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The use of EPR in The use of EPR in Nitric Oxide ResearchNitric Oxide Research
Neil Hogg,Neil Hogg,Medical College of WisconsinMedical College of Wisconsin
SFRBM 2005SFRBM 2005Austin, TXAustin, TX
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A STABLE free radical A STABLE free radical gasgas
N O
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Direct Detection of NO Direct Detection of NO by EPRby EPR
Broad ugly looking spectrumBroad ugly looking spectrum Need high concentrationNeed high concentration Unsuitable for biological detectionUnsuitable for biological detection
→ Spin ‘Trapping’
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Strategies used for the EPR Strategies used for the EPR detection of Nitric Oxidedetection of Nitric Oxide
Nitronyl Nitroxides
Fe/Dithiocarbamate
Hemoglobin/Myoglobin DNIC
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The Nitronyl NitroxidesThe Nitronyl Nitroxides
CPTIO
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Brief HistoryBrief History
First reported by Osieki and Ullman First reported by Osieki and Ullman (1968) JACS, 90, 1078(1968) JACS, 90, 1078
Proposed use as ‘NO dosimeter’ by Proposed use as ‘NO dosimeter’ by Nadeau and Boocock (1977) Anal. Nadeau and Boocock (1977) Anal. Chem. 49, 1672Chem. 49, 1672
Role as Biological NO spin trap. Role as Biological NO spin trap. Joseph et al (1993), BBRC, 192, 926Joseph et al (1993), BBRC, 192, 926
Antagonism of EDRF. Akaike et al Antagonism of EDRF. Akaike et al (1993) Biochemistry. 32, 827 (1993) Biochemistry. 32, 827
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MechanismMechanism
Joseph et al (1993), BBRC, 192, 926Joseph et al (1993), BBRC, 192, 926
NNOINO
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NO detectionNO detection
Joseph et al (1993), BBRC, 192, 926Joseph et al (1993), BBRC, 192, 926
∙NO
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Origin of EPR spectrum Origin of EPR spectrum (NNO with two equivalent N (NNO with two equivalent N
centers)centers)
1 2 3 2 1
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Origin of EPR spectrum Origin of EPR spectrum (INO with two inequivalent (INO with two inequivalent
N centers)N centers)
1 1 2 1 2 1 1
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Spectral Overlap of NNO Spectral Overlap of NNO and INOand INO
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Overlap Minimized on low-Overlap Minimized on low-field lines (Left side of field lines (Left side of
spectrum)spectrum)
Hogg et al (1995), Free Rad. Res., 22, 47Hogg et al (1995), Free Rad. Res., 22, 47
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Better way: Multiple Linear Better way: Multiple Linear regression uses all the dataregression uses all the data
MLR
0
10
20
30
40
50
60
0 10 20 30 40 50 60 70 80 90 100
Time (min)
[Rad
ical
] (µ
M)
(Simulation using WinSim)
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Reaction characteristics:Reaction characteristics:
Reaction of NO converts one radical to Reaction of NO converts one radical to another therefore not spin-trapping in the another therefore not spin-trapping in the traditional sense.traditional sense.
Rate const of ~1000 MRate const of ~1000 M-1-1ss-1-1 therefore fast therefore fast enough to compete with oxygen but not fast enough to compete with oxygen but not fast enough to compete with (e.g.) superoxide.enough to compete with (e.g.) superoxide.
Cannot use the ‘DMPO’ trick of using huge Cannot use the ‘DMPO’ trick of using huge amounts of trap to offset a small rate amounts of trap to offset a small rate constant due to the fact that the trap itself constant due to the fact that the trap itself has an EPR spectrumhas an EPR spectrum
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Problem with too much Problem with too much trap..trap..
If we used 10 mM CPTIO, then CPTIO spectrum would be
40 times bigger but CPTI would be
the same size!
250 uM CPTIO/200 uM Spermine NONOate
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Reaction stoichiometry?Reaction stoichiometry?
~2 NOs consumed per CPTIO
Hogg et al (1995), Free Rad. Res., 22, 47Hogg et al (1995), Free Rad. Res., 22, 47
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NO/CPTIO generates NO/CPTIO generates nitrosating intermediates.nitrosating intermediates.
Zhang et al (2004), Am.J.Physiol., 287, L467Zhang et al (2004), Am.J.Physiol., 287, L467
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CPTIO/CPTI are redox CPTIO/CPTI are redox active nitroxides – makes active nitroxides – makes
for many problems in for many problems in complex systemscomplex systems
● CPTIO○ CPTI■TEMPOL (control)
SIN-1 and CPTIO
Singh et al (1999), Arch. Biochem. Biophys.., 361, 331Singh et al (1999), Arch. Biochem. Biophys.., 361, 331
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Advantages/Advantages/DisadvantagesDisadvantages
Clear NO-dependent change in EPR Clear NO-dependent change in EPR spectrum allows quantification of kinetics of spectrum allows quantification of kinetics of NO formation.NO formation.
