The use of EPR in Nitric Oxide Research

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

A STABLE free radical gas

N O

Direct Detection of NO by EPR

 Broad ugly looking spectrum  Need high concentration  Unsuitable for biological detection → Spin ‘Trapping’

Strategies used for the EPR detection of Nitric Oxide

Fe/ Dithiocarbamate Nitronyl Nitroxides Hemoglobin/ Myoglobin DNIC

The Nitronyl Nitroxides

CPTIO

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

NNO

Mechanism

INO Joseph et al (1993), BBRC, 192, 926

NO detection

∙ NO Joseph et al (1993), BBRC, 192, 926

Origin of EPR spectrum (NNO with two equivalent N centers)

1 2 3 2 1

Origin of EPR spectrum (INO with two inequivalent N centers)

1 1 2 1 2 1 1

Spectral Overlap of NNO and INO

Overlap Minimized on low-field lines (Left side of spectrum)

Hogg et al (1995), Free Rad. Res., 22, 47

Better way: Multiple Linear regression uses all the data

MLR (Simulation using WinSim) 60 50 40 30 20 10 0 0 10 20 30 40 50

Time (min)

60 70 80 90 100

Reaction characteristics:

   Reaction of NO converts one radical to another therefore not spin-trapping in the traditional sense.

Rate const of ~1000 M with (e.g.) superoxide.

-1 s -1 therefore fast enough to compete with oxygen but not fast enough to compete 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

Problem with too much trap..

250 uM CPTIO/200 uM Spermine NONOate

If we used 10 mM CPTIO, then CPTIO spectrum would be 40 times bigger but CPTI would be the same size!

Reaction stoichiometry?

~2 NOs consumed per CPTIO Hogg et al (1995), Free Rad. Res., 22, 47

NO/CPTIO generates nitrosating intermediates.

Zhang et al (2004), Am.J.Physiol., 287, L467

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

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.

Dinitrosyl Iron Complexes (DNIC) g = 2 region

2.1

2.08

2.06

2.04

2.02

2 1.98

1.96

1.94

1.92

    NO + Staph Aureus ‘g=2.04’ signal indicates presence of DNIC Endogenous signal from NO in all cell types Likely derives from NO interaction with Iron Sulfur clusters

g Value

Stadler et al (1993), Arch.Biochem.Biophys., 302, 4

Dithiocarbamates

R C SH S NO Fe 2+ HS C S R Heat killed yeast loaded with Dethyldithiocarbamate/Fe Mordvintcev, P et al (1991), Anal.Biochem., 199, 142

Hydrophilic Alternative

 MGD (N-methyl-D-glucamine dithiocarbamate not Miller Genuine Draft) N-Methyl-D-glucamine Tsuchiya et al (2002), Biochem. J., 367, 771

In vivo NO spin trapping

Time Course of S-band EPR signal from MGDFe 2+ NO in the tail of a rat Komarev et al (1993), BRRC, 195, 1191

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

.

Magn Reson Med. 1996 36:212-8.

Problems….?

Tsuchiya et al (2002), Biochem. J., 367, 771

Iron/Dithiocarbamates Advantages/Disadvantages

     Actually traps the NO – therefore 15 N 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.

Hemoglobin/Myoglobin

 Reacts with NO with rate constant > 10 7 M -1 s -1  Cheap and plentiful.

 The reaction is accompanied by a UV-vis spectral change and a change in EPR spectrum

Reactions of •NO with Hb

HbO

2  •

NO

 3 .



x



M

  1

s

  1 

metHb



NO

3 

Hb

 •

NO

 2 .

  1

x

10 7 

M

  1

s

 

HbNO

Reaction of NO with MbO

2

  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

g~6

EPR of metHb

g~2

EPR: metHb at 4 K (He)

-10000

Determination of metHb concentration using correlation

18000 17000 16000 15000 14000 13000 12000 11000 y = 0.2481x + 12487 R 2 = 0.8237

  IF the shape of the line does not change then don’t double integrate.

Plot spectrum against that of a standard and the slope will immediately give you the concentration.

10000 -5000 9000 0 5000 10000 15000

metHb standardization

Sensitivity of ~ 100 nM 1.2

1 0.8

0.6

0.4

0.2

0 0 y = 1.1507x - 0.0741

R 2 = 0.9285

0.2

0.4

0.6

[expeted metHb] (µM)

0.8

1 1.2

metHb during NO inhalation

0.12

0.1

0.08

0.06

250 0.04

0.02

200 0 2h rP e 1h rP re 0h rP re 1h rN o 2h rN O 150 3h rN O 100 4H rN O 20 m in P os t 40 m in P os t 1h rP os t 2h rP os t 50 0 2h rP e 1h rP re 0h rP re 1h rN o 2h rN O 3h rN O 4H rN O 20 m in P os t 40 m in P os t 1h rP os t 2h rP os t

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 15 N experiments.

Reactions of •NO with Hb

HbO

2  •

NO

 3 .



x



M

  1

s

  1 

metHb



NO

3 

Hb

 •

NO

 2 .

  1

x

10 7 

M

  1

s

 

HbNO

EPR: deoxyHb with NEM at 77 K

100 G N Fe 2+ NO

EPR: deoxyHb with IP6 at 77K

100 G N Fe 2+ NO

Analysys of HbNO spectra

C

60000 40000 20000 0 -20000 -40000 -60000 -80000

A

i 50 G iii 2.50E+08 2.00E+08 1.50E+08 1.00E+08 5.00E+07 0.00E+00 0

B

10 20 30

[HbNO] (µM)

40 50 60 100 G ii 60000

D

40000 20000 0 -20000 -40000 -60000 -80000 50 G Piknova et al (2005), JBC.(in Press)

HbNO in blood after NO inhalation

Artery Artery 100 G Vein Vein 100 G Piknova et al (2005), JBC.(in Press)

3 2.5

2 1.5

1 Vein 1 Artery 1 Vein 2 Artery 2 Vein 3 Artery 3 0.5

-2 -1 0 0 1 2

Time (h)

0.9

3 0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 Piknova et al (2005), JBC.(in Press) NO Inhalation stopped 4 5 Artery 6 6-Coord Alpha 6-Coord Beta 5-Coord Alpha Vein

Advantage/Disadvantages of HbNO detection

 Complex multi-component signal.

  Sensitive at liquid N 2 temperatures NO is trapped and so can do 15 N experiments.

 Needs to be deoxygenated!!

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.

Acknowledgements

Barbora Piknova Yanhong Zhang Agnes Keszler Netanya Spencer Ravinder Singh

National Biomedical EPR Center Medical College of Wisconsin (EB001980)

Raman Kalyanaraman Bill Antholine Brian Bennett Jim Hyde Mark Gladwin Alan Schechter Chris Reiter Dany Kim-Shapiro Ron Mason ..many others who’s work I have used