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

A STABLE free radical A STABLE free radical gasgas

N O

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’

Strategies used for the EPR Strategies used for the EPR detection of Nitric Oxidedetection of Nitric Oxide

Nitronyl Nitroxides

Fe/Dithiocarbamate

Hemoglobin/Myoglobin DNIC

The Nitronyl NitroxidesThe Nitronyl Nitroxides

CPTIO

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

MechanismMechanism

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

NNOINO

NO detectionNO detection

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

∙NO

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

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

Spectral Overlap of NNO Spectral Overlap of NNO and INOand INO

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

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)

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

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

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

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

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

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.

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

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

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

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

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.

Problems….?Problems….?

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

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.

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

Reactions of •NO with Reactions of •NO with HbHb

HbNONOHb

NOmetHbNOHbOsMx

sMx

117

117

106.2

3107.3

2

•

•

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

EPR of metHbEPR of metHb

g~6

g~2

EPR: metHb at 4 K (He)EPR: metHb at 4 K (He)

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

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

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

)

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.

Reactions of •NO with Reactions of •NO with HbHb

HbNONOHb

NOmetHbNOHbOsMx

sMx

117

117

106.2

3107.3

2

•

•

EPR: deoxyHb with NEM at EPR: deoxyHb with NEM at 77 K77 K

Fe2+ NON

EPR: deoxyHb with IP6 at EPR: deoxyHb with IP6 at 77K77K

Fe2+ NON

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)

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)

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

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!!

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.

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