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12th Annual Meeting of SRFBM

12th Annual Meeting of SRFBM. Pre-Meeting Workshop I: Rigorous Detection and Identification of Free Radicals in Biology and Medicine. Spin Trapping of Protein Radicals To Investigate Oxidative Mechanisms. Ohara Augusto, PhD Departamento de Bioquímica Instituto de Química

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12th Annual Meeting of SRFBM

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  1. 12th Annual Meeting of SRFBM Pre-Meeting Workshop I: Rigorous Detection and Identification of Free Radicals in Biology and Medicine Spin Trapping of Protein Radicals To Investigate Oxidative Mechanisms Ohara Augusto, PhD Departamento de Bioquímica Instituto de Química Universidade de São Paulo São Paulo, Brazil

  2. RADICAL AND OXIDANT DETECTION IN BIOLOGY DIRECT -EPR-electron paramagnetic resonance INDIRECT (UP TO THE 90’s) -Use of scavengers (DMSO, dimethylurea, etc) -Use of antioxidant enzymes (mimetics and inhibitors) -Quantification of end products of lipid peroxidation (TBA, chemiluminescence, etc) -Spin trapping INDIRECT (MORE RECENTLY) -Knock-outs/super-expression of antioxidant enzymes and/or radical/oxidant producer enzymes -Characterization/quantification of radical products from biotargets (lipids, proteins, DNA) (stable isotope-dilution LC/ESI/MS/MS-immunodetection) -Spin trapping (LC/MS-immunodetection) -Use of fluorescent/chemiluminescent probes Augusto, 2005 (fast results?)

  3. Association of nitrotyrosine levels with cardiovascular disease and modulation by statin therapy Shishehbor et al, JAMA 289, 1675, 2003 BIOMARKERS OF OXIDATIVE DAMAGE & DISEASE Stable isotope-dilution LC/ESI/MS/MS analyses of oxidant/radical products of biomolecules (P-13C/15N, etc) Augusto, 2005

  4. BIOMARKERS OF OXIDATIVE DAMAGE & DISEASE Stable isotope-dilution LC/ESI/MS/MS analyses of oxidant/radical products of biomolecules Red cell membrane and plasma linoleic acid nitration products: synthesis, clinical identification and quantitation Parker et al, PNAS 101, 11577, 2004 Table 1. Biologically active nitrogen oxide derivatives in human blood: Comparison with nitrated linoleic acid Species Compartment Fraction Concentration, nM - NO Plasma Total 205 ± 21 2 RSNO Plasma Total 7.2 ± 1.1 3-Nitro- Plasma Total 0.74 ± 0.30 tyrosine LNO Plasma Free 79 ± 35 2 Esterified 550 ± 275 Total 630 ± 240 Hb-NO Blood Total <50 Hb-SNO Blood Total 0-150 LNO Red cells Free 50 ± 17 2 Esterified 199 ± 121 Total 249 ± 104 * * LNO Whole blood Total 477 ± 128 2 Augusto, 2005

  5. Biomarkers of oxidative damage & early biomarkers exogenous sources endogenous sources antioxidant defences high levels NO2• NO• low levels O2•- N2O3 CO3•- H2O2 •OH ONOO- Impaired physiology Homeostasis Impaired physiology (oxidative stress response) biomolecule damage redox signaling < proliferative response < microbicidal activity repair cell/tissue damage Normal metabolism growth Augusto, 2005 PHYSIOLOGICAL ROLES OF RADICALS/OXIDANTS

  6. Spin-trapping of P• EARLY BIOMARKERS & OXIDATIVE MECHANISMS UNDER SCRUNITY -Detection of small increases in oxidant/radical production improved probes/spin traps under development (this symposium) -Monitoring GSSG/2 GSH levels (Schafer & Beuttner FRBM 30, 1191) may not reflect localized redox unbalance (Go et al. JBC 279, 5837) -Characterizing/quantifying products/radicals of protein/lipids/DNA (this symposium) not excluding ionic products whose formation is likely to occur (P-TyrNO2) or may occur by radical mechanisms (PS-NO, PSOH (redox signaling?), PSO2H, PSO3H (transition from signaling to damage?)) Augusto, 2005

  7. ADVANTAGES OF PROTEIN RADICALS TO EXAMINE OXIDATIVE MECHANISMS -Proteins are abundant (organ, cell and plasma level) -Proteins are central players of physiological processes including as producers of radicals/oxidants and as cell signaling mediators. -Protein radicals are known biological intermediates of some enzymatic reactions(cell homeostasis) and of some protein damaging processes (protein peroxides, cross-linking reactions, protein oxidation, nitration and backbone cleavage) (cell injury). -Several protein-amino acid radicals have been characterized by EPR and spin trapping combined with other methodologies. (Davies & Hawkins FRBM 36,1072 (review) Augusto, 2005

