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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
Cys93
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