J. Biol. Chem.-1996-Karoui-6000-9

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Frjaville, Paul Tordo and B. KalyanaramanHakim Karoui, Neil Hogg, ClaudineOXYGEN UPTAKE STUDIES

ANDPeroxynitrite: ESR-SPIN TRAPPINGOxidation of Thiols and Sulfite byRadical Intermediates Formed during theCharacterization of Sulfur-centeredCell Biology and Metabolism:

doi: 10.1074/jbc.271.11.60001996, 271:6000-6009.J. Biol. Chem.

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Characterization of Sulfur-centered Radical Intermediates Formedduring the Oxidation of Thiols and Sulfite by Peroxynitrite

ESR-SP IN TRAPP ING AND OXYGEN U PTAKE STUD IES*

(Received for publication, November 15, 1995, and in r evised form, December 19, 1995)

Hakim Karoui, Neil Hogg, Claudine Frejaville, Paul Tordo, and B. Kalyanaraman

F r o m t h e Bi ophysics Research I nstit ute, Medical Coll ege of Wisconsin, M il wau kee, Wisconsin 53226 and the Labor at oir eSt r uct ur e et R eacti vi te des Especes Par am agn eti qu es, CN RS U RA 1412 , U ni versi te de Prov ence, M ar seil l e, F r an ce

Using a novel phosphorylated spin trap, 5-diethoxy-phosphoryl-5-methyl-1-pyrrolineN-oxide(DEPMPO), ananalogof thecommonly used trap 5,5-dimethyl-1-pyrro-lineN-oxide(DMPO), wehave investigated thereactionsof sulfur-centered radicals produced from the oxidationof thiols and sulfite by peroxynitrite. The predominantspecies trapped in all cases are the corresponding sul-fur-centered radicals, i .e. glutathionyl radical (GS) fromglutathione (GSH), N-acetyl-DL-penicillamine thiyl radi-cal (S-NAP) from N-acetyl-DL-penicillamine (NAP) andsulfite anion radical (SO3.) from sulfite. These radicalsconsume molecular oxygen forming either peroxyl orsuperoxide anion radicals.GS, S-NAP, and SO3

.-derivedradicals react with ammonium formate to form the car-bon dioxideanion radical (CO2

.).F urther support of spinadduct assignments and radical reactions are obtainedfromphotolysis ofS-nitrosoglutathioneand S-nitroso-N-acetyl-DL-penicillamine. We conclude that the direct re-action of peroxynitrite with thiols and sulfite formsthiyl and sulfite anion radicals, respectively, by a hy-droxyl radical-independent mechanism. Pathologicalimplications of thiyl radical formation and subsequentoxyradical-mediated chain reactions are discussed. Ox-ygen activation by thiyl radicals formed during per-oxynitrite-mediated oxidation of glutathione may limit

theeffectivenessof GSH against peroxynitrite-mediatedtoxicity in cellular systems.

The reaction between nitric oxide (NO) and superoxide an-ion (O

2

. ) generates peroxynitrite1 at a near diffusion controlledrate (Equation 1) (1, 2).

NOO2

.O

k1 4.3 6.7 109

M1 s1

ONOO (Eq. 1)

This rea ction h as been implicated in man y pa thological condi-tions including reperfusion injury (3), atherosclerosis (4), andneurodegenerative diseases (5). There a re ma ny potentia l ta r-gets for peroxynitrite in biological systems including t hiols,

l ipid s, a nd DNA (6 8). Alt hough fr ee r ad ical int er m ed iat es

ha ve been proposed in peroxynitrite-mediated oxidat ion reac-t ions, t hey have been d ir ect ly d et ect ed in only a few cases(913). B ecause of its role in pa thophysiology, further under-st and ing of t he fr ee r ad ical r eact ions of per oxynit r i t e w it hpotentia l biological ta rgets is essential.

The chemistry of decomposition of peroxynitrite is complex(14). ONOO is s t able in a lkaline solut ions. How ever , uponprotonation (pK

a 6.9), ONOOH decays r a pidly (t12 1 s at pH

7.4) (15). The decay of ONOOH has been shown to have some

free radical chara cter as evidenced by malondialdehyde forma-t ion fr om d eoxyr ibose by a pr ocess inhibit able by hyd r oxylradical scavengers and aromatic hydroxylation of sodium ben-zoate (15). This a ctivity ha s been shown to ha ve man y similar-ities to the oxidant produced in the Fenton rea ction and is oftenreferred to as a hydroxyl radical-like species. Koppenol et al.(14) have shown that homolytic decomposition of ONOOH isunlikely and that the oxidant is probably an active conformerpr od uced d ur ing int er nal r ear r angem ent t o nit r ic ac id . Theoxidat ive effects of peroxynitrite are not confined t o this spe-cies, however, as ONOO and ONOOH can act as bot h one-electron and two-electron oxidants (6, 10, 16). For example,ONOO will oxidize gluta thione (G SH) to gluta thione disulfide(GSSG) (6).

In this study w e have att empted to cha ra cterize the reactions

of sulfur-centered ra dicals formed fr om the one-electron oxida -tion of thiols by peroxynitrite using the spin-trapping tech-nique. Previous electron spin resonance (ESR) studies of thedecomposition of peroxynitrite a t physiological pH ha ve used5,5-dimethyl-1-pyrroline N-oxide (DMPO)2 a s t h e s p i n t r a pand t he r esult s a r e cont r over sial . S hi et al . (13) reported theformation of 5,5-dimet hy l-1-pyrr olidin-2-one-1-oxy, a furt heroxidat ion product of DMP O, at high concentra tion of peroxyni-trite and concluded that free hydroxyl radical is not released.However, Augusto et al. (9) reported the formation of DMPO/OH from DMP O using low peroxynitrite concentra tions an d ast r ong and per sist ent DM PO/OH signal a t higher concent r a-t ions only in t he pr esence of GS H. R ecent ly , Pou et al . (11)reported that the yield of formation of HO from peroxynitrite

decomposition was about 1 to 4%based on the methane sulfinicacid assay . A m or e r ecent s t ud y fr om Lem er cier et a l . (12)claim ed t hat DM PO/OH adduct detected during the reactionb e t w e e n D M P O a n d O N O O is formed from a molecule-in-duced homolysis mechanism and not from trapping of HO .

