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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 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/MSimmunodetection) -Spin trapping (LC/MS-immunodetection) -Use of fluorescent/chemiluminescent probes (fast results?) Augusto, 2005 BIOMARKERS OF OXIDATIVE DAMAGE & DISEASE Stable isotope-dilution LC/ESI/MS/MS analyses of oxidant/radical products of biomolecules (P-13C/15N, etc) Association of nitrotyrosine levels with cardiovascular disease and modulation by statin therapy Shishehbor et al, JAMA 289, 1675, 2003 Augusto, 2005 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 NO2 Plasma Total 205 ± 21 RSNO Plasma Total 7.2 ± 1.1 3-NitroPlasma Total 0.74 ± 0.30 tyrosine LNO2 Plasma Free 79 ± 35 Esterified 550 ± 275 Total 630 ± 240 Hb-NO Blood Total <50 Hb-SNO Blood Total 0-150 LNO2 Red cells Free 50 ± 17 Esterified 199 ± 121 Total 249 ± 104 * * LNO2 Whole blood Total 477 ± 128 Augusto, 2005 PHYSIOLOGICAL ROLES OF RADICALS/OXIDANTS Biomarkers of oxidative damage & early biomarkers endogenous sources exogenous sources antioxidant defences high levels • NO 2 O2 •H2O2 •OH CO3 N2-O3 ONOO •- low levels Impaired physiology NO• Homeostasis Impaired physiology (oxidative stress response) < proliferative response < microbicidal activity redox signaling Normal metabolism growth biomolecule damage repair cell/tissue damage Augusto, 2005 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 Spin-trapping of P• 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 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, crosslinking 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 SPIN TRAPPING OF PROTEIN RADICALS TO EXAMINE OXIDATIVE MECHANISMS Typical experiment: P-Tyr• + spin trap Proteolysis P-radical adduct LC/MS/peptide mapping Proteolysis Tyr• + DBNBS-N=O Tyr-N-DBNBS O• Tyr-N-DBNBS O• EPR spectra 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 EPR SPECTRA COMPARISON MAY HINT TARGETS EPR spectra features known Davies & Hawkins FRBM 36,1072 (review) DBNBS/MNP radical adducts DMPO radical adducts H N N N H CH3 aH = 15.4 G O H N O Histidine C-2 radical OH CH2R Tyrosine phenoxyl radical O N N CH2 O CH2 CH2R Tyrosine C-3 radical CH3 CH2 CH2R CH2R Cys93 DMPO/•CysHb human erythrocytes CH3 H HN aH = 8.8 G dialyzed hemolysates + NEM Tyr 24,42,140 36,130,145 DMPO/•TyrHb Lysine N-centered radical N O H aH = 16.0 G N Tryptophan C-3 radical CH2R H N Augusto et al FRBM 36, 1224 (review) CH3 H O CH2R Protein-Cys as a relevant target of peroxynitrite-derived radicals N O Tryptophan C-6 radical H S CH3 N CH2 O R CH3 Cysteine S-centered radical human plasma Cys34 DMPO/ •CysHSA aH ~15 - 16 G Augusto, 2005 SPIN TRAPPING/PROTEOLYSIS/MS/PEPTIDE MAPPING TO IDENTIFY P-RADICAL RESIDUE(S) -Simplified scheme: P-Tyr• + spin trap Proteolysis P-radical adduct LC/MS/peptide mapping Proteolysis Tyr• + DBNBS-N=O Tyr-N-DBNBS 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 access?/ rate constant? Trp• Tyr Trp Tyr• Augusto, 2005 SPIN TRAPPING/PROTEOLYSIS/MS/PEPTIDE MAPPING TO IDENTIFY P-RADICAL RESIDUE (S) Hen lysozyme treated with MPO/H2O2/NO2 Spin Trapping: -DMPO-no EPR signals -DBNBS-mainly a DBNBS/•Tyr-lysozyme signal (inhibited by pre-iodination) Peptide mass fingerprints: (MALDI-TOF-trypsin digests) -Trp123-DMPO adduct -no DBNBS adduct (so far) -Trp62/63-NO2/Trp108/111-NO2/Tyr20-NO2 native lysozyme Trp123 + DMPO iodo-lysozyme unpublished 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? 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 occur by radical mechanisms. Jourd'heuil et al JBC, 2003; Schrammel et al FRBM, 2003 (100 mM) Fibroblasts +SperNO (0.1 mM) RSNO (pmols/106 cells) GSH (1 mM) + SperNO (0.1 mM) pH 7.4 ambient air (10 mM) Jourd'heuil et al JBC, 2003 Augusto, 2005 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 12 OH P-cysS• 8 + N O• OH •NO P-cysSNO N2O3 HCO3- + NO2-+ + N O •NO + O2 4 ONOO- 0 0 12.5 50 PBN (mM) Augusto, 2005 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 H 2 O2 ONOO-/ONOOH PSOH HSA(0.5 mM) + ONOO-(0.4 mM) Pi, pH= 7.4 HSA-cysSONDB -HCO3- (25 mM) +HCO3- (25 mM) PSNO •NO •2/CO3 (ONOO /CO2) PS• O2 •NO Rapid mixing EPR/spin trapping showed that GS• is a GSO• precursor Bonini & Augusto JBC 2001 GSH/ONOO-/CO2 PSO• XH GSO • DMPO/•SG +DMPO PSOH Augusto, 2005 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 Augusto et al FRBM 2004 mediators of redox signaling. -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 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-. (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-exposed BSA-tyr HCO3-(50 mM) • solvent-unexposed BSA-tyr • 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 Cu,Zn-SOD peroxidase activity in the presence of HCO3and NO2- poduces diffusible CO3•- and NO2• that oxidize BSA to solvent-exposed and -unexposed BSA-Tyr•. Bonini et al, 2004 Biochemistry BSA BSA tyr• Kalyanaraman and co-workers, Fridovich and co-workers 1999-2003 BSA-cysS• was not trapped! Radical tyr• •NO CO3•- 2 CO3•- HCO3-/CO2 NO2•OH NO2• AA k (M-1.s-1) Cys 4.6 x 107 Tyr 4.5 x 107 Trp 7.0 x 108 Cys 5.0 x 107 Gly-Tyr 3.2 x 105 Gly-Trp 1.0 x 106 Cu(II) Augusto, 2005 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) Reduced thiol (mM) 1.0 BSAcysS-NDB BSAcysSO-NDB 0.5 -HCO3+HCO310 20 30 Time (min) 350 450 Wavelength (nm) Effects of HCO3- in accelerating H2O2-mediated BSA-cysSH oxidation were attributed to peroxycarbonate (HCO4-) formation. (Bonini et al, 2004 Biochemistry) 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- BSA-cysSH BSA-cysSOH (Bonini et al, 2004 Biochemistry; Trindade et al, 2005 unpublished) Keq= 0.32 (25 oC) e- (reducing agent) CO3•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 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 spin trap access?/ rate constant? Trp• Tyr Trp Tyr• radicals can be useful to examine biological oxidative 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 PHYSIOLOGICAL ROLES OF RADICALS/OXIDANTS Biomarkers of oxidative damage & early biomarkers endogenous sources exogenous sources antioxidant defences high levels • NO 2 O2 •H2O2 •OH CO3 N2-O3 ONOO •- low levels Impaired physiology NO• Homeostasis Impaired physiology (oxidative stress response) < proliferative response < microbicidal activity redox signaling Normal metabolism growth biomolecule damage repair cell/tissue damage Augusto, 2005