Works best in simple chemical systems as Works best in simple chemical systems as both reactant and product nitroxides are both reactant and product nitroxides are easily reduced by cellular reductants.easily reduced by cellular reductants.
The nitroxides are good oxidants and so care The nitroxides are good oxidants and so care must be taken to examine if the redox must be taken to examine if the redox properties of the nitroxides are altering the properties of the nitroxides are altering the chemistry of the systemchemistry of the system
Nitrogen dioxide is a product of the reaction Nitrogen dioxide is a product of the reaction and so these compounds my inhibit NO but and so these compounds my inhibit NO but enhance nitrosation/nitration reactions.enhance nitrosation/nitration reactions.
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Dinitrosyl Iron Complexes Dinitrosyl Iron Complexes (DNIC)(DNIC)
g = 2 region
1.921.941.961.9822.022.042.062.082.1
g Value
NO + Staph AureusNO + Staph Aureus ‘‘g=2.04’ signal g=2.04’ signal
indicates presence indicates presence of DNICof DNIC
Endogenous signal Endogenous signal from NO in all cell from NO in all cell typestypes
Likely derives from Likely derives from NO interaction with NO interaction with Iron Sulfur clustersIron Sulfur clusters
Stadler et al (1993), Arch.Biochem.Biophys., 302, 4Stadler et al (1993), Arch.Biochem.Biophys., 302, 4
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DithiocarbamatesDithiocarbamates
Mordvintcev, P et al (1991), Anal.Biochem., 199, 142Mordvintcev, P et al (1991), Anal.Biochem., 199, 142
SH
S
Fe2+C
HS
S
CR R
NO
Heat killed yeast loaded with Dethyldithiocarbamate/Fe
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Hydrophilic AlternativeHydrophilic Alternative
MGD (N-methyl-D-glucamine MGD (N-methyl-D-glucamine dithiocarbamate not Miller Genuine dithiocarbamate not Miller Genuine Draft)Draft)
N-Methyl-D-glucamine
Tsuchiya et al (2002), Biochem. J., 367, 771Tsuchiya et al (2002), Biochem. J., 367, 771
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In vivo NO spin trappingIn vivo NO spin trapping
Komarev et al (1993), BRRC, 195, 1191Komarev et al (1993), BRRC, 195, 1191
Time Course of S-band EPR signalfrom MGDFe2+ NO in the tail of a rat
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EPR imaging of NO using EPR imaging of NO using MGDMGD
Magn Reson Med. 1996 36:212-8.
Spatial mapping of nitric oxide generation in the ischemic heart using electron paramagnetic resonance imaging.
Kuppusamy P, Wang P, Samouilov A, Zweier JL.
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Problems….?Problems….?
Tsuchiya et al (2002), Biochem. J., 367, 771Tsuchiya et al (2002), Biochem. J., 367, 771
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Iron/DithiocarbamatesIron/DithiocarbamatesAdvantages/DisadvantagesAdvantages/Disadvantages
Actually traps the NO – therefore Actually traps the NO – therefore 1515N experiments N experiments can be used to identify the source of the signal.can be used to identify the source of the signal.
Use in in vivo NO spin trapping and EPR imaging.Use in in vivo NO spin trapping and EPR imaging. Potential for signal from sources other than NO Potential for signal from sources other than NO
(S-nitrosothiols/nitrite/HNO)(S-nitrosothiols/nitrite/HNO) Dithiocarbamates are good metal chelators and Dithiocarbamates are good metal chelators and
may inhibit metal ion-dependent enzymes (SOD, may inhibit metal ion-dependent enzymes (SOD, NOS etc).NOS etc).
A Cu/dithiocarbamate signal overlaps the Fe/NO A Cu/dithiocarbamate signal overlaps the Fe/NO signal and can cause problems in situations where signal and can cause problems in situations where copper is present. copper is present.
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Hemoglobin/MyoglobinHemoglobin/Myoglobin
Reacts with NO with rate constant > Reacts with NO with rate constant > 101077 M M-1-1ss-1-1
Cheap and plentiful.Cheap and plentiful. The reaction is accompanied by a The reaction is accompanied by a
UV-vis spectral change and a change UV-vis spectral change and a change in EPR spectrumin EPR spectrum
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Reactions of •NO with Reactions of •NO with HbHb
HbNONOHb
NOmetHbNOHbOsMx
sMx
117
117
106.2
3107.3
2
•
•
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Reaction of NO with Reaction of NO with MbOMbO22
Major spectral Major spectral changes going changes going from oxyMb to from oxyMb to metMb.metMb.
Watch out for Watch out for mixing artifacts mixing artifacts when using pure when using pure NO solutions!NO solutions!
Zhang and Hogg (2002), FRBM., 32, 1212Zhang and Hogg (2002), FRBM., 32, 1212
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EPR of metHbEPR of metHb
g~6
g~2
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EPR: metHb at 4 K (He)EPR: metHb at 4 K (He)
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Determination of metHb Determination of metHb concentration using concentration using
correlationcorrelation IF the shape of the IF the shape of the
line does not line does not change then don’t change then don’t double integrate.double integrate.