The use of DM PO d oes not a l low easy d iscr im inat ion be-

* This research wa s supported by National Inst i tutes of Heal thGrants RR01008, HL 45058, and HL 47250 from the National Heart,Lung and Blood Institute. The costs of publication of this article weredefrayed in part by the payment of page charges . This art ic le musttherefore be hereby marked advertisement in accordance wi th 18U.S .C. S ection 1734 solely to indicate t his fa ct.To whom correspondence should be addressed: Biophysics Research

Institute, Medical College of Wisconsin, Milwaukee, WI 53226. Tel.:414-456-4035; F a x: 414-266-8515; E -ma il: B ala ra ma @post.its.m cw.edu.

1 Peroxynitrite has been used throughout this manuscript where noconsideration is given to the st at e of protonat ion or to t he conformat ionof this molecule. Where specificity is implied, ONOO and ONOOHhave been used.

2 The abbreviat ions used are : DMPO, 5,5-dimethyl-1-pyrroline N-oxide; CO

2

. , carbon dioxide radical anion; DEPMPO, 5-diethoxyphospho-ryl-5-methyl-1-pyrroline N-oxide; ESR, electron spin resonance; GSH,reduced glutathione; GSNO, S-nitrosoglutathione; HCO

2NH

4, ammo-

nium formate; NAP, N-acetyl-DL -penicillamine; ONOO, peroxynitrite;SNAP, S-nitroso-N-acetyl-DL -penicillamine; S-NAP, N-acetyl-DL -peni-cillaminyl radical; SO

3

. , sul fite a nion ra dical ; G , gauss .

THE J OURNAL OF B IOLOGICALC HEMISTRY Vol. 271, No. 11, Issu e of Ma rch 15, pp. 60006009, 1996 1996 by The American S ociety for B iochemistry and Molecular B iology, Inc. P r i nted i n U .S .A .

6000


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t w een DM PO/O H a n d D M P O /S G , t he r a d ical a d d uct for m edfrom trapping of the glutathionyl radical (GS ). To obviat e th isproblem, we ha ve used a novel spin tra p, 5-diethoxyphospho-ryl-5-methyl-1-pyrrolineN-oxide (DE P MP O) (1719, 45), to ex-amine the oxidative reactions of peroxynitrite. The phosphorusnucleus (I 1/2) in DE P MPO spin a dducts ha s a large hyper-fine splitting (a P between 45 and 53 G) that is sensitive to the

nat ur e of t he r ad ical (R ) t r apped and t o t he nit r oxid e spinad d uct confor mat ion (S chem e 1) (18). For t he DEP M PO/Rspectrum, the extra hyperfine coupling to phosphorus (I 1/2)results, in most cases, in a spectrum tha t resembles a juxtapo-sit ion of t w o DM PO/R spin adduct spectra.

I n t his paper , using ES R , gas chr om at ogr aphy, a nd oxygenuptake studies, we have characterized the reactions of sulfur-cent er ed r ad icals for m ed fr om t he r eact ion of per oxynit r it ewith sodium bisulfite and severa l thiols including gluta thione.Biological implications of thiyl radical production from per-oxynitrite in cellular systems a re discussed.

EXP ER IME NTAL P R OCE DUR ES

ChemicalsG S H , G S S G , C h el ex 100, N-acetyl-DL -penicillamine(NAP), ferrous sul fate , EDTA, diethylenetriaminepentaa cet ic acid

(DTPA), and manga nese dioxide w ere obtained from S igma. Sodiumnitrite, hydrogen peroxide, and sodium hydroxide were obtained fromFischer Scientific (Pittsbur gh, P A). S-Nitrosoglutathione (GSNO) andS-nitroso-N-acetyl-DL -penicillamine (SNAP) were synthesized accord-ing to the published procedure (20). 2,2,5,5-Tetra methy l-3-pyrr oline-1-oxyl-3-carboxamide w as obtained fr om Molecular Pr obes I nc. (Eugene,OR). The manganese macrocyclic superoxide dismutase mimic, SC-52608 (21), w as obtained fr om the Monsanto C o. (St . Louis, MO).

Peroxynitr ite SynthesisPeroxynitri te was synthesized by mixingice-cold solutions of 1 M NaNO

2 a n d 1 M H

2O2

in 0.3 M H2

S O4

a ndrapidly quenched by 1.4 M Na OH (15). E xcess hydr ogen peroxide wa sremoved by adding a sma ll amount (1 mg)of MnO

2. After filtering, the

solut ion was kept frozen at 20 C. The yellow t op layer conta ined170250 m M peroxynitrite a s determined by the a bsorbance at 302 nm( 1670 M1 cm1). Peroxynitrite solutions w ere prepared by dilutionto the desired concentrat ion in 0.1 M N aO H .

