The Pecking Order of Free Radicals and Antioxidants

1
The Pecking Order of Free Radicals and
Antioxidants
GR Buettner, FQ Schafer Free Radical Radiation
Biology and ESR Facility The University of
Iowa Iowa City, IA 52242-1101
2
The Road Ahead - Overview
Free Radicals Classes of Antioxidants Donor,
Chain Breaking Antioxidants tocopherol, vitamin
C Metals, Adventitious Sacrificial Chain Breaking
Antioxidants ?NO
3
What is a Free Radical?
A free radical an atom or group of atoms
possessing one or more unpaired electrons."
The word "free" in front of "radical" is in
this era considered unnecessary.
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Free Radical Notation?
A. Superscript dot to the right, usually B.
Examples (Note dot, then charge) H?, Cl?,
HO? O2?? or O22? dioxygen, the O2 you are
breathing now. H3C? O2?- , CO2?-, Asc?-, PQ?
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Types of radicals we have
Sigma, ? pi-delocalized, ? Mixture of sigma and
pi Carbon-centered, H3C? O2centered,
H3COO? Sulfur-centered, GS?
Nitrogen-centered, R2NO? Reducing radicals,
CO2? -, PQ? Oxidizing radicals, HO?, LOO?
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Classes of Antioxidants
There are two broad classes Preventive Chain-
breaking
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Lipid Peroxidation
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Preventive Antioxidants
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Preventive Antioxidants
Preventative Antioxidants reduce the rate of
chain initiation. Targets are 1. Metals
- Fe, Cu 2. Hydroperoxides  
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Preventive Antioxidants Metals
Chelates EDTA, DETAPAC, Desferal
(deferrioxamine), Phytate ? Proteins
metals Transferrin, Fe Ferritin, Fe Heme
peroxidases, Fe Hemoglobin, Fe Myoglobin, Fe C
aeruloplasmin, Cu
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Preventive Antioxidants Metals
Key to prevention is slowing rxns 1 and 2
Fe(III)chelate O2?- ? Fe(II)chelate O2
1 (or AscH-, GSH, cellular reducing
agents)   Fe(II)chelate H2O2 ? HO?
Fe(III)chelate 2 Fe(II)chelate LOOH ? LO?
Fe(III)chelate The coordination environment of
the metal determines the thermodynamics and
kinetics of these rxns.
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Preventive Antioxidants Peroxides
Hydroperoxides H2O2, ROOH, LOOH
Catalase GPx GPx1, GPx2,GPx3, GPx4 ( PhGPx)
Peroxiredoxins via Trx and TR
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Chain Breaking Antioxidants
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Chain Breaking Antioxidants, How do they work?
Intercept L? ? L?
Antiox-H ¾¾ Antiox? L-H Where Antiox-H
is a donor antioxidant.
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Kinetics rules
The competition 3 x 108 M-1 s-1 L O2
¾¾¾ LOO Propagation 10-50
M-1 s-1 LOO L-H ¾¾¾ L LOOH
Cycle Vitamin E 8 x 104 M-1 s-1 LOO
TOH ¾¾¾ TO L-H
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Tocopherol in Action
 
 
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Ascorbate as a Donor Antioxidant
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C and E as Co-Antioxidants
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C and E as Co-Antioxidants
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Thermodynamics
Both, kinetics and thermodynamics are involved in
the control of antioxidant reactions.
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The Pecking Order
Redox Couple (one-electron reductions)
E'/mV HO?, H/H2O 2310 RO?,
H/ROH (aliphatic alkoxyl radical)
1600 ROO?, H/ROOH (alkyl peroxyl radical)
1000 GS?/GS? (glutathione) 920 PUFA?,
H/PUFA-H (bis-allylic-H) 600 TO?, H/TOH
480 H2O2, H/H2O, HO? 320 Asc??,
H/AscH- 282 CoQH?,
H/CoQH2 190 Fe(III) EDTA/ Fe(II) EDTA
120 O2/ O2?? -
160 CoQ/CoQ?- - 230 Paraquat/
Paraquat? - 448 Fe(III)DFO/ Fe(II)DFO
- 450 RSSR/ RSSR?? (GSH) - 1500 H2O/
e?aq - 2870 Buettner GR (1993) The pecking
order of free radicals and antioxidants Lipid
peroxidation, ?-tocopherol, and ascorbate. Arch
Biochem Biophys. 300 535-543.
