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Free Radical Biology & Medicine, Vol. 22, No. 5, pp. 885 – 888, 1997 Copyright q 1997 Elsevier Science Inc.
Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00 THE ORIGIN OF THE HYDROXYL RADICAL OXYGEN IN THE Roger V. Lloyd,* Phillip M. Hanna and Ronald P. Mason National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709 (Received 5 June 1996; Revised 27 July 1996; Accepted 14 August 1996) Abstract—There is an ongoing discussion in the chemical literature regarding the nature of the highly reactive
hydroxyl radical formed from the reaction between ferrous iron and hydrogen peroxide (the Fenton reaction). How-
ever, the fundamental experiment of directly determining the source of the hydroxyl radicals formed in the reaction
has not yet been carried out. In this study, we have used both hydrogen peroxide and water labeled with 17O, together
with ESR spin trapping, to detect the hydroxyl radicals formed in the reaction. ESR experiments were run in
phosphate buffer with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, and either H2O2 or H2O labeled with
17O. The hydroxyl radical was generated by addition of Fe2/ ion to H2O2, or as a control, by photolysis of H2O2 in
the ESR cavity. Observed ESR spectra were the sum of DMPO/ i16OH and DMPO/ i17OH radical adduct spectra.
Within experimental accuracy, the percentage of 17O-labeled hydroxyl radical trapped by the DMPO was the same
as in the original hydrogen peroxide, for either method of hydroxyl radical generation, indicating that the trapped
hydroxyl radical was derived exclusively from hydrogen peroxide and that there was no exchange of oxygen atoms
between H2O2 and solvent water. Likewise, the complementary reaction with ordinary H2O2 and 17O-labeled water
also showed that none of the hydroxyl radical was derived from water. Our results do not preclude the ferryl
intermediate, [Fe O]2/ reacting with DMPO to form DMPO/ iOH if the ferryl oxygen is derived from H2O2 rather
than from a water ligand.
Copyright q 1997 Elsevier Science Inc. Keywords—Hydroxyl radical, Fenton reaction, Spin trapping, Electron spin resonance, Electron paramagnetic res-
onance, Free radicals
It has been argued, however, that some, if not all of thehydroxyl radical produced in the Fenton reaction may There is an ongoing discussion in the chemical litera- remain bound at the iron center, either as the ture regarding the nature of the highly-reactive hy- [Fe···iOH]3/ or the [Fe O]2/ intermediate (Eq. 2).
droxyl radical formed from the reaction between fer-rous iron and hydrogen peroxide (the Fenton reaction).1The classical Fenton mechanism (Eq. 1, or with ligands included, Eq. 1a) predicts that hydrogen peroxide isreduced at the iron center with generation of free hy-droxyl radical.
These intermediates are proposed to have oxidizingproperties similar to, but distinguishable from, free hy- droxyl radical, and have been promoted based on com-parison of Fenton reaction kinetics with that of the hy- droxyl radical generated independently of iron.
Recently Sawyer et al.2 studied ‘‘Fenton reagents'' with hydrocarbons as radical scavengers. From product Address correspondence to: Ronald P. Mason, National Institute analysis, they concluded that free iOH is not the dom- of Environmental Health Sciences, National Institutes of Health, P.O.
inant reactant at all, and that with chelated iron, a nu- Box 12233, Research Triangle Park, NC 27709.
*Permanent Address of Roger V. Lloyd: Department of Chemistry, cleophilic adduct reacts directly with substrates. Wink University of Memphis, Memphis, TN 38152.
et al.3 used stopped-flow kinetics and competition stud- / 2b25 2390 Mp
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R. V. LLOYD et al. ies to probe the reaction of a Fenton intermediate with Croft et al.12 have shown, however, that the radicals N-nitrosodimethylamine. They proposed a reversible formed from alcohols in the Fenton reaction can react reaction between Fe2/ and H2O2 to an intermediate X, with both iron(II) and iron(III) to yield kinetic results whose reactivity patterns were consistent with an iron that can appear to differ from the hydroxyl radical.