ESR M easurementsESR spectra were recorded a t room tempera-

ture on a Varian E109 spectrometer at 9.5 GHz employing 100 kHz fieldmodulation. Reaction mixtures were prepared in Chelex-treated 0.2 Mphosphate buffer, pH 7.4. After the addi t ion of peroxyni tri te to thesolution mixture, the solution wa s incubated for 30 s, transferred into a100-l capillar y (Corning, NY) and placed in a 4-mm qua rtz t ube insidethe ESR cavity. The final pH was 7.47.5. The phosphate buffer usedfor the spin-trapping experiments was saturated with argon. Authenticspin adducts w ere genera ted by (i)photolysis of nitrosothiols, the qua rtztube containing the capi l lary was i rradiated wi th vis ible l ight ( 350550 nm) inside the ESR cavity; and (ii) a Fenton reaction systemconsisting of H

2O2

(1 m M), Fe2 (0.05 m M), and EDTA (1 m M). AuthenticDE P M P O /OOH w as generat ed by a nucleophilic addi t ion of H

2O2

t oDEPMPO in pyridine. Data were collected using the VIKING software,developed a t t he Medical College of Wisconsin, and simulated with theESR softwa re developed by D. D uling from the Labora tory of MolecularBiophysics, National Institute of Environmental Health Sciences, NC.Simula tions were used to deconvolute the complex ES R spectra derived

from D EP MPO. As wi th al l f i t t ing procedures , we recognize that thecomputer simulations may not represent a unique solution to the data.ESR spectra were approximately quantified by double integration andcomparison to a nitroxide standard (2,2,5,5-tetramethyl-3-pyrroline-1-

oxyl-3-carboxamide). The relative contribut ion of th e different ra dicaladducts to the total integrated area was derived from the s imulatedspectrum.

Gas ChromatographyC O2

ana lysis wa s performed using a Varian3700 Gas Chromatograph equipped with a 6 feet 1/8 inch P ora pak Qcolumn a nd t hermal conductivi ty detector operat ing at 30 C wi th aflow rate of 5 ml/min. Sa mples w ere prepared in a sealed vial a ndperoxynitrite wa s injected through the seal immediat ely prior to mea s-urement. Headspace gas (100 l) was injected into the gas chromato-graph w ith a n a irt ight Ha mil ton syringe. The CO

2re tention t ime w as

2.12.3 m in.Oxygen UptakeOxygen uptake experiments w ere performed using

a YSI Oxygen electrode (Yellow Springs Instruments, Columbus, OH)at 37 C . The electrode wa s cal ibrated using air-satura ted w ater (240M oxygen) and sodium di thioni te-treated water (0 M oxygen). Allexperiments were performed using phospha te buffer (200 m M, pH 7.4)with a f inal volume of 5 ml . Data are g iven a s mean S . D. (n 3).

RESULTS

S pi n- t r appi ng of H O and G S by D E P M P O Fig. 1, aa nd b,show s t he ES R spect r a of aut hent ic DEP M PO/OH (aN 14.0G , aH 13.2 G, a H 0 .27 G (3H ) , a n d a P 47.3 G) andD E P M P O /S G (aN 14.1 G, aH 1 4. 9 G , a n d a P 45.8 G)ad d uct s gener at ed by a Fent on syst em and by phot olysis ofGSNO, respectively. There is a clear difference between thesetwo spin adduct spectra that is mainly due to the variations inphosphorus hyperfine split t ings. A simulated spectrum of am i xt u r e o f D E P M P O /OH (20%) a nd DEP M PO/SG (80%) isshown in Fig. 1c. The majority of the spectral lines overlap andcannot be used as dia gnostic indicators of either ra dical. H ow-ever , t he cent r al por t ion of t he spect r um clear ly show s t hecontributions of DE PMP O/OH and DEPM PO/SG t o the signal.

S C H EM E 1 .Spin trapping oftransient radical (R) by DMPO andDEPMPO. The asymmetric carbon atoms are marked by an asterisk(*).

F IG . 1.ESR spectra oftheauthentic hydroxyl andglutathionylradical adductsof DEPMPO. a, spectrum obtained upon incubatingDEP MPO (20 mM), H

2O2

(0.2 m M), E DTA (0.2 m M), and FeS O4

(0.1 m M);b, spectrum obta ined upon photolysis of GS NO (1 mM) in the presenceof DEPMPO (20 m M) and DTPA (0.1 mM); c, computer simulation of amixture of DEP MPO/SG (80%) and D EP MPO/OH (20%) ad du cts. Thespectra in a a nd b were obtained at room temperature in phosphatebuffer (0.2 M , pH 7.4). S pectrometer sett ings: microwave power, 5 mW;modulation a mplitude, 0.5 G; time consta nt, 0.128 s; gain, 5 104; scanrange, 200 G; and scan time, 240 s.

Thiyl Radicals from Peroxynitr i te 6001


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Spin -tra ppin g of Radi cals Form ed from th e Reaction betw een

P er o xy n i t r i t e, G l u t a t h i on e, a n d D E P M P O I n cu b a t i on o fDEP MPO w ith peroxynitrite in phosphat e buffer (200 mM, pH7.4) did not result in an y E SR detectable spin adduct (Fig. 2a).

This is in contra st to previous studies with DMP O wherein theinvestigators reported formation of DMPO/OH during the re-action between DMPO and peroxynitrite (9, 11, 12). The addi-tion of HO scavengers (ethanol, 1.8 M; Me

2SO, 1.4 M; am m o-

nium formate, 1 M ; mannitol, 1 M , or t-buta nol, 1.1M ) also gaveno ESR detecta ble adduct. In th e presence of GSH (1 mM), th espectrum shown in Fig. 2bwa s observed. This signa l is complexan d consists of several spin a dducts. The predominant speciesi s D E P M P O /SG which corresponds t o 55 to 60%of the signa l.This represents a concentra tion of 12 t o 14 M D E P M P O /S Gindicating that the efficiency of the one-electron oxidation ofG S H t o G S by peroxynit rite is a bout 12%. The hig h perform-ance l iquid chr om at ogr aphy d a t a ind icat e t hat DEP M PO d oesnot affect the yield of GSSG during ONOO-dependent oxida-t ion suggest ing t hat GS is a minor product from t he reaction

