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DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES

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Chemical structures and names of superoxide spin traps ... V squez-Vivar et al. Methods in Enzymology 1999, 301: 169 ... In: Methods in Enzymology 301: 169-177. ... – PowerPoint PPT presentation

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Title: DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES


1
DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED
NITRONES Jeannette Vásquez Vivar,
Ph.D. Medical College of Wisconsin Milwaukee,
Wisconsin 53226 jvvivar_at_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
O2 O2 O2 H2O2 O2
2H
NO
GSH/GPx
XOD
Xanthine
ONOO-
Uric Acid
GSSG
CO2
RSH
Y
C(O)NH2
Cys-SH
CO3
Y RS
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-oxide 2,2-Dimethy
    l-3,4-dihydro-2H-pyrrole 1-oxide
  • DEPMPO
  • 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-
    N-oxide 2-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-pyr
    role-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
Application I Detection of Hydroxyl radical in
Superoxide-driven Reactions
Aconitase 4Fe-4S2 (active)
Aconitase 3Fe-4S1 (inactive)
Vásquez-Vivar et al. J Biol Chem 2000, 27514064
8
EPR Spin Trapping Detection of Superoxide with
DEPMPO
  • Characteristics
  • Unique EPR spectrum cis- and trans-DEPMPO-OOH
    (19) and
  • conformers exchange
  • Formation of persistent superoxide
    DEPMPO-OOH loss of signal
  • adduct (t1/215 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 (I1/2
    and large hyperfine
  • coupling constant 49 G) decreases
    sensitivity 0.2 nmol superoxide
  • Purification is difficult

10
EPR Spin Trapping Detection of Superoxide with
5-Ethoxycarbonyl-5-Methyl-1-Pyrroline N-oxide
(EMPO) and 15N-EMPO
O2
O2
(15N) I½
(14N) I1
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-EMPOlt0.05 nmoles
    superoxidegt14N-EMPO
  • Limitation
  • Purification
  • t½lt DEPMPO-OOH

12
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
L-Arginine, L-NAME and BH4 Effects on Superoxide
Release from eNOS
Vásquez-Vivar et al. Circulation 2000, 102 II-63
14
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, 362733
15
EPR Spin Trapping Detection of Superoxide with
BMPO
BH4-free nNOS
BH4 (10 nM)
Zhang et al Free Radical Biol Med 2001, 31599
Porter et al. Chem Res Toxicol 2005, 18864
  • 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
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,R6CH3 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
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, 145
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-CD 96.0
  • Protection against reduction (Ascorbate, GSH,
    GSH/GPx)
  • EMPOgtDEPMPOgtDMPO-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, 451043
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. Scans100, timelt4 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, 351149
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
    701198-1203
  • Clement et al. (2003) Deuterated analogues of the
    free radical trap DEPMPO synthesis and EPR
    studies. Org Biomol Chem 11591-1597
  • Frejaville et al. (1995) 5-(Diethoxyphosphory)l-5m
    ethyl-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 351149-1157
  • Karoui Tordo (2004) ESR-spin trapping in the
    presence of cyclodextrins. Tetrahedron Lett.
    451043-1045
  • 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
    18864-843
  • Roubaud et al. (1997) Quantitative measurement of
    superoxide generation using the spin trap
    5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxid
    e. 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 27514064-14069
  • Vásquez-Vivar et al. (2000) EPR spin trapping of
    superoxide from nitric oxide synthase Analusis
    (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
    362733-739
  • 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 31599-606
  • 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
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