Transcript DETECTION OF SUPEROXIDE WITH DMPO AND IMPROVED NITRONES Jeannette Vásquez Vivar, Ph.D. Medical College of Wisconsin Milwaukee, Wisconsin 53226 [email protected].

DETECTION OF SUPEROXIDE WITH DMPO
AND IMPROVED NITRONES
Jeannette Vásquez Vivar, Ph.D.
Medical College of Wisconsin
Milwaukee, Wisconsin 53226
[email protected]
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
Sources of Superoxide and other Reactive Species
•OH
BH4-deficient NOS
•NO
Fe2+
NO2–
NADPH Oxidase
Cl-/Br-
Mitochondria
O2
Drug metabolism
Aconitase
O2•– + O2•–
2H+
Uric Acid
•C(O)NH2
RSH
HOCl
HOBr
MPO
H2O2
NO
XOD
Xanthine
2
+
O2
GSH/GPx
ONOOGSSG
CO2
Y
Cys-SH
Y•
RS•
CO3•–
Cys-SOH
Selection of the Spin Trap
• Stable and easy to purify
• Radical adduct is persistent
• Radical adducts present distinctive EPR spectra
• EPR spectra is simple
Nitrones Commonly Used for Detection of Superoxide
H3C
H3C
•
DMPO
5,5-Dimethyl-1-pyrroline-N-oxide
2,2-Dimethyl-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-pyrrole-1-oxide
5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide
•
BMPO
5-Tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide
N
O
O
(EtO)2P
H3 C
O
N
O
EtOC
H3 C
N
O
O
t-BuOC
H3C
N
O
EPR Spin Trapping Detection of Superoxide with
5-Diethoxyphosphoryl-5-Methyl-1-Pyrroline N-oxide (DEPMPO)
(EtO) 2(O)P
H3C
H
N
2 min
O
O2•–
H
15 min
H
OOH
(EtO) 2(O)P
H 3C
N
H
20 min
O
DEPMPO-OOH
SOD
DEPMPO-OH
20 G
Frejaville et al. J Med Chem 1995, 38: 258
Application I: Detection of Hydroxyl radical in
Superoxide-driven Reactions
O
OOH
(EtO)2P
H3C
.
O
N
Aconitase
[4Fe-4S]2+
H
(active)
Aconitase
[3Fe-4S]1+
O
OH
(EtO)2P
H3C
.
N
(inactive)
H
O
g=
2.018
20 G
Vásquez-Vivar et al. J Biol Chem 2000, 275:14064
EPR Spin Trapping Detection of Superoxide with DEPMPO
Characteristics:
• Unique
EPR spectrum: cis- and trans-DEPMPO-OOH (1:9) and
conformers exchange
H3 C
OOH
H
H
(EtO)2(O)P
O
N
H
H
H
(EtO)2(O)P
H
O
CH3
trans-DEPMPO
• Formation of persistent superoxide
adduct (t1/2~15 min)
OOH
H
N
H
H
H
cis-DEPMPO
• DEPMPO-OOH loss of signal
is not followed by DEPMPO-OH
appearance
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
EPR Spin Trapping Detection of Superoxide with
5-Ethoxycarbonyl-5-Methyl-1-Pyrroline N-oxide (EMPO) and 15N-EMPO
O
EtOC
O
EtOC
15
H3C
N
H3C
O
N
O
O2•–
O2•–
EtO(O)C
H3 C
OOH
14
N
H
O
OOH
15
N
H
O
(14N) I=1
10 G
EtO(O)C
H3 C
(15N) I=½
10 G
Olive et al. Free Radical Biol Med 1999, 28: 403
Zhang H et al.FEBS Lett 2000, 473: 58
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
• Sensitivity:
EMPO-OH
15N-EMPO
Limitation:
• Purification
• t½< DEPMPO-OOH
<0.05 nmoles superoxide>14N-EMPO
Application II.