  8. Typical experiment: Proteolysis P-Tyr• + spin trap P-radical adduct LC/MS/peptide mapping Proteolysis Tyr-N-DBNBS + DBNBS-N=O O• Tyr-N-DBNBS O• EPR spectra Tyr• SPIN TRAPPING OF PROTEIN RADICALS TO EXAMINE OXIDATIVE MECHANISMS Useful to: -Hint P-amino acid targets of oxidants/radicals -Identify P-radical residue(s) (combined with MS) -Discriminate radical from non radical reactions -Hint “new” biological oxidants? Augusto, 2005

  9. EPR SPECTRA COMPARISON MAY HINT TARGETS EPR spectra features known Protein-Cys as a relevant target of peroxynitrite-derived radicals Davies & Hawkins FRBM 36,1072 (review) Augusto et al FRBM 36, 1224 (review) aH = 15.4 G Cys93 human erythrocytes DMPO/•CysHb Tyr 24,42,140 aH = 8.8 G 36,130,145 dialyzed hemolysates + NEM DMPO/•TyrHb aH = 16.0 G Cys34 human plasma DMPO/ •CysHSA aH ~15 - 16 G Augusto, 2005

  10. SPIN TRAPPING/PROTEOLYSIS/MS/PEPTIDE MAPPING TO IDENTIFY P-RADICAL RESIDUE(S) -Simplified scheme: Proteolysis P-Tyr• + spin trap P-radical adduct LC/MS/peptide mapping Trp Trp• Proteolysis Tyr-N-DBNBS + DBNBS-N=O O• Tyr-N-DBNBS O• -Actual situation: Intramolecular long-range electron transfer from one residue to other(s) occurs and different P• radicals may be produced but not trapped. Little is still known about trapping efficiency particularly in proteins. spin trap Tyr• Tyr• Tyr access?/ rate constant? Augusto, 2005

  11. SPIN TRAPPING/PROTEOLYSIS/MS/PEPTIDE MAPPING TO IDENTIFY P-RADICAL RESIDUE (S) Hen lysozyme treated with MPO/H2O2/NO2 - Spin Trapping: Peptide mass fingerprints: (MALDI-TOF-trypsin digests) -DMPO-no EPR signals -DBNBS-mainly a DBNBS/•Tyr-lysozyme signal (inhibited by pre-iodination) -Trp123-DMPO adduct -no DBNBS adduct (so far) -Trp62/63-NO2/Trp108/111-NO2/Tyr20-NO2 native Trp123 + DMPO lysozyme iodo-lysozyme unpublished

  12. SPIN TRAPPING OF PROTEIN RADICALS TO EXAMINE OXIDATIVE MECHANISMS Useful to: -Hint P-amino acid targets of oxidants/radicals based on EPR spectra comparison. -Identify P-radical residue(s) (combined with MS) -Discriminate radical from non radical reactions based on product yield inhibition by spin traps. -Hint “new” biological oxidants?

  13. SPIN TRAPS TO DISCRIMINATE RADICAL FROM NON RADICAL MECHANISMS Inhibition of product yield by spin traps has been a classical approach. A recent and relevant example was the demonstration that GSNO formation from NO•/O2 may occurby radical mechanisms. Jourd'heuil et al JBC, 2003; Schrammel et al FRBM, 2003 Fibroblasts +SperNO (0.1 mM) GSH (1 mM) + SperNO (0.1 mM) pH 7.4 ambient air (100 mM) RSNO (pmols/106 cells) (10 mM) Jourd'heuil et al JBC, 2003 Augusto, 2005

  14. SPIN TRAPPING TO DISCRIMINATE RADICAL FROM NON RADICAL MECHANISMS PcysSNO by recombination of PcysS• and •NO Tempol diverts ONOO-/CO2 reactivity towards proteins and cells from P-cys oxidation (20-50% inhibition) and P-tyr nitration (70-90% inhibition) to P-cys nitrosation (200-400% increase). (Fernandes et al FRBM, 2005) Tempol inhibits PBN/•ScysBSA PBN inhibits BSA-cysNO yield P-cysS•/P-cysSOH/P-tyrNO2/others P-cysSNO PBN/•ScysBSA P P ONOO-/CO2 CO3•- + •NO2 N2O3 12 OH P-cysS• 8 + •NO + O2 N •NO nitrosocysteine (M) O• 4 OH HCO3- +NO2-+ P-cysSNO ONOO- + 0 N 0 12.5 50 PBN (mM) O Augusto, 2005

  15. SPIN TRAPPING TO DISCRIMINATE RADICAL FROM NON RADICAL MECHANISMS PcysSOH formation by radical mechanisms? HSA is oxidized to HAS-cysSOH by ONOO-/ONOOH (2 e- mechanism predominates) and ONOO-/CO2 (1 e- mechanism predominates). Carballal et al, Biochemistry 2003 PSH HSA(0.5 mM) + ONOO-(0.4 mM) Pi, pH= 7.4 H2O2 ONOO-/ONOOH •NO2/CO3•- (ONOO-/CO2) HSA-cysSONDB PSOH Rapid mixing EPR/spin trapping showed that GS• is a GSO• precursor PS• -HCO3- (25 mM) O2 Bonini & Augusto JBC 2001 +HCO3- (25 mM) •NO PSNO GSH/ONOO-/CO2 GSO• PSO• DMPO/•SG XH +DMPO PSOH Augusto, 2005