between GSH and ONOO

.3

The origin of the remaining signalin Fig . 2b is uncertain, but the whole spectrum can be simu-lat ed (Fig . 2b, dot t ed l i ne) by assum ing t he pr esence of t w oradical adducts with hyperfine split t ing constants characteris-tic of ca rbon-centered a dducts (17) (aN 14.5 G, aH 21.5 G,a n d a P 45.9 G; aN 14.8 G, aH 20.7 G, and a P 47.8 G )and a species ( from 10 t o 19%) w it h par am et er s s im ilar t oD E P M P O /OH (aN 14.0 G , aH 13.4 G , aH 0.6 G (3H), an da P 47.5 G). Bot h DE PM P O/S G a n d D E P M P O /OH a d d uct sd eca y e d w i t h t i m e l ea v i n g o n l y t h e s i gn a l a s s i gn ed t o t h ecarbon-centered radical adducts (Fig. 2c). This spectrum can besimulated (Fig. 2c, dotted l ine) using the same ESR pa ra meters

for t he car bon-cent er ed r a d icals show n in Fig . 2b. We alsoobser ved a s im ilar behavior af t er t he d ecay of t he DEP M PO/

S G a d d u ct ob t a i n ed d u ri n g p h ot ol y si s o f G S N O (d a t a n otshown). Augusto et a l . (9) also reported the detection of anunidentified carbon-centered radical during oxidation of GSHby peroxynitrite in the presence of DMPO. The source of thesecarbon-centered radical is unknown but Grierson et al .(22) an dZhao et al . (23) have reported formation of a carbon-centeredradical from an intra molecular rea rra ngement of the glutath io-nyl r a d ical .

I n or d er t o exam ine w het her t he DEP M PO/OH spin adductin Fig . 2b is formed from trapping of HO , the effects of HO

scavengers were investigat ed. Fig. 3 shows th at a large excessof t-butanol (1.1 M) (Fig. 3b), mannitol (1 M) (Fig. 3c), and alsoethanol (1.8 M) a n d M e

2SO (1.4 M) (d at a not show n) had only

m inor e ffect s on t he DEP M PO/OH com ponent of t he E S R

spectrum in Fig. 3a. The effects of ethanol and Me2S O w er eid ent ical ir r espect ive of DEPM PO concent r at ion (20 t o 100m M). This suggest s t ha t t he signal w it h DEP M PO/OH par am -eters (Figs. 2ba n d 3a) does not a rise from the tr apping of freehydroxyl radical.

An a lt er nat e r out e by w hich DEP M PO/OH can be formed isvia decomposition of DEPMPO/O OH t o D E P M P O /OH. How -ever, unlike DMP O/OOH, t he DEP M PO/OOH adduct does notdecay sponta neously to DE PMP O/OH (18, 24). It is conceiva blet hat t his conver sion is fac i l i t a t ed by high concent r at ions ofGS H. This reaction is similar to the G SH-dependent reductionof hydroperoxide to alcohol (Equation 2). In support of this,GS H w a s show n t o fac ili t a t e t he conver sion of chem ically orenzym at ically synt hesized DEPM PO/O OH t o D E P M P O /OH(data not shown).3 H. Karoui , N. Hogg, and B. Kalyanaraman, unpublished data.

F IG . 2. The ESR spectra obtained after addition of peroxyni-trite to GSH in the presence of DEPMPO at pH 7.4. a, spectrumobtained upon incubat ing D EP MPO (50 mM), ONOO (0.8 m M) , andDTPA (0.1 m M)in phosphate buffer (0.2M , pH 7.4)a t room temperat ure;b, a s a but obtained immediately in the presence of GSH (1 m M) an dONOO; an d c, the spectrum obta ined aft er 10 min. The dotted l i nesinb a nd c show a computer simulation of the respective spectra. Spec-trometer settings: microwa ve power, 5 mW; modulat ion amplitude, 1 G;time constant , 0.128 s; gain, 8 104; scan ra nge, 200 G; a nd scan time,120 s . Contributions of the various radical adducts to the s imulatedspectra are described in the text.

F IG . 3. E ffect of hydroxyl r adical scavengers on the formationof DEPMPO/SG adduct. a, spectrum obtained upon incubat ingDEPMPO (20 m M), GSH (1 m M), peroxynitrit e (0.8 m M), an d D TP A (0.1m M) at room temperature in phosphate buffer (0.2 M, pH 7.4); b, a s awith t he addi t ion of tert-but yl a lcohol (1.1 M); c, a s awi th the addi t ionof man nitol (1 M ); and d, a s cin the a bsence of GSH (1 mM). Spectrom-eter settings: microwave power, 5 mW; modulat ion amplitude, 1 G; t imeconstant , 0 .128 s ; gain , 8 104; s c an r a n g e, 2 00 G ; an d s can t i m e,120 s.

Thiyl Radicals from Peroxynitr i te6002


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2 G SH G SS G H 2O

DE P M P O /OO H OOO3 DE P M P O / OH (E q . 2)

To fur t her ver ify t hat DEPM PO/OH is actually formed fromD E P M P O /OOH, we investigated the effect of superoxide dis-muta se mimic in peroxynitrite/G SH system.4 The spectrum

obt ained in t he absence of super oxid e d ism ut ase m im ic w a ss im u la t e d a s s u m in g t h e p re se nce o f D E P M P O /SG (44%),D E P M P O /OH (19%), a nd tw o ca rbon-centered a dducts (37%)(Fig. 4a) . No ES R spect r um w as obt ained in t he absence ofperoxynitrite. The spectrum obtained in the presence of super-oxide dismutase mimic (1 m M) shows no evidence for the pres-ence of t he DEPM PO/OH ad d uct (Fig . 4b) as d et er m ined bysim ulat ion using DEP M PO/SG (88%) and on e carb on-center edad duct (12%) (Fig. 4b, dot t ed l i ne). The spectral intensity ofD E P M P O /SG wa s enha nced in t he presence of superoxide dis-muta se mimic (Fig. 4b), perhaps indicating a superoxide-medi-at ed destruction of the DEP MPO/SG adduct. This is consistentw it h a pr evious r epor t w her e i t w as show n t hat ad d it ion ofsuperoxide dismuta se enhanced the signal int ensity of DMPO/SG adduct formed dur ing h orseradish peroxidase/H

2

O2

-cata-lyzed oxidation of GSH (25).

