The use of EPR in Nitric Oxide Research

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The use of EPR in Nitric Oxide Research

• Neil Hogg,
• Medical College of Wisconsin
• SFRBM 2005
• Austin, TX

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A STABLE free radical gas
N
O
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Direct Detection of NO by EPR

• Broad ugly looking spectrum
• Need high concentration
• Unsuitable for biological detection

?
Spin Trapping
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Strategies used for the EPR detection of Nitric
Oxide
Fe/ Dithiocarbamate
Nitronyl Nitroxides
Hemoglobin/ Myoglobin
DNIC
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The Nitronyl Nitroxides
CPTIO
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Brief History

• First reported by Osieki and Ullman (1968) JACS,
  90, 1078
• Proposed use as NO dosimeter by Nadeau and
  Boocock (1977) Anal. Chem. 49, 1672
• Role as Biological NO spin trap. Joseph et al
  (1993), BBRC, 192, 926
• Antagonism of EDRF. Akaike et al (1993)
  Biochemistry. 32, 827

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Mechanism
INO
NNO
Joseph et al (1993), BBRC, 192, 926
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NO detection
NO
Joseph et al (1993), BBRC, 192, 926
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Origin of EPR spectrum (NNO with two equivalent N
centers)
1 2 3
2 1
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Origin of EPR spectrum (INO with two inequivalent
N centers)
1 1 2 1 2
1 1
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Spectral Overlap of NNO and INO
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Overlap Minimized on low-field lines (Left side
of spectrum)
Hogg et al (1995), Free Rad. Res., 22, 47
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Better way Multiple Linear regression uses all
the data
MLR
(Simulation using WinSim)
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Reaction characteristics

• Reaction of NO converts one radical to another
  therefore not spin-trapping in the traditional
  sense.
• Rate const of 1000 M-1s-1 therefore fast enough
  to compete with oxygen but not fast enough to
  compete with (e.g.) superoxide.
• Cannot use the DMPO trick of using huge amounts
  of trap to offset a small rate constant due to
  the fact that the trap itself has an EPR spectrum

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Problem with too much trap..
If we used 10 mM CPTIO, then CPTIO spectrum would
be 40 times bigger but CPTI would be the same
size!
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Reaction stoichiometry?
2 NOs consumed per CPTIO
Hogg et al (1995), Free Rad. Res., 22, 47
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NO/CPTIO generates nitrosating intermediates.
Zhang et al (2004), Am.J.Physiol., 287, L467
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CPTIO/CPTI are redox active nitroxides makes
for many problems in complex systems
SIN-1 and CPTIO
? CPTIO ? CPTI TEMPOL (control)
Singh et al (1999), Arch. Biochem. Biophys..,
361, 331
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Advantages/Disadvantages

• Clear NO-dependent change in EPR spectrum allows
  quantification of kinetics of NO formation.
• Works best in simple chemical systems as both
  reactant and product nitroxides are easily
  reduced by cellular reductants.
• The nitroxides are good oxidants and so care must
  be taken to examine if the redox properties of
  the nitroxides are altering the chemistry of the
  system
• Nitrogen dioxide is a product of the reaction and
  so these compounds my inhibit NO but enhance
  nitrosation/nitration reactions.

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Dinitrosyl Iron Complexes (DNIC)

• NO Staph Aureus
• g2.04 signal indicates presence of DNIC
• Endogenous signal from NO in all cell types
• Likely derives from NO interaction with Iron
  Sulfur clusters

Stadler et al (1993), Arch.Biochem.Biophys., 302,
4
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Dithiocarbamates
NO
Heat killed yeast loaded with Dethyldithiocarbama
te/Fe
Mordvintcev, P et al (1991), Anal.Biochem., 199,
142
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Hydrophilic Alternative

• MGD (N-methyl-D-glucamine dithiocarbamate not
  Miller Genuine Draft)

N-Methyl-D-glucamine
Tsuchiya et al (2002), Biochem. J., 367, 771
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In vivo NO spin trapping
Time Course of S-band EPR signal from MGDFe2 NO
in the tail of a rat
Komarev et al (1993), BRRC, 195, 1191
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EPR imaging of NO using MGD

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.?
Tsuchiya et al (2002), Biochem. J., 367, 771
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Iron/DithiocarbamatesAdvantages/Disadvantages

• Actually traps the NO therefore 15N experiments
  can be used to identify the source of the signal.
• Use in in vivo NO spin trapping and EPR imaging.
• Potential for signal from sources other than NO
  (S-nitrosothiols/nitrite/HNO)
• Dithiocarbamates are good metal chelators and may
  inhibit metal ion-dependent enzymes (SOD, NOS
  etc).
• A Cu/dithiocarbamate signal overlaps the Fe/NO
  signal and can cause problems in situations where
  copper is present.

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Hemoglobin/Myoglobin

• Reacts with NO with rate constant gt 107 M-1s-1
• Cheap and plentiful.
• The reaction is accompanied by a UV-vis spectral
  change and a change in EPR spectrum

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Reactions of NO with Hb
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Reaction of NO with MbO2

• Major spectral changes going from oxyMb to metMb.
• Watch out for mixing artifacts when using pure NO
  solutions!

Zhang and Hogg (2002), FRBM., 32, 1212
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EPR of metHb
g6
g2
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EPR metHb at 4 K (He)
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Determination of metHb concentration using
correlation

• IF the shape of the line does not change then
  dont double integrate.
• Plot spectrum against that of a standard and the
  slope will immediately give you the concentration.

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metHb standardization
Sensitivity of 100 nM
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metHb during NO inhalation
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Advantage/Disadvantages of metHb detection

• Simply easily analyzable signal.
• Highly sensitive at liquid He temperatures
• Not necessarily specific for NO (peroxynitrite
  and other oxidants could do the same thing)
• NO is not trapped and so cannot do 15N
  experiments.

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Reactions of NO with Hb
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EPR deoxyHb with NEM at 77 K
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EPR deoxyHb with IP6 at 77K
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Analysys of HbNO spectra
Piknova et al (2005), JBC.(in Press)
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HbNO in blood after NO inhalation
Piknova et al (2005), JBC.(in Press)
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Piknova et al (2005), JBC.(in Press)
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Advantage/Disadvantages of HbNO detection

• Complex multi-component signal.
• Sensitive at liquid N2 temperatures
• NO is trapped and so can do 15N experiments.
• Needs to be deoxygenated!!

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In conclusion

• EPR is a phenomenally useful tool in NO research
  for both in vitro, ex vivo and in vivo studies
• EPR direct detection of NO is possible after its
  stabilization by association with metal centers.
• EPR can also be detected by reactions that form
  or destroy paramagnetic species.
• Homework Design a non-metallic, non-redox active
  NO spin-trap. Send compounds to Neil Hogg,
  Department of Biophysics, Medical College of
  Wisconsin, Milwaukee, WI.

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Acknowledgements
Barbora Piknova Yanhong Zhang Agnes
Keszler Netanya Spencer Ravinder Singh Raman
Kalyanaraman Bill Antholine Brian Bennett Jim
Hyde Mark Gladwin Alan Schechter Chris
Reiter Dany Kim-Shapiro Ron Mason ..many others
whos work I have used
National Biomedical EPR Center Medical College of
Wisconsin (EB001980)