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The Pecking Order
Redox Couple (one-electron reductions)
E'/mV HO?, H/H2O 2310 RO?, H/ROH
(aliphatic alkoxyl radical) 1600 ROO?, H/ROOH
(alkyl peroxyl radical) 1000 GS?/GS?
(glutathione) 920 PUFA?, H/PUFA-H
(bis-allylic-H) 600 TO?, H/TOH
480 H2O2, H/H2O, HO? 320 Asc??,
H/AscH- 282 CoQH?-,
H/CoQH2 190 Fe(III) EDTA / Fe(II) EDTA
120
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The Pecking Order
Redox Couple (one-electron reductions)
E'/mV HO?, H/H2O 2310 RO?, H/ROH
(aliphatic alkoxyl radical) 1600 ROO?, H/ROOH
(alkyl peroxyl radical) 1000 GS?/GS?
(glutathione) 920 PUFA?, H/PUFA-H
(bis-allylic-H) 600 TO?, H/TOH
480 H2O2, H/H2O, HO? 320 Asc??,
H/AscH- 282 CoQH?,
H/CoQH2 190 Fe(III) EDTA/ Fe(II) EDTA
120
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Trouble, when?
When a reaction produces a product that jumps
up in the Pecking Order.
HO?, H/H2O 2310 ROO?, H/ROOH (alkyl
peroxyl radical) 1000 PUFA?, H/PUFA-H
(bis-allylic-H) 600 H2O2, H/H2O, HO?
320 Note the reaction of L? (PUFA?) with O2
will result in a species higher in the Pecking
Order (ROO? above) likewise with the Fenton Rxn,
HO?.
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Iron, a bit of history

• Iron contaminates buffers, 0.1 1 or more ?M
• Choice of chelating agent can change
  observations
• DETAPAC (DTPA) introduced to free radical
  community
• Iron a big player in spin trapping
• Everything goes better with DETAPAC.
• Buettner, G.R. and Oberley, L.W. (1978)
  "Considerations in the spin trapping of
  superoxide and hydroxyl radicals in aqueous
  systems using 5,5-dimethyl-1-pyrroline-1-oxide."
  Biochem. Biophys. Res. Commun. 83 69-74. ( and
  the Pinawa Meeting, 1977)
• Buettner, G.R., Oberley, L.W., and Leuthauser,
  S.W.H.C. (1978) "The effect of iron on the
  distribution of superoxide and hydroxyl radicals
  as seen by spin trapping and on the superoxide
  dismutase assay." Photochem. Photobiol. 28
  693-695. ( and the Pinawa Meeting, 1977)
• "Citation Classics", selection by the Institute
  for Scientific Information, the publishers of
  Current Contents

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Iron, how much is there?
Buettner GR. (1988) In the absence of catalytic
metals, ascorbate does not autoxidize at pH 7
Ascorbate as a test for catalytic metals. J
Biochem Biophys Meth 16 20-40.
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Ascorbate radical, a tool
Buettner GR, Jurkiewicz BA. (1993) The ascorbate
free radical as a marker of oxidative stress An
EPR study. Free Rad Biol Med 14 49-55.
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Ascorbate radical, a tool
500 uM AscH-
Buettner, G.R. and Jurkiewicz, B.A. (1993) Free
Rad. Biol. Med., 14, 49-55.
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Asc??, marker of oxidative stress
Human plasma 58 ?M AscH-
Buettner, G.R. and Jurkiewicz, B.A. (1993) The
ascorbate free radical as a marker of oxidative
stress An EPR study. Free Rad Biol Med,
14 49-55
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Iron Catalyzes Ascorbate Oxidation
Fe-EDTA
Fe-Desferal
Buettner, G.R. (1990) Ascorbate oxidation UV
absorbance of ascorbate and ESR spectroscopy of
the ascorbyl radical as assays for iron. Free
Rad Res Commns, 10 5-9.