Quantification of Superoxide from Nitric Oxide Synthase
Electron acceptor
Reduced
FMN
FAD
O2•─
HEME
BH4
L-Arg
NADPH
• 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
Reductase
Domain
Oxygenase
Domain
Vásquez-Vivar et al. Methods in Enzymology 1999, 301: 169
L-Arginine, L-NAME and BH4 Effects on Superoxide Release from eNOS
Ca2+/CaM
EtO(O)C
H3 C
OOH
14
N
H
O
L-Arg (0.1 mM)
L-NAME (1.0 mM)
7,8-BH2 (0.1 mM)
L-Arg/BH4 (2 µM)
Vásquez-Vivar et al. Circulation 2000, 102: II-63
Tetrahydrobiopterin Coordinates the Inhibition of Superoxide and
the Stimulation of NO Formation from eNOS
140
12
120
+L-Arginine
10
100
8
80
6
60
4
-L-Arginine
40
2
20
0
0
0.0001 0.001
0.01
0.1
1
10
nmol min-1 mg protein-1
14
•NO
EMPO-OOH (nmoles min -1mg protein -1)
97.7 nmoles O2•– min-1 mg protein-1 BH4 IC50 0.15 µM
100
BH 4(mM)
Vásquez-Vivar et al. Biochem J 2002, 362:733
EPR Spin Trapping Detection of Superoxide with BMPO
O
t-BuOC
H3C
N
BH4-free nNOS
O
O2•–
O
t-BuOC
H3C
OOH
N
+ BH4 (10 nM)
H
O
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
Superoxide Spin Trapping in the Presence of ß-Cyclodextrins
OR3
R2O
Ramdom-ß-cyclodextrin
(RM-ß-CD)
R3O
O
O
OR6
R2O
OR2
O
O
OR3
OR6
O
R2, R3, R6= H and CH3
R6O
O
O
R3O
OR6
O
6
R6O
R6O
R2O
R6O
O
O
OR2
Dimethyl-ß-cyclodextrin
(DM-ß-CD)
OR2
2
5
O
O
O
O
1
4
3 OR3
R2,R6=CH3
R3 =H
OR2
R3O
R3O
Bardelang et al. J Phys Chem B 2005, 109: 10521
Karoui et al. Chem Commun 2002, 24: 3030
Hydrophobic core
OOH
OOH
6.5 A
N
H3
H3
H5
OOH
N O
H5
O
H3
O
C
O
6A
O
C
O
H5
R
H3
H3
H5
H5
N O
O
H5
C
O
R
R
H3
Superoxide Spin Trapping with BMPO
in DM-ß-Cyclodextrin Containing Solutions
O
O
Control
6 mM
t-BuOC
t-BuOC
H3C
OOH
N
H
H3C
O
O
12 mM
N
KNITROXIDE =660 M-1
KNITRONE =230 M-1
25 mM
100 mM
H3
H3
H5
H5
10 G
BMPO-OOH / DM-b-CD
Karoui et al. EPR-2005 Abstracts 2005, 1:45
Properties of the Superoxide Radical Adduct
and in ß-Cyclodextrin Inclusion Complex
Characteristics:
• Enhanced persistence
Superoxide Radical Adduct t½ (min)
DMPO-OOH
EMPO-OOH
DEPMPO-OOH
0.8
4.6
14.0
Inclusion complex
t½ (min)
DMPO-OOH/RM-ß-CD
5.9
EMPO-OOH/ RM-ß-CD ~38.0
DEPMPO-OOH/ RM-ß-CD 96.0
• 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
Quantification of Superoxide Using Spin Trapping Methodology
Considerations:
• Spin trapping is a kinetic method
• Calibration curve
• Baseline
• Simulation and identification of radical adduct
species
Superoxide Spin Trapping: Kinetic Analysis
BIOLOGICAL PROCESS
(E)
k1
Nitrone + O
•–
2
k2
P
kd
[Nitroxide-OOH]
k3
Other
d ( Nitroxide OOH )
 k2 ( Nitrone)(O2 )  [k3 ( Nitroxide OOH )  kd ( Nitroxide OOH )2 ]
dt
Under steady-state concentrations of superoxide and saturating concentrations of
the nitrone, then
d (O2 )
 k1 ( E )  k2 ( Nitrone)(O2 )  0
dt
and,
thus,
k2 ( Nitrone)(O2 )  k1( E )
d ( Nitroxide  OOH )
 k1 ( E )  [k3 ( Nitroxide  OOH )  kd ( Nitroxide  OOH )2 ]
dt
Quantification of Superoxide Using Spin Trapping Methodology
A. 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
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
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
33
14.15
14.09
11.34
11.78
1.58
0.17
-
-
[14N] EMPO-OOH
54
46
12.8
12.8
12.1
8.6
0.15
-
-
-
[15N] EMPO-OOH
55
45
17.9
17.8
12.0
8.7
0.3
-
-
BMPO-OOH
55
45
13.4
13.37
12.1
9.42
-
-
-
DEPMPO-OOH
50
50
13.4
13.2
11.9
10.3
0.8
0.9
52.5
48.5
0.4
0.43
-
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
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 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 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
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