Application Techniques of Electron Spin Resonance Ronald P. Mason and JinJie Jiang National Institute of Environmental Health Sciences, NIH Research Triangle Park, NC 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 17 O in O 2. - generated during steady-state oxidation of xanthine catalyzed by xanthine oxidase. Both the 11-line spectrum from 17 O 17 O. - and the six- line spectrum from 17 O 16 O. - 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, , 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., , 1999

Steady-State Condition Is When the Rate of Formation Is Equal to the Rate of Decay X R.R. R-R R. + R. 2  Ms -1 8 X 10 5 M -1 s Time (S) R. (  M) 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, , 1993 Mendes, P Time (S) R-R (mM)

Detection of Nitrobenzene Anion Radical in An Anaerobic Microsomal Incubation NADP + Glucose-6-phosphate Glucose-6-phosphate dehydrogenase KCl-Tris-MgCl 2 buffer: 150 mM KCl, 20 mM Tris (pH7.4), and 5 mM MgCl 2 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 mixture 1.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-MgCl 2 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 handling 1.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 , 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, 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 X R.R. R-R R. + R. 2  s -1 5 X 10 9 M -1 s R. (  M) Time (S) X (mM) Time (S) R-R (mM) Time (S)

Steady-State Condition Is Unsustainable with Rapid Substrate Depletion X R.R. R-R R. + R. 200  Ms -1 5 X 10 9 M -1 s -1 Time (S) R-R (mM) X (mM) Time (S) R. (  M) Time (S)

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/H 2 O 2 and Bovine Lactoperoxidase/H 2 O 2 Fischer, V., Harman L.S., West P.R., and R.P. Mason, Chem.-Biol. Interactions, 60, , 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: 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 CCl 4 1.Fast the rats for 20 h. 2.Homogenize CCl 4, 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 handling 1.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 CCl 4 Hanna, P.M., Kadiiska, M.B., Jordan, S.J., and Mason, R.P., Chem. Res. Toxicol., 6, , 1993.

Protocol 3. Biliary Detection of Radical Adduct of Halothane-Derived Free Radical Metabolite Equipment and reagents Male rats: 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 BrClCHCF 3 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 BrClCHCF 3 i.g.

Protocol 3. (continue) B. Collection and treatment of bile 1.Collect bile every 15 min into plastic Eppendorf tubes. 2.Freeze immediately on dry ice and store at –70 o C 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 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 BrClCHCF 3 -derived radical adducts.

Bile samples collected every 20 min for 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: , 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 FH 2 F FH. RNO 2 RNO 2 -. O2O2 O2.-O2.-

Steady-State Metronidazole Anion Radical under Anaerobic Conditions X R.R. R-R R. + R. 2  Ms -1 8 X 10 5 M -1 s Time (S) R. (  M) Time (S) X (mM) Time (S) R-R (mM)

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 B0B0

Kinetic Simulation of DMPO Superoxide Adduct and Metronidazole Anion Radical Appearance and Disappearance R. (  M) Time (s) O 2 (  M) Time (s) DMPO/O 2. - (  M) Time (s) X R.R. R-R R. + R. 80  Ms -1 8 X 10 5 M -1 s -1 DMPO + O 2. - DMPO/ O 2. - DMPO x R. + O X 10 6 M -1 s -1 X + O 2. - O O X 10 5 M -1 s -1 O 2 + H 2 O X 10 2 M -1 s X s -1 DMPO/ O

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