  16. PROTEIN-CysS• AS SIGNALING INTERMEDIATES? -A hypothesis based on simple experiments (EPR, EPR spin trapping, product analyses) that indicated PcysS• as precursor of PcysSNO and PcysSOH both of which are considered to be mediators of redox signaling. Augusto et al FRBM 2004 -A likely possibility based on solid and elegant data (molecular biology,structural protein analyses, NMR and fluorescence) to support that Rascys118S• (itself) participates in the mechanism of Ras regulation by redox agents. Campbell, Heo & co-workers Biochemistry 2004, JBC 2005a,b, Biochemistry 2005, JMB 2005 RasGDP Rascys118S• Ras + GDPox Augusto, 2005

  17. (Bonini et al, 2004 Biochemistry) Direct EPR & spin trapping Cu,Zn-SOD (2.5 mg/ml) + H2O2 (2.5 mM) + BSA (100 mg/ml) solvent-unexposed BSA-tyr • solvent-exposed BSA-tyr • HCO3-(50 mM) NO2-(50 mM) 8.6 G + DMPO + DMPO + DNBS + DNBS Augusto, 2005 LACK OF BSA-cysS• TRAPPING USED TO PROPOSE PEROXYCARBONATE AS A BIOLOGICAL OXIDANT BSA as a target of the oxidants produced during Cu,Zn-SOD peroxidase activity in the presence of HCO3- or NO2-.

  18. BSA-cysS• was not trapped! Radical AA k (M-1.s-1) CO3•- Cys 4.6 x 107 Tyr 4.5 x 107 Trp 7.0 x 108 NO2• Cys 5.0 x 107 Gly-Tyr 3.2 x 105 Gly-Trp 1.0 x 106 Augusto, 2005 LACK OF BSA-cysS• TRAPPING USED TO PROPOSE PEROXYCARBONATE AS A BIOLOGICAL OXIDANT Cu,Zn-SOD peroxidase activity in the presence of HCO3- and NO2- poduces diffusible CO3•- and NO2• that oxidize BSA to solvent-exposed and -unexposed BSA-Tyr•. Kalyanaraman and co-workers, Fridovich and co-workers 1999-2003 Bonini et al, 2004 Biochemistry BSA BSA tyr• tyr• •NO2 HCO3-/CO2 CO3•- NO2- •OH Cu(II)

  19. LACK OF BSA-cysS• TRAPPING USED TO PROPOSE PEROXYCARBONATE AS A BIOLOGICAL OXIDANT BSA-CysSH was fast oxidized to BSA-CysSOH by H2O2 and the process was accelerated by HCO3-. BSA-cysSH (1 mM SH)+ H2O2 (2.5 mM) 1.0 BSAcysS-NDB BSAcysSO-NDB -HCO3- 0.5 Reduced thiol (mM) Absorbance +HCO3- 350 450 10 20 30 Wavelength (nm) Time (min) Effects of HCO3- in accelerating H2O2-mediated BSA-cysSH oxidation were attributed to peroxycarbonate (HCO4-) formation. (Bonini et al, 2004 Biochemistry) Augusto, 2005

  20. e- (reducing agent) BSA-cysSH BSA-cysSOH CO3•- (Bonini et al, 2004 Biochemistry; Trindade et al, 2005 unpublished) Bonini et al, 2004 JBC We proposed HCO4- as potentially relevant biological oxidant that could act by two-electron mechanisms or as a precursor of the CO3•-. The latter view has been supported by other investigators. (Liochev & Fridovich PNAS, 2004; Ramirez, Mejiba, Mason JBC, 2005) Augusto, 2005 LACK OF BSA-cysS• TRAPPING USED TO PROPOSE PEROXYCARBONATE AS A BIOLOGICAL OXIDANT Peroxycarbonate was known from chemical literature as a two-electron oxidant. (Richardson & co-workers JACS 2000, 2003, FRBM 2004) H2O2 + HCO3- H2O + HCO4- Keq= 0.32 (25 oC)

  21. Trp Trp• Tyr• Tyr SPIN TRAPPING/MS OF PROTEIN RADICALS TO EXAMINE OXIDATIVE MECHANISMS -In spite of the many questions that remain to be answered, (particularly in regard to intramolecular electron transfer from one P-residue• to other residues and to trapping efficiency),spin trapping of protein radicals can be useful to examine biological oxidative spin trap access?/ rate constant? mechanisms (hint P-amino acid targets, identify P-radical residues, discriminate radical from non radical mechanisms) among other applications that will be discussed in the following presentations. Augusto, 2005

  22. PHYSIOLOGICAL ROLES OF RADICALS/OXIDANTS Biomarkers of oxidative damage &early biomarkers exogenous sources endogenous sources antioxidant defences high levels NO2• NO• low levels O2•- N2O3 CO3•- H2O2 •OH ONOO- Impaired physiology Homeostasis Impaired physiology (oxidative stress response) biomolecule damage redox signaling < proliferative response < microbicidal activity repair cell/tissue damage Normal metabolism growth Augusto, 2005

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