1 / 26

DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES Jeannette V á squez Vivar, Ph.D.

DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES Jeannette V á squez Vivar, Ph.D. Medical College of Wisconsin Milwaukee, Wisconsin 53226 jvvivar@mcw.edu. Outline. Chemical structures and names of superoxide spin traps Superoxide spin trapping with cyclic nitrones

janicepatel
Download Presentation

DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES Jeannette V á squez Vivar, Ph.D.

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES Jeannette Vásquez Vivar, Ph.D. Medical College of Wisconsin Milwaukee, Wisconsin 53226 jvvivar@mcw.edu

  2. Outline • Chemical structures and names of superoxide spin traps • Superoxide spin trapping with cyclic nitrones • Experimental considerations and applications • Quantification of superoxide from radical adduct data

  3. Sources of Superoxide and other Reactive Species •OH •NO2 HOCl HOBr Fe2+ BH4-deficient NOS NADPH Oxidase Mitochondria NO2– Cl-/Br- Aconitase MPO Drug metabolism O2O2•– + O2•–H2O2 + O2 2H+ NO GSH/GPx XOD Xanthine Uric Acid ONOO- GSSG CO2 RSH Y •C(O)NH2 Cys-SH Y• RS• CO3•– Cys-SOH

  4. Selection of the Spin Trap • Stable and easy to purify • Radical adduct is persistent • Radical adducts present distinctive EPR spectra • EPR spectra is simple

  5. Nitrones Commonly Used for Detection of Superoxide • DMPO 5,5-Dimethyl-1-pyrroline-N-oxide2,2-Dimethyl-3,4-dihydro-2H-pyrrole 1-oxide • DEPMPO 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide2-Diethylphosphono-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide(2-Methyl-3,4-dihydro-1-oxide-2H-pyrrol-2-yl) diethylphosphonate • EMPO 2-Ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide • BMPO 5-Tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide

  6. EPR Spin Trapping Detection of Superoxide with 5-Diethoxyphosphoryl-5-Methyl-1-Pyrroline N-oxide (DEPMPO) 2 min 15 min 20 min O2•– DEPMPO-OOH SOD 20 G DEPMPO-OH Frejaville et al. J Med Chem 1995, 38:258

  7. g= 2.018 20 G O O OOH OH (Et (Et O O ) ) P P 2 2 H H C C H H N N . . 3 3 O O Application I: Detection of Hydroxyl radical in Superoxide-driven Reactions Aconitase [4Fe-4S]2+ (active) Aconitase [3Fe-4S]1+ (inactive) Vásquez-Vivar et al. J Biol Chem 2000, 275:14064

  8. EPR Spin Trapping Detection of Superoxide with DEPMPO • Characteristics: • Unique EPR spectrum: cis- and trans-DEPMPO-OOH (1:9) and • conformers exchange • Formation of persistent superoxide • DEPMPO-OOH loss of signal • adduct (t1/2~15 min) is not followed by DEPMPO-OH • appearance trans-DEPMPO cis-DEPMPO

  9. EPR Spin Trapping Detection of Superoxide with DEPMPO Limitations: • Substitution with 5-methyl group with 31P (I=1/2 and large hyperfine • coupling constant ~49 G) decreases sensitivity ~0.2 nmol superoxide • Purification is difficult

  10. 10 G 10 G EPR Spin Trapping Detection of Superoxide with 5-Ethoxycarbonyl-5-Methyl-1-Pyrroline N-oxide (EMPO) and 15N-EMPO O2•– O2•– (15N) I=½ (14N) I=1 Olive et al. Free Radical Biol Med 1999, 28: 403 Zhang H et al.FEBS Lett 2000, 473: 58

  11. EPR Spin Trapping Detection of Superoxide with EMPO • Characteristics: • Distinctive EPR spectra EMPO-OOH composite of two conformers • EMPO-OOH is more persistent than DMPO-OOH • EMPO-OOH EMPO-OH • Sensitivity: 15N-EMPO<0.05 nmoles superoxide>14N-EMPO • Limitation: • Purification • t½< DEPMPO-OOH

  12. HEME FMN BH4 FAD L-Arg NADPH Application II. Quantification of Superoxide from Nitric Oxide Synthase O2•─ Electron acceptor Reduced • Electron acceptors such as • cytochrome c, lucigenin and NBT are • directly reduced by NOS • • In the case of redox-active compounds, • this reaction increases superoxide • generation • • BH4 reduces cytochrome c • Spin trapping is the ideal technique to detect superoxide from NOS Oxygenase Domain Reductase Domain Vásquez-Vivar et al. Methods in Enzymology 1999, 301: 169