complex and not the iOH radical. However, Walling According to existing mechanisms, the oxygen atom and Amarnath,4 in a study of the oxidation of a man- in both the ferryl intermediate and the hydroxyl radical delic acid – iron complex by H2O2, had previously con- originate from hydrogen peroxide, but this has not been cluded that both an FeIV species and the hydroxyl rad- tested. Furthermore, if free hydroxyl radical reacts with ical were involved.
water, an exchange of oxygen atoms would occur. A For reactions involving free radical intermediates, more likely possibility is that iron-bound hydroxyl rad- ESR spectroscopy would appear to be the method of ical species would undergo exchange of oxygen with choice, but the iOH radical in solution cannot be di- water. In any case, the fundamental experiment of di- rectly detected by ESR. Thus, indirect techniques must rectly determining the source of the hydroxyl radicals be used, such as spin trapping with 5,5-dimethyl-1-pyr- formed has not yet been carried out. In this study, we roline-N-oxide (DMPO). Based on relative yields in have used both hydrogen peroxide and water labeled competitive trapping experiments, Yamazaki and with 17O, together with ESR spin trapping, to detect the Piette5,6 presented evidence for the presence of addi- hydroxyl radicals formed in the reaction. We show that, tional oxidizing species other than hydroxyl radical and within experimental accuracy, in the Fenton reaction stated that, although the iOH radical is present, it is not there is no exchange of oxygen atoms between 17O- all free in solution. However, it has been shown that, labeled H2O2 and solvent water or vice-versa.
under reaction conditions commonly employed, theDMPO/ iOH adduct can arise as an artifact, such as by MATERIALS AND METHODS
oxidation of the DMPO itself followed by reaction with Experiments were run in 100 mM phosphate buffer water, or by reaction of a precursor DMPO/superoxide (pH 7.4) with 100 mM 5,5-dimethyl-1-pyrroline N-ox- ide (DMPO, Sigma Chemical Co., St. Louis, MO) Although the reactivity of iron complexes has been added as a spin trap. The DMPO was purified twice by recognized,2 the existence of ‘‘secondary'' reactions vacuum distillation at room temperature and stored at having different reaction rates for different complexes 0807C before use, and the buffer was treated with has not always been considered. Burkitt7 has shown Chelexw 100 ion-exchange resin (Bio-Rad Laborato- that such reactions can involve complexed iron with ries, Hercules, CA) to remove trace heavy metal con- spin trap-hydroxyl radical adducts under commonly taminants. The 17O-labeled H employed reaction conditions. For example, the 2O2 was from Isotec, Inc.
(Miamisburg, OH), and was received as a 2% aqueous FeIIIDTPA (diethylenetriaminepentaacetic acid) com- solution, 82 atom % 17O. For each experiment it was plex, in particular, can be reduced by oxidation of the diluted fourfold to a final concentration of 0.5%. In the DMPO/ iOH spin adduct. If not recognized, these re- complementary experiment, the Fenton reaction was actions can confuse kinetic comparisons and stoichio- run with normal H metric calculations in the Fenton reaction.
2O2 in 17O-enriched water (52.7 atom %, Isotec, Inc.). ESR spectra were obtained on a Varian Scavenging experiments can also be difficult to in- E-109 ESR spectrometer with a TM terpret if the radicals formed from the scavenger mol- quartz aqueous flat cell. Spectra were stored on a com- ecules are capable of further reaction. Rush and Kop- puter for later analysis. The hydroxyl radical was gen- penol9,10 studied the Fenton reaction of FeIIEDTA, erated by addition of 1 mM Fe2/ ion (as FeSO FeIIDTPA, and FeIIHEDTA with alcohol scavengers reaction mixture. As a control, the hydroxyl radical was and recognized that the nature of the chelator was an also generated directly by photolysis in the ESR cavity important factor. Results with FeIIHEDTA suggested with a 1-kW Hanovia Xe/Hg compact-arc lamp in a an intermediate other than hydroxyl radical, but, in the Schoeffel housing.13 The solution for photolysis exper- other two cases, the properties of the intermediate were iments was identical to the Fenton solution except with- reported to be similar to the hydroxyl radical. Rahhaland Richter11 reported that FeIIDTPA reacted with H out added iron. Experiments were run at least in trip- to yield an oxidizing species whose properties in scav- licate. ESR hyperfine splittings and relative intensities enging experiments with tert-butyl alcohol were not were obtained by means of the program WINSIM.14 consistent with the iOH radical (Eq. 3).