Plot spectrum Plot spectrum against that of a against that of a standard and the standard and the slope will slope will immediately give immediately give you the you the concentration.concentration.
y = 0.2481x + 12487
R2 = 0.8237
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
-10000 -5000 0 5000 10000 15000
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metHb standardizationmetHb standardization
y = 1.1507x - 0.0741
R2 = 0.9285
0
0.2
0.4
0.6
0.8
1
1.2
0 0.2 0.4 0.6 0.8 1 1.2
[expeted metHb] (µM)
[mea
sure
d m
etH
b](
µM
)
Sensitivity of ~ 100 nM
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metHb during NO metHb during NO inhalationinhalation
0
0.02
0.04
0.06
0.08
0.1
0.12
Pla
sma
met
Hb
(µ
M)
0
50
100
150
200
250[m
etH
b](
µM
)
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Advantage/Disadvantages of Advantage/Disadvantages of metHb detectionmetHb detection
Simply easily analyzable signal.Simply easily analyzable signal. Highly sensitive at liquid He Highly sensitive at liquid He
temperaturestemperatures Not necessarily specific for NO Not necessarily specific for NO
(peroxynitrite and other oxidants (peroxynitrite and other oxidants could do the same thing)could do the same thing)
NO is not ‘trapped’ and so cannot do NO is not ‘trapped’ and so cannot do 1515N experiments.N experiments.
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Reactions of •NO with Reactions of •NO with HbHb
HbNONOHb
NOmetHbNOHbOsMx
sMx
117
117
106.2
3107.3
2
•
•
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EPR: deoxyHb with NEM at EPR: deoxyHb with NEM at 77 K77 K
Fe2+ NON
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EPR: deoxyHb with IP6 at EPR: deoxyHb with IP6 at 77K77K
Fe2+ NON
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Analysys of HbNO Analysys of HbNO spectraspectra
0.00E+00
5.00E+07
1.00E+08
1.50E+08
2.00E+08
2.50E+08
0 10 20 30 40 50 60
[HbNO] (µM)
Are
a o
f A
bs
orb
tio
n
Sp
ec
tru
m (
Arb
itra
ry U
nit
s)
ii
iiii
Sig
nal I
nten
sity
-80000
-60000
-40000
-20000
0
20000
40000
60000
50 G
Sig
nal I
nten
sity
-80000
-60000
-40000
-20000
0
20000
40000
60000
50 G
A B
C
D
Piknova et al (2005), JBC.(in Press)Piknova et al (2005), JBC.(in Press)
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HbNO in blood after NO HbNO in blood after NO inhalationinhalation
Piknova et al (2005), JBC.(in Press)Piknova et al (2005), JBC.(in Press)
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0
0.5
1
1.5
2
2.5
3
-2 -1 0 1 2 3 4 5 6
Time (h)
Hb
NO
(µM
)
Vein 1
Artery 1
Vein 2
Artery 2
Vein 3
Artery 3
NO Inhalation stopped
Piknova et al (2005), JBC.(in Press)Piknova et al (2005), JBC.(in Press)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Artery Vein
Fra
cti
on
of
tota
l Hb
NO
6-Coord Alpha
6-Coord Beta
5-Coord Alpha
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Advantage/Disadvantages of Advantage/Disadvantages of HbNO detectionHbNO detection
Complex multi-component signal.Complex multi-component signal. Sensitive at liquid NSensitive at liquid N22 temperatures temperatures NO is trapped and so can do NO is trapped and so can do 1515N N
experiments.experiments. Needs to be deoxygenated!!Needs to be deoxygenated!!
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In conclusion…In conclusion… EPR is a phenomenally useful tool in NO EPR is a phenomenally useful tool in NO
research for both in vitro, ex vivo and in vivo research for both in vitro, ex vivo and in vivo studiesstudies
EPR direct detection of NO is possible after EPR direct detection of NO is possible after its stabilization by association with metal its stabilization by association with metal centers.centers.
EPR can also be detected by reactions that EPR can also be detected by reactions that form or destroy paramagnetic species.form or destroy paramagnetic species.
Homework: Design a non-metallic, non-redox Homework: Design a non-metallic, non-redox active NO spin-trap. Send compounds to Neil active NO spin-trap. Send compounds to Neil Hogg, Department of Biophysics, Medical Hogg, Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI. College of Wisconsin, Milwaukee, WI.
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AcknowledgementsAcknowledgements
National Biomedical EPR CenterMedical College of Wisconsin(EB001980)
Barbora PiknovaYanhong ZhangAgnes KeszlerNetanya SpencerRavinder Singh
Raman KalyanaramanBill AntholineBrian BennettJim Hyde
Mark GladwinAlan SchechterChris ReiterDany Kim-ShapiroRon Mason
..many others who’s work I have used