  13. Ca2+/CaM L-Arg (0.1 mM) L-NAME (1.0 mM) 7,8-BH2 (0.1 mM) L-Arg/BH4 (2 µM) L-Arginine, L-NAME and BH4 Effects on Superoxide Release from eNOS Vásquez-Vivar et al. Circulation 2000, 102: II-63

  14. ) -1 140 14 mg protein 12 120 -1 10 100 •NO nmol min 8 80 6 60 -1 40 4 EMPO-OOH (nmoles min mg protein 2 20 0 0 -1 0.0001 0.001 0.01 0.1 1 10 100 BH ( m M) 4 +L-Arginine -L-Arginine Tetrahydrobiopterin Coordinates the Inhibition of Superoxide and the Stimulation of NO Formation from eNOS 97.7 nmoles O2•– min-1 mg protein-1 BH4 IC50 0.15 µM Vásquez-Vivar et al. Biochem J 2002, 362:733

  15. O2•– EPR Spin Trapping Detection of Superoxide with BMPO BH4-free nNOS + BH4 (10 nM) Zhang et al Free Radical Biol Med 2001, 31:599 Porter et al. Chem Res Toxicol 2005, 18:864 • Characteristics: • More persistent superoxide radical adducts • BMPO-OOH BMPO-OH • More sensitive measurements ~ 0.01 nmoles superoxide • Solid readily purified by recrystallization in MeOH

  16. O O O O O O H H H H H H 3 3 3 H H H 3 3 3 N N N O O O H H H 5 5 5 H H H 5 5 5 O O O C C C O O O R R R Superoxide Spin Trapping in the Presence of ß-Cyclodextrins Ramdom-ß-cyclodextrin (RM-ß-CD) R2, R3, R6= H and CH3 Dimethyl-ß-cyclodextrin (DM-ß-CD) R2,R6=CH3 R3 =H Bardelang et al. J Phys Chem B 2005, 109: 10521 Karoui et al. Chem Commun 2002, 24: 3030 Hydrophobic core 6.5 A 6 A

  17. H 3 H 3 H 5 H 5 10 G Superoxide Spin Trapping with BMPO in DM-ß-Cyclodextrin Containing Solutions Control 6 mM 12 mM KNITROXIDE =660 M-1 KNITRONE =230 M-1 25 mM 100 mM BMPO-OOH/ DM-b-CD Karoui et al. EPR-2005 Abstracts 2005, 1:45

  18. Properties of the Superoxide Radical Adduct and in ß-Cyclodextrin Inclusion Complex • Characteristics: • Enhanced persistence • Superoxide Radical Adduct t½ (min) Inclusion complex t½ (min) • DMPO-OOH 0.8 DMPO-OOH/RM-ß-CD 5.9 • EMPO-OOH 4.6 EMPO-OOH/ RM-ß-CD~38.0 • DEPMPO-OOH 14.0 DEPMPO-OOH/ RM-ß-CD96.0 • Protection against reduction (Ascorbate, GSH, GSH/GPx) • EMPO>DEPMPO>DMPO-OOH • Limitations: • • Changes in hyperfine coupling constant of the radical adduct in inclusion complex Bardelang et al. J Phys Chem B 2005, 109: 10521 Karoui & Tordo Tetrahedron Lett. 2004, 45:1043

  19. Quantification of Superoxide Using Spin Trapping Methodology • Considerations: • Spin trapping is a kinetic method • Calibration curve • Baseline • Simulation and identification of radical adduct species

  20. Superoxide Spin Trapping: Kinetic Analysis BIOLOGICAL PROCESS (E) P kd k1 k2 k3 Nitrone + O2•– [Nitroxide-OOH] Other Under steady-state concentrations of superoxide and saturating concentrations of the nitrone, then thus, and,

  21. Quantification of Superoxide Using Spin Trapping Methodology • Data acquisition: • i. Static scanning of spectra- 2D data set • ii. Rapid scan of spectra- 3D data set Kinetics of DMPO-OOH formation in incubations containing DMPO (10 mM), Xanthine (0.5 mM), Xanthine Oxidase (50 mU/ml) in phosphate buffer 50 mM, pH 7.4 and DTPA 0.1 mM. #Scans=100, time<4 s EPR Spectra after SVD (identification total components and isolation of the main component) and Spectral Analysis Keszler et al. Free Radical Biol Med 2003, 35:1149