E f f ect of A m m oni um F or m at e (H C O 2

N H4) on Sp i n A d d u ct

Formati on d ur ing the React ion between Peroxynitr i te, GSH,and D E P M P O As mentioned a bove, the rea ction between per-oxynitrite (0.8 m M) a n d H C O

2NH

4 (1 M) in t he pr esence of

DEPMPO (20, 50, and 100 m M) generated no ESR signa l. How-ever, in the presence of GSH (1 m M) , DEPM PO/C O

2

w as ob-served (Fig. 5, ac). Lower concentrations of HCO

2NH

4 (100

m M) g a v e a n E S R s pe ct r u m t h a t co ns is t ed of a m i xt u r e o fD E P M P O /SG (marked E) a n d D E P M P O /C O

2

(marked ) ad -

ducts (Fig. 5a). At higher concentrations of HCO2

NH4

(0.4 a nd

1 M ), t he DEP M PO/C O2

adduct domina ted the spectrum (Fig.5, b a nd c, respectively). The dotted l ine signal represents thecomputer simulat ion of the spectrum 5c which is a mixture ofD E P M P O /C O

2

(80%) and D EP MP O/SG (20%). The a ut hen ticD E P M P O /C O

2

spin adduct (a N 14.5 G, a H 17.3 G, a nd a P

51.6 G) was generated by the Fenton reaction in the presenceof DEP M PO (20 mM) a n d H C O

2NH

4(1 M) (Fig. 5d). The gen-

eration of GS from photolysis of GSNO (1 m M) in th e presenceof DEP MPO (20 m M) a n d H C O

2NH

4(400 m M) led t o a time-de-

pend ent for m at ion of DEPM PO/C O2

(Fig. 6, ae). These re-sult s indicat e t ha t G S generated from reaction of peroxynitritew i t h G S H r e a c t s w i t h H C O

2NH

4 t o f o r m t h e C O

2

. r ad ical(Equations 3 and 4).

G S H ONOOOk3 10

3 104M1 s1

G S NO 2H O

(Eq. 3)

G S HCOOOk4 10

4M1 s1

G S H CO2

. (Eq. 4)

CO2

.O2O

k5 109

M1 s1

C O2 O2. (Eq. 5)

Glutathionyl Radical-mediated CO2

FormationI n t he ab-sence of DEPMPO, CO

2

. will react with O2

to give CO2

a n d t h esuperoxide anion, O

2

. (Equa tion 5) (26). No detecta ble CO2

w a sformed from the reaction between peroxynitrite and HCO

2NH

4.

However, in the presence of GSH , CO2

formation w as observed(Fig. 7). DEP MP O (30 mM) completely inhibited C O

2formation

in this reaction system (fig. 7), suggesting that GS is respon-

4 Unlike superoxide dismuta se, the superoxide dismutase mimic doesnot react directly wit h peroxynitrite (persona l communicat ion w ith D r.Ran dy Weiss of the Monsanto C o.).

F IG . 4. Effect of superoxide dismutase mimic on the radical

adduct formation during the reaction between GSH, ONOO

,and DEPMPO.a , spectrum obtained upon incubating DEPMPO (20m M) , G S H ( 1 m M), peroxynitrite (0.75 m M) , and DTPA (0.1 m M) inphosphat e buffer (0.2 M, pH 7.4) at room temperature ; b, a s a in thepresence of superoxide dismutase mimic (1000 M). The dotted l inesina a nd b show a computer simulation of the respective spectra. Instru-ment set t ings : microwa ve power, 5 mW; modulat ion ampli tude, 1 G ;time consta nt, 0.128 s; gain, 8 104; scan ra nge, 200 G; a nd scan time,120 s . Contributions of the various radical adducts to the s imulatedspectra are described in the text.

F IG . 5. Effect of ammonium formate on formation of spin ad-ducts during oxidation of GSH by ONOO.a , spectrum obtainedupon incubating DEPMPO (20 m M), G SH (1 mM), HCO

2NH

4(100 m M),

ONOO (0.8 m M), and DTPA (0.1 mM) in phosphate buffer (200 m M, pH7.4);b, a s abut in the presence of HCO

2NH

4(400 m M).c, same as ab ut

containing HCO2

NH4

(1 M). The d o t t ed l in e in c shows a computersimulation of the spectrum; and d, the authentic DEPMPO/C O

2

spinadduct prepared by oxidat ion of formate anion in a Fenton system.Spectrometer sett ings: microwa ve power, 5 mW; modulation a mplitude,1 G; t ime consta nt, 0.128 s; gain, 8 104 (2.5 104 for d); scan range,2 0 0 G ; an d s c an t i m e , 1 2 0 s . Open a nd closed circl es indicate thelow-field lines of D EP MPO/S G an d DE P M P O /C O

2

, respectively.



