration of spin trap and CCl4

1. Fast the rats for 20 h.2. Homogenize CCl4, PBN, or both with corn oil.3. Administer by stomach tube.4. with nitrogen gas for 20 sec.

Protocol 2. (continue)

B. Folch extraction and sample handling1. Kill treated rats after 2 h.2. Immediately remove livers and homogenize in chloroform-

methanol (2:1, v/v) in glass according to reference.3. Dry sample with anhydrous sodium sulfate.4. Remove chloroform layer and evaporate solvent under nitrogen

gas until volume is reduced to 0.5 ml.5. Transfer sample to 3 mm quartz tube and slowly bubble with

nitrogen gas for 3 min using long needle or tubing.6. Mount sample and tune and operate ESR spectrometer to obtain

six-line spectrum of the PBN-trichloromethyl radical adduct.

Spin Trapping in Vivo of the Trichloromethyl Radical Metabolite of CCl4

Hanna, P.M., Kadiiska, M.B., Jordan, S.J., and Mason, R.P., Chem. Res. Toxicol., 6, 711-717, 1993.

Protocol 3. Biliary Detection of Radical Adduct of Halothane-Derived Free Radical Metabolite

Equipment and reagents

• Male rats: 350-400 g• Halothane• PBN: 50 mg/kg dissolved in deionized

water at 140 mM• Oxygen and nitrogen tanks• Eppendorf tubes

• Dry ice• Potassium ferricyanide• Polyethylene tubing (0.28 mm i.d. and

0.61 mm o.d.)• ESR spectrometer

A. Administration of spin trap and BrClCHCF3

1. Fast the rats for 20 h.2. Anaesthetize rat with Nembutal.3. Cannulate bile duct with a segment of polyethylene tubing.4. Inject PBN i.p. and BrClCHCF3 i.g.

Protocol 3. (continue)

B. Collection and treatment of bile1. Collect bile every 15 min into plastic Eppendorf tubes.2. Freeze immediately on dry ice and store at –70oC until ESR

analysis (within a few days).3. Thaw bile and transfer to quartz flat cell.4. Bubble with oxygen to oxidize reduced radical adducts and then

with nitrogen to narrow the spectral line width (or add 0.1-1 mM potassium ferricyanide).

5. Mount the flat cell in the microwave cavity with aqueous cell holders.

6. Tune and operate ESR spectrometer to obtain spectrum of two BrClCHCF3-derived radical adducts.

Bile samples collected every 20 minfor 2 h in tube containing DP and BC

Free Radical Metabolism of Halothane in Vivo: Radical Adducts Detected in Bile

Knecht, K.T., DeGray, J.A., and Mason, R.P., Mol. Pharmacol. 41: 943-949, 1992.

Rapid Sampler Technique with Gilford Rapid Sampler

Mason, R.P.: Assay of in situ radicals by electron spin resonance. Meth. Enzymol. 105:416‑422, 1984

Rapid Sampler Technique with Commercial Bruker Auto-Sampler and AquaX

Metronidazole Anion Radical

Perez-Reyes, E., Kalyanaraman, B., and Mason, R.P., Mol. Pharmacol. 17:239‑244, 1980

NADP+

NADPH

FH2

F

FH.

RNO2

RNO2-

.O2

O2

.-

Steady-State Metronidazole Anion Radical under Anaerobic Conditions

XR

.

R-RR.+ R

.

2 Ms-1

8 X 105 M -1s-1

0.1

1

10

0 200 400 600 800 1000 1200Time (S)

R. (

M)

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10

100

0 200 400 600 800 1000 1200

Time (S)

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ESR Spectrum of Metronidazole Anion Radical and Computer Simulation

DMPO Superoxide Radical Adduct Formed by Futile (Redox) Cycling of Metronidazole Anion Radical

Time Course of DMPO Superoxide Adduct and Metronidazole Anion Radical

B0

Kinetic Simulation of DMPO Superoxide Adduct and Metronidazole Anion Radical Appearance and

Disappearance

0.0

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)

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DM

PO

/O2

. - (M

)

Time (s)

X R.

R-RR. + R.

80 Ms-1

8 X 105 M -1s-1

DMPO + O2.- DMPO/ O2

.-

DMPOx

R. + O2

7.8 X 106 M -1s-1

X + O2.-

O2.- + O2

.-2 X 105 M -1s-1

O2 + H2O2

1.7 X 102 M -1s-1

1.2 X 10-2 s-1

DMPO/ O2.-

0.8

Summary of How to Catch A Radical

Stop decay by freezing

1) Freeze quench (millisecond)

2) Snap freeze (seconds)

Steady-state by continuous generation

1) Flat cells with ample substrates

2) Rapid sampling for kinetics on second time scale

3) Fast-flow for radicals with diffusion-limited second-order decay

Spin trapping

1) Has a higher steady-state concentration than direct ESR because of the slower decay rate of the radical adduct

2) In vivo spin trapping is possible for extremely stable radical adducts