  22. Calculating Initial Rates of Superoxide Radical Adduct Formation B. Standard reactions- known rates of superoxide flux (µM/min) - Xanthine Oxidase and hypoxanthine, xanthine or acetaldehyde - Spin trap concentration (10-100 mM), buffers (concentration, pH) C. Simulation and integration - Corrects baseline - Identify major component of analysis D. Calculating initial rates of superoxide radical formation - Use results with standard reaction to calculate superoxide concentration

  23. Superoxide Radical Adduct Data Analysis: Simulation - Simulation rationale: correction baseline drifting and analysis of one species only. Public EPR Software (WinSim) Table I. EPR Parameters of Superoxide Radical Adducts Radical Adduct Conformers Hyperfine coupling constant (G) (%) aN aHß aH aP aH DMPO-OOH 67 14.15 11.34 1.58 - - 33 14.09 11.78 0.17 [14N]EMPO-OOH 54 12.8 12.1 0.15 - - 46 12.8 8.6 - [15N] EMPO-OOH 55 17.9 12.0 0.3 - - 45 17.8 8.7 - BMPO-OOH 55 13.4 12.1 - - - 45 13.37 9.42 DEPMPO-OOH 50 13.4 11.9 0.8 52.5 0.4 50 13.2 10.3 0.9 48.5 0.43

  24. References-I • Bardelang et al. (2005) Inclusion complexes of PBN-type nitrones spin traps and their superoxide spin adducts with cyclodextrin derivatives: parallel determination of the association constants by NMR-titrations and 2D-EPR simulations. J Phys Chem B 109: 10521-10530 • Clement et al. (2005) Assignment of the EPR spectrum of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) superoxide spin adduct. J Org Chem 70:1198-1203 • Clement et al. (2003) Deuterated analogues of the free radical trap DEPMPO: synthesis and EPR studies. Org Biomol Chem 1:1591-1597 • Frejaville et al. (1995) 5-(Diethoxyphosphory)l-5methyl-1-pyrroline N-oxide: A new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen centered radicals. J Med Chem 38:258-265 • Keszler et al. (2003) Comparative investigation of superoxide trapping by cyclic nitrone spin traps: the use of singular value decomposition and multiple linear regression analysis. Free Radical Biol Med 35:1149-1157 • Karoui & Tordo (2004) ESR-spin trapping in the presence of cyclodextrins. Tetrahedron Lett. 45:1043-1045 • Karoui et al. (2002) Spin trapping of superoxide in the presence of ß- cyclodextrins. Chem Commun 24: 3030-3031 • Olive et al. (1999) 2-Ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide: evaluation of the spin trapping properties Free Radical Biol Med 28: 403-408

  25. References-II • Porter et al. (2005) Reductive activation of Cr(VI) by nitric oxide synthase. Chem Res Toxicol 18:864-843 • Roubaud et al. (1997) Quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide. Anal Biochem 247: 404-411 • Vásquez-Vivar et al. (1999) ESR Spin-trapping detection of superoxide generated by neuronal nitric oxide synthase. In: Methods in Enzymology 301: 169-177. • Vásquez-Vivar et al. (2000) Mitochondrial aconitase is a source of hydroxyl radical. J Biol Chem 275:14064-14069 • Vásquez-Vivar et al. (2000) EPR spin trapping of superoxide from nitric oxide synthaseAnalusis (Eur J Anal Chem)28: 487-492 • Vásquez-Vivar et al. (2000) BH4/BH2 ratio but not ascorbate controls superoxide and nitric oxide generation by eNOS. Circulation 102: II-63 • Vasquez-Vivar et al. (2002) The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study. Biochem J 362:733-739 • Zhang H et al. (2000) Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications. FEBS Lett 473: 58-62 • Zhao et al. (2001) Synthesis and biochemical applications of a solid cyclic nitrone spin trap: a relatively superior spin trap for detecting superoxide anions and glutathiyl radicals. Free Radical Biol Med 31:599-606 • Public EPR Software and Data Base: http://epr.niehs.nih.gov/pest.html

  26. Acknowledgements • B. Kalyanaraman • Joy Joseph • Hakim Karoui • Neil Hogg • Hao Zhang • Hongtao Zhao • Medical College of Wisconsin: • Free Radical Research Center • National Biomedical EPR Center

More Related