6/11

sible for CO2

formation. GSNO photolysis in the presence ofH C O

2NH

4 a lso r esult ed in t he evolut ion of CO

2 w h i c h w a s

inhibited by DEPMPO (data not shown).Spin-trappi ng of Radicals Formed dur ing th e Reaction between

N-Acetyl-DL -penici l lami ne and Peroxynitr i teThe DEP MPO/N-acetyl-DL -penicillaminyl spin adduct (DEPMPO/S-NAP)5 ex-hibits a distinctly different ES R spectrum tha n other DEP MPO

t h i yl a d d u ct s . Au t h en t i c D E P M P O /S -N AP a d d u ct w a s ob -

t a i n ed b y p h ot ol y si s of S N AP (1 m M) i n t h e p re se nce ofDEP M PO (50 m M) an d D TP A (0.1 mM) in Chelex-treated phos-phat e buffer (Fig . 8a) . The spect r um show n in Fig . 8a w a ssimulated by assuming the presence of two diastereoisomericD E P M P O /S -NAP ad d uct s w it h sl ight ly d if fer ent hyper f inesplit t ing consta nts (aN 14.3 G, aH 16.5 G, a nd a P 44.9 G(32%); aN 14.4 G, aH 15.2 G, a nd a P 46.2 G (38%)) an da carbon-centered radical adduct (a N 14.8 G, a H 20.8 G ,a n d a P 48.0 G (30%)) (Fi g. 8a, dotted l ine).

The incuba tion of NAP (10 m M) w it h DEP M PO (50 m M) andperoxynitrit e (0.8 m M) in phospha te buffer led to the forma tionof a spectrum (Fig. 8b) w hich w as essent ial ly id ent ical t o t hespectr um obta ined by photolysis of SNAP (Fig. 8a). No evidencefor DEP M PO/OH a dduct wa s observed and the addit ion of HO

scavengers such as ethanol (1.8 M) or M e2SO (1.4 M) h a d n oeffect on the signal. DE P MPO/OH wa s also not observed whenN-acetyl-D L-cysteine was used (data not shown ). Addition ofperoxynitrite to a mixture conta ining DE PMP O (50 mM), NAP(10 m M), and ammonium formate produced an ESR spectrum(Fig. 8c) consist ing of bot h DEPM PO/S - NAP and DEPM PO/C O

2

ad d uct s . The spect r um w a s sim ulat ed by a ssum ing t hepr esence of t w o d iast er eoisomer s of DE PM P O/S -NAP (33%)a n d D E P M P O /C O

2

(67%) (F ig. 8c, dotted l i ne). These result sind icat e t hat S - NAP also r eact s w it h am m onium for m at e t oform CO

2

. .

5 The a bbreviat ions SNAP and S-NAP indicate S-nitroso-N-acetyl-DL -penicillamine and the N-acetyl-DL -penicillamine thiyl radical, re-spectively. Note tha t t he S in SNAP r efers to the S-nitroso functional

group which is lost upon conversion to S-NAP. In S-NAP the S indicates the presence of a thiyl radical.

F IG . 6. E ffect of ammonium formate on radical adduct forma-tion fromGSNO photolysis. a, spectrum obtained immediat ely uponcontinuous i rradiat ion of GSNO (1 m M) a n d D E P M P O ( 2 0 m M) inphosphate buffer (0.2M , pH 7.4);b, a s abut in the presence of HCO

2NH

4

(400 m M);c, spectrum baft er 5 min;d ,a s aa f ter 7 min; and e,a s aaf ter15 min. Sym bols E a nd indicat e low-field line positions of D EP MPO/S G a n d D E P M P O /CO

2

, respectively. S pectrometer settings: micro-wa ve power, 5 mW; modulat ion amplitude, 1 G; time constant , 0.128 s;gain , 8 104; scan range, 200 G; and scan time, 120 s.

F IG . 7. Effect of GSH on CO2 formation in the presence ofammonium formateand peroxynitrite. Forma tion of CO

2wa s mon-

i tored by G C in the headspace gas of the vial containing a mixture ofammonium formate (1 M ), GSH (1 m M), DE PMP O (30 mM), or peroxyni-tri te as indicated. Resul ts ar e presented a s mean S . D. (n 3).

F IG . 8. The ESR spectra obtained after addition of peroxyni-trite to NAP in the presence of DEP MPO. a , spectrum obtainedupon photolysis of SNAP (1 m M), DEPMPO (50 m M), an d D TP A (0.1 mM)in phosphate buffer (200 m M, pH 7.4); b, spectrum obtained uponincubat ing DEP MPO (50 mM), NAP (10 m M), peroxynitrite (0.72 m M),and DTPA (0.1 m M) in phosphate buffer; c, s a m e a s b but containingH CO

2NH

4 (100 m M). The dotted l i nes in a a nd c show a computer

simulation of the r espective spectra. Spectrometer sett ings: microwa vepower, 5 mW; modulat ion amplitude, 1 G ; time constant , 0.128 s; gain,5 104 (a) and 8 104 (ba nd c); scan ra nge, 200 G; a nd scan time, 120s. Contributions of the var ious radical addu cts to the simulated spectraare described in the text.



7/11

Thi yl Rad ical-mediat ed Oxygen Upt akeDecomposition ofperoxynitrite results in t he evolution of O

2 (7). The concentra-

tion of oxygen formed w as an approximately linear function ofperoxynitrite concentration up to 1 m M (10% yield) and wa sunaffected by DEPMPO (20 m M). H owever, in t he presence ofG S H (1 m M), rapid consumption of O

2 wa s observed (Fig. 9).

The addition of DEPMPO (20 m M) inhibited O2

consum ption by62 2%(Fig. 9, inset), indicating the possible involvement oft hiy l r a d icals .

The O2

upt ake d ur ing t he r eact ion bet w een per oxynit r i t eand NAP in phosphat e buffer (200 m M, p H 7. 4) a t 3 7 C i sshown in Fig. 10. The O

2upta ke increa sed as a function of NAP

concentration (Fig. 10, inset). U nder similar experimenta l con-ditions, the O

2 upt ake w it h NAP w as m uch higher t han w it h

GSH, suggesting differences in the reaction mechanism of thecorresponding thiyl radicals.