RESULTS AND DISCUSSION
The observed spectra were the sum of the DMPO/ OH and DMPO/ i17 OH radical adduct spectra. The / 2b25 2390 Mp
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17O-labeling of the hydroxyl radical Table 1. ESR Parameters for the DMPO/ iOH Radical Adduct Hyperfine Coupling Constants, % DMPO/ i17OH Radicala a The sum of the 17OH and 16OH radical intensities was normalized to 100%.
b Literature value for 17O Å 4.66 G.15c Average { 1 range.
results of the experiments are given in Table 1. Typical nary H2O2 and 17O-labeled water also showed that, in experimental spectra with simulations based on the pa- the complementary reaction, none of the hydroxyl rad- rameters in Table 1 are shown in Figure 1.
ical was derived from the water solvent. In either case, Within the limits of experimental uncertainty, the trapping with DMPO was sufficiently rapid to prevent percentage of 17O-labeled hydroxyl radical trapped by any significant scrambling of the hydroxyl radical with the DMPO was the same as in the original hydrogen the solvent water.
peroxide for either method of hydroxyl radical gener- Because photolysis of hydrogen peroxide produces ation, indicating that the trapped hydroxyl radical was hydroxyl radicals by homolytic cleavage, these exper- derived exclusively from hydrogen peroxide and not iments indicated an insignificant exchange of 17O be- from water. Likewise, the Fenton reaction with ordi- fore trapping by DMPO (Table 1). Comparison of thephotolysis results with those for hydroxyl radical gen-erated via the Fenton reaction, shows that, in the latterreaction, the labeled radical was also trapped beforeany isotopic exchange could occur. Equations 2 and 3above imply that a significant fraction of the hydroxylradicals formed are bound in an iron complex. If thisis the case, dissociation of the radical as the original17O-labeled species must not be in competition withintramolecular electron transfer involving bound watermolecules, resulting in isotope scrambling. In addition,our results do not preclude the ferryl intermediate[Fe O]2/ reacting directly with DMPO to formDMPO/ iOH if the ferryl oxygen is derived from H2O2and does not exchange with a water ligand, but do ex-clude an exchangeable oxygen in any iron complexwith hydroxyl radical-like reactivity or that such a spe-cies dissociates to hydroxyl radical. Finally, the pos-sibility of a DMPO cation radical intermediate that re-acts with hydroxide anion to form DMPO/iOH8,16 isexcluded in the generation of DMPO/ iOH by the Fen-ton reaction or by the photolysis of H2O2.
1. Sutton, H. C.; Winterbourn, C. C. On the participation of higher Fig. 1. A: First-derivative ESR spectrum of the DMPO/ iOH adduct; oxidation states of iron and copper in Fenton reactions. Free Rad. H2 O2, 1 mM FeSO4, 100 mM DMPO, 100 mM phosphate Biol. Med. 6:53–60; 1989.
buffer (Chelex treated, pH 7.4). B: Computer simulation of spectrum 2. Sawyer, D. T.; Kang, C.; Llobet, A.; Redman, C. Fenton reagents A, based on the parameters in Table 1. C: Stick diagram. (----), (1:1 FeIILx/HOOH) react via [LxFeIIOOH(BH/)] (1) as hydrox- Spectrum of the DMPO/ i16OH radical. ( — ), Spectrum of the ylases (RH ! ROH), not as generators of free hydroxyl radicals DMPO/ i17OH radical.
(HOi). J. Am. Chem. Soc. 115:5817–5818; 1993.
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R. V. LLOYD et al. 3. Wink, D. A.; Nims, R. W.; Saavedra, J. E.; Utermahlen, W. E., aminecarboxylate complexes and hydrogen peroxide: An inves- Jr.; Ford, P. C. The Fenton oxidation mechanism: Reactivities of tigation of the reaction intermediates by stopped flow spectro- biologically relevant substrates with two oxidizing intermediates photometry. J. Inorg. Biochem. 29:199–215; 1987.
differ from those predicted for the hydroxyl radical. Proc. Natl. 11. Rahhal, S.; Richter, H. W. Reduction of hydrogen peroxide by Acad. Sci. USA 91:6604–6608; 1994.
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