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Iron Catalyzes Ascorbate Oxidation
Fe-EDTA
Fe-Desferal
Buettner, G.R. (1990) Ascorbate oxidation UV
absorbance of ascorbate and ESR spectroscopy of
the ascorbyl radical as assays for iron. Free
Rad Res Commns, 10 5-9.
32
Iron from Syringes
Buettner, G.R. (1990) Ascorbate oxidation UV
absorbance of ascorbate and ESR spectroscopy of
the ascorbyl radical as assays for iron. Free
Rad Res Commns, 10 5-9.
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The Ascorbate Test
Ascorbic acid solution (3.5 ?L of 0.100 M) is
added to 3.00 mL of near-neutral buffer solution
Absorbance is followed for 15 min at 265 nm
(AscH- ?265 14,500 M-1cm-1) A loss of more
than 0.5 in this time indicates significant
metal contamination goal lt0.05. Tips use
AscH2, not NaAscH- Do not interrogate the
solution continuously, photochemistry Clean,
clean, clean ground glass is a disaster
Buettner GR. (1988) In the absence of catalytic
metals, ascorbate does not autoxidize at pH 7
Ascorbate as a test for catalytic metals. J
Biochem Biophys Meth 16 20-40.
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Iron, mechanisms
Fe(III)chelate O2?? ??
Fe(II)chelate O2 or Fe(III)chelate AscH?
?? Fe(II)chelate Asc?? Then,
Fe(II)chelate H2O2 ?? HO?
Fe(III)chelate or LOOH ?? LO? or Fe(II)
O2 ?? Oxidants
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Iron and DioxygenSpin Trapping with POBN
Qian SY, Buettner GR. (1999) Iron and dioxygen
chemistry is an important route to initiation of
biological free radical oxidations An electron
paramagnetic resonance spin trapping study. Free
Radic Biol Med, 261447-1456.
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Iron and Dioxygen
What is O2H2O2 in tissue? O2 10 20
uM H2O2 1 100 nM Thus, O2H2O2 gt
1001
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Iron O2, mechanism?
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Chelates, some surprises
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Chelates, can drive Fe(II) oxidation
E?? (Fe(III)Desferal/Fe(II)Desferal) - 450
mV Kstability Fe(III) 1030.6 Kstability Fe(II)
107.2
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Chelates, can be great for Fenton rxn
E?? (Fe(III)EDTA/Fe(II)EDTA) 120 mV k
(Fe(II)EDTA H2O2) 104 M-1 s-1
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EDTA, Coordination of Fe(III)
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pH Iron problems
?, pH5.5 ?, pH6.0 ?, pH6.5 ?, pH7.0 ?,
pH7.5
POBN/Ld?. Cells were treated with Photofrin (9
?g/mL) for 45 min, then washed and resuspended in
PBS (pH 7.5-5.5) containing 100 ?M ascorbate and
5 ?M ferrous iron. Schafer FQ, Buettner GR.
(2000) Acidic pH amplifies iron-mediated lipid
peroxidation in cells. Free Radic Biol Med.
281175-1181.
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Desferal reduces UV light-induced radical
formation in skin
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Lipid Peroxidation and Nitric Oxide
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?NO Inhibits Iron-InducedLipid Peroxidation
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?NO and Fe2 vs Time
Kelley EE, Wagner BA, Buettner GR, Burns CP.
(1999) Nitric oxide inhibits iron-induced lipid
peroxidation in HL-60 cells. Arch. Biochem.
Biophys. 370 97-104.
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E vs ?NO Kinetics
1. Look at ?NO rate inhibition by
?NO kNO ?NO LOO? 2 x 109 M-1s-1
? (8 ? 45) ?10-9 M LOO?
720 s-1 LOO?
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E vs ?NO Kinetics
2. Look at TOH, i.e. E rate inhibition
by TOH kTOH TOH LOO?
8 ? 104 M-1s-1 ? 400
? 10-6 M LOO? 32 s-1 LOO?
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E vs ?NO Kinetics
3. Compare rate (?NO) 720 s-1
LOO? rate (TOH) 32 s-1 LOO?
? 20/1
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E vs ?NO Kinetics
4. When are rates 11? If TOH 400
?M Then when ?NO 2 nM Rates ? 1/1