S pi n- t r appi ng of S ul f i t e R adi cal A ni on F or m ed dur i ng t he

React ion between Sodiu m Bisul f i te and Peroxynitr i teThe re-act ion bet w een per oxynit r it e and NaHS O

3 in the presence of

DEPM PO r esult ed in t he for m at ion of DEPM PO/S O3

adduct(aN 13.3 G, aH 14.9 G, and a P 48.9 G) (Fig. 11a). Forcom par ison, t he DEP M PO/S O

3

adduct formed from HO a ndN a H S O

3 i s s h o w n i n F i g . 1 1c. Hyd r oxyl r ad ical scavenger s

(ethanol, 1.8 M, and M e2

SO 1.4 M) had only a slight effect (5 to10%) on t he ES R signa l intensity shown in Fig. 11a indicatingt h a t S O

3

. probably a rises from a direct reaction betw een HSO3

and per oxynit r i t e. The signal int ensit y of DEP M PO/

S O3.

a d-duct wa s diminished in oxygen satu rat ed buffer. This supportstrapping of free SO

3

. b y D E P M P O t o f or m t h e D E P M P O /S O3

ad d uct , as S O3

. radical reacts ra pidly with oxygen (27).Sulf i te Radical A nion-mediat ed Oxygen Upt akeAddition of

peroxynitrite to va rious concentra tions of NaH SO3

(from 0.1 to1 m M) caused a concentration-dependent increase in O

2 con-

sum pt ion (Fig . 12). Dur ing t he r eact ion bet w een 200 MN a H S O

3an d 300Mperoxynitrite, DE PMP O (20 mM)inhibited

oxygen consum ption by 69 6%(Fig. 12, inset). The inhibitionin O

2upta ke observed in the presence of DEP MPO is probably

due to effective tra pping of SO3

. b y D E P M P O .E f f ect of A m m oni um F or m at e on R adi cal F or m at i on dur i ng

the React ion between Peroxynitr i te and Sulf i teAddition ofammonium formate to the incubation mixture (sodium bisul-

fite, DEPMPO, and peroxynitrite) but containing a lower con-ce nt r a t ion of D E P M P O (1 m M) g a v e t h e D E P M P O /C O

2

(marked ) a n d t h e D E P M P O /S O3

(marked f) adducts (Fig.13). The dotted l inesin ba nd c show computer simulations of am i x t u r e o f D E P M P O /C O

2

(56% for b a nd 64% for c) a n dD E P M P O /SO

3

(44% for ba nd 36% for c) adducts. At higherconcentrations of DEPMPO (i.e.20 m M) we did not observe the

F IG . 9. Oxygen consumption during thereaction between GSHand peroxynitrite:effect of spin trap. Peroxynitri te was added intoa chamber of an oxygen electrode containing GSH (1 m M) and DTPA(0.1 m M) in phosphate buffer (0.2 M , pH 7.4) and the tota l O

2consumed

was recorded. Inset, O2

uptake tra ces obtained by the addi t ion of GSH(1 m M) alone (), with peroxynitrite (0.3 m M) in the absence of DEP MPO

() an d i n t h e p r e s e n c e o f DE P M P O ( 2 0 m M) (). The a r r o w (2)indicat es the point of a ddition of peroxynitrite.

F IG . 10. Oxygen consumption during the reaction betweenNAP and peroxynitrite:effect of spin trap. Peroxynitrite (280 M)wa s a dded into a chamber of an oxygen electrode containing var yingconcentrations of NAP and DTPA (0.1 m M) in phosphate buffer (0.2 M,pH 7.4) and the tota l O

2consumed was recorded. Inset,O

2uptake tra ces

obtained by the addition of NAP (1 m M) alone (), with peroxynitrite(280 M) i n t h e a b s e n c e o f D E P M P O () an d i n t h e p r e s e n c e o fDEPMPO (30 m M) (). The a r r o w (2) indicat es the point of ad dition ofperoxynitrite.

F IG . 11. Addition of peroxynitrite to sodium bisulfite in thepresence of DEPMPO. a, spectrum obtained upon the incubation ofDEP MPO (20 mM), NaHS O

3(1 m M), peroxynitrit e (0.36 m M), and D TPA

(0.1 mM) in phosphate buffer (200 m M, pH 7.4); b, a s a in 100% O2-

satura ted buffer; c, the ESR spectrum of an authentic DEPMPO/S O3

obtained in a Fenton reaction (H2

O2

(0.1 m M), NaH SO3

(30 m M), EDTA(0.1 m M), and FeSO

4(0.05 m M)). The dotted l inein cshows a computer

simulation of the spectrum ; and d, a s ain t he a bsence of peroxynitrite.Spectrometer sett ings: microwa ve power, 5 mW; modulation a mplitude,1 G; t ime consta nt, 0.128 s; gain, 8 104; scan range, 200 G; a nd scantime, 120 s.



8/11

D E P M P O /C O2

ad d uct . This is l ikely t o be d ue t o t he m or eeffective tr apping of S O

3

. under these conditions.The SO

3

. is a reducing ra dical a nd probably does not oxidizeH C O

2

t o C O2

. . On t he ot her hand , t he OOSO3

radical formedby the reaction between O

2 a n d S O

3

. can a bst r act a hyd r ogenatom from HCO

2

to give CO2

. (Equa tion 6) which is t ra pped byD E P M P O .

O3S OO H C O23O3SOOH C O2

. (Eq. 6)

It is likely that SO4

. formed from SO3

. a n d O2

could a lso oxidize

for m at e a nion t o t he CO2. radical.

