1. The use of EPR in Nitric Oxide Research Neil Hogg, Medical College of Wisconsin SFRBM 2005 Austin, TX

2. A STABLE free radical gas N O

3. Direct Detection of NO by EPR Direct Detection of NO by EPR Broad ugly looking spectrum Broad ugly looking spectrum Need high concentration Need high concentration Unsuitable for biological detection Unsuitable for biological detection → Spin ‘Trapping’

4. Strategies used for the EPR detection of Nitric Oxide Nitronyl Nitroxides Fe/ Dithiocarbamate Hemoglobin/ Myoglobin DNIC

5. The Nitronyl Nitroxides CPTIO

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

7. Mechanism Joseph et al (1993), BBRC, 192, 926 NNO INO

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

9. Origin of EPR spectrum (NNO with two equivalent N centers) 1 2 3 2 1

10. Origin of EPR spectrum (INO with two inequivalent N centers) 1 1 2 1 2 1 1

11. Spectral Overlap of NNO and INO

12. Overlap Minimized on low-field lines (Left side of spectrum) Hogg et al (1995), Free Rad. Res., 22, 47

13. Better way: Multiple Linear regression uses all the data MLR (Simulation using WinSim)

14. Reaction characteristics: Reaction of NO converts one radical to another therefore not spin-trapping in the traditional sense. Reaction of NO converts one radical to another therefore not spin-trapping in the traditional sense. Rate const of ~1000 M -1 s -1 therefore fast enough to compete with oxygen but not fast enough to compete with (e.g.) superoxide. Rate const of ~1000 M -1 s -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 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

15. 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!

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

17. NO/CPTIO generates nitrosating intermediates. Zhang et al (2004), Am.J.Physiol., 287, L467

18. CPTIO/CPTI are redox active nitroxides – makes for many problems in complex systems ● CPTIO ○ CPTI ■TEMPOL (control) SIN-1 and CPTIO Singh et al (1999), Arch. Biochem. Biophys.., 361, 331

19. Advantages/Disadvantages Clear NO-dependent change in EPR spectrum allows quantification of kinetics of NO formation. 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. 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 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. Nitrogen dioxide is a product of the reaction and so these compounds my inhibit NO but enhance nitrosation/nitration reactions.

20. Dinitrosyl Iron Complexes (DNIC) 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 Stadler et al (1993), Arch.Biochem.Biophys., 302, 4

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

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

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

24. EPR imaging of NO using MGD 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. Kuppusamy PWang PSamouilov AZweier JL

25. Problems….? Tsuchiya et al (2002), Biochem. J., 367, 771

26. Iron/Dithiocarbamates Advantages/Disadvantages Actually traps the NO – therefore 15 N experiments can be used to identify the source of the signal. 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. Use in in vivo NO spin trapping and EPR imaging. Potential for signal from sources other than NO (S- nitrosothiols/nitrite/HNO) 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). 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. A Cu/dithiocarbamate signal overlaps the Fe/NO signal and can cause problems in situations where copper is present.

27. Hemoglobin/Myoglobin Reacts with NO with rate constant > 10 7 M -1 s -1 Reacts with NO with rate constant > 10 7 M -1 s -1 Cheap and plentiful. Cheap and plentiful. The reaction is accompanied by a UV-vis spectral change and a change in EPR spectrum The reaction is accompanied by a UV-vis spectral change and a change in EPR spectrum

28. Reactions of NO with Hb

29. 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

30. EPR of metHb g~6 g~2

31. EPR: metHb at 4 K (He)

32. Determination of metHb concentration using correlation 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.

33. metHb standardization Sensitivity of ~ 100 nM

34. metHb during NO inhalation

35. Advantage/Disadvantages of metHb detection Simply easily analyzable signal. Simply easily analyzable signal. Highly sensitive at liquid He temperatures Highly sensitive at liquid He temperatures Not necessarily specific for NO (peroxynitrite and other oxidants could do the same thing) 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. NO is not ‘trapped’ and so cannot do 15 N experiments.

36. Reactions of NO with Hb

37. EPR: deoxyHb with NEM at 77 K Fe 2+ NO N

38. EPR: deoxyHb with IP6 at 77K Fe 2+ NO N

39. Analysys of HbNO spectra ii iiii AB C D Piknova et al (2005), JBC.(in Press)

40. HbNO in blood after NO inhalation Piknova et al (2005), JBC.(in Press)

42. Advantage/Disadvantages of HbNO detection Complex multi-component signal. Complex multi-component signal. Sensitive at liquid N 2 temperatures Sensitive at liquid N 2 temperatures NO is trapped and so can do 15 N experiments. NO is trapped and so can do 15 N experiments. Needs to be deoxygenated!! Needs to be deoxygenated!!

43. In conclusion… EPR is a phenomenally useful tool in NO research for both in vitro, ex vivo and in vivo studies 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 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. 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. 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.

44. Acknowledgements National Biomedical EPR Center Medical College of Wisconsin (EB001980) 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 who’s work I have used