D I S C U S S I O N

Peroxynitr i te-mediated Oxidat ion of DEPMPO and DMPO

Our d at a show t hat incubat ing DEPM PO w it h per oxynit r i t ed oes not for m t he DEPM PO/OH ad d uct (Fig . 2a). This is incontra st to previous findings using D MPO (9, 11, 12). S everalinvestigators have detected DMPO/OH a d d uct d ur ing t he r e-action betw een DMP O a nd peroxynitrite (9, 11, 12). P roposedm echanism s of for m at ion of DM PO/OH include oxidation ofD M P O t o D M P O r a d i ca l ca t i on f ol low e d b y h y d rol y si s, a sshown below (12):

H

DM P O ONOO3DM P O.NO 2H O (Eq. 7)

DM P O.H 2O 3DM P O /OH H (Eq. 8)

The inability of peroxynitrite to oxidize DEPMPO is of interest.The oxid at ion pot ent ials of DM P O a nd D EP M PO w er e m eas-ured t o be about 1.87 V (versusNHE ) and 2.24 V (versusNHE ),respectively.6 It is likely t ha t t he 400 mV difference in oxida-t i on p ot e n t ia l cou l d a c co un t f or t h e l a ck of ox id a t i on ofDEPM PO by per oxynit r i t e . The pr esent d at a show t hat t heD E P M P O /OH a dduct tha t is formed from the rea ction betweenperoxynitrite and DEPMPO is likely to be derived from GSH-dependent decomposition of D EP MPO/OOH.

P er oxyni t r i t e-dependent S ul f ur R adi cal F or m at i on Thiyl

radical formation in peroxynitrite-mediated oxidation of thiolshas been previously detected using DMPO (9, 12, 13). Thisobservation is now confirmed using DEPMPO, as thiyl radicaladducts of G SH, cysteine, an d NAP were observed. The forma-tion of thiyl adducts was independent of the hydroxyl radical-like reactivity of peroxynitrite, as hydroxyl radical scavengersd id not af fect t he DE PM P O-t hiy l r ad ical spect r a . I t has beenpreviously reported that ONOO reacts with t hiols to generat ethiol disulfide (6). It is not clear wh ether one-electron oxida tionof GS H by per oxynit r i t e is an int er m ed iat e st ep in d isulf id e

formation or represents an independent pathway of oxidationby one of the conformers of ONOOH.

As discussed earlier, the DEPMPO-dependent inhibition ofO2

uptake observed during peroxynitrite-mediated oxidation ofGS H a nd NAP is d ue t o

RS O2Ok9 10

9M1 s1

RSOO (Eq. 9)

RS D E P M P OOk10 10

6 107M1 s1

DEP MPO/SR

(Eq. 10)

t r apping of t hiy l r a d icals (Equa t ions 9 a nd 10). M ost t hiy lr a d i ca l s , s u ch a s G S , r e a c t w i t h O

2 at nearly diffusion-con-

t r olled r a t e (1091010 M1 s1) (22, 23). Since peroxynitrite6 P. Tordo, unpublished data.

F IG . 12. Oxygen consumption during the reaction betweenNaHSO3 and peroxynitrite:effect of spin trap. Peroxynitrite (300M) was added into a chamber of an oxygen electrode containing vary-ing concentrations of NaHSO

3and DTPA (0.1 m M) in phospha te buffer

(0.2 M, p H 7 . 4 ) an d t h e t o t a l O2

consumed was recorded. Inset, O2

uptake traces obtained by the addition of peroxynitrite (300 M) alone

(), NaHS O3(0.2 m M) and DTPA (0.1 m M) alone (), with peroxynitrite(300 M) i n t h e a b s e n c e o f D E P M P O (f) an d i n t h e p r es en ce ofDEPMPO (20 m M) (). Ar r o w (2) indicates the point of a ddi t ion ofperoxynitrite.

F IG . 13. ESR spectra obtained after adding peroxynitrite tosodium bisulfite and formate in the presence of DEP MPO. a ,spectrum obtained upon incubat ing DEP MPO (1 mM), NaHS O

3(1 m M),

peroxynitrite (0.7 m M), and DTPA (0.1 m M) in phosphate buffer (200m M, pH 7.4); b, a s a in the presence of HCO

2NH

4 (1 M) in buffer

saturated wi th argon; c, s am e as bbut in buffer sa tura ted wi th 100%O2; d, the ESR spectrum of an a uthentic DEP MPO/CO

2

obtained in aFenton rea ction (H

2O2

(0.1 m M), HCO2NH

4(1 M ), EDTA (0.1 m M), and

FeSO4(0.05 m M)); a nd e, a s ain the absence of peroxynitrite. The dott ed

l inesi n b a nd c show a computer simulation of the respective spectra(mixture of DEPMPO/S O

3

an d DE P M P O /C O2

add ucts). Spectrometersettings: microwave power, 20 mW(5 mWfor d); modulat ion amplitude,1 G; t ime consta nt, 0.128 s; gain, 8 104 (2.5 104 for d); scan range,200 G; and scan t ime, 120 s . Open a nd closed squares indicate the

low-field lines of DEPMPO/C O2

an d DE P M P O /S O3

, respectively. Con-tributions of the va rious ra dical adducts to t he s imulated spectra aredescribed in the text.



9/11

decomposition was unaffected by DEPMPO,7 the nearly com-plete inhibition O

2upta ke observed at higher concentra tions of

D E P M P O ( 1 0 0 m M) could be explained i f k9

[O2

] k10

[DEPMPO], where k9 109 M1 s1, [O

2] 2.4 104 M, a n d

[DEPM PO] 1 1 01 M. H ence, k10

can be est im at ed t o begr eat er t ha n 106107 M1 s1 (25, 29).

Peroxynitrite oxidized sulfite by an one-electron mechanismt o g e n er a t e t h e r a d i ca l . Th i s a g a i n a p p ea r s t o b e a d ir ectreaction as hydroxyl radical scavengers did not affect the ESRspect r al int ensit y of DEP M PO/S O

3

spect r um and t he signalwa s diminished in the presence of 100%oxygen.

F or m at i on of D E P M P O / C ar bon- cent er ed A dduct s dur i ng

Peroxynitr i te-mediat ed Oxid at ion of GSH an d N APOur re-s ul t s i nd ica t e t h a t b ot h D E P M P O /S R a n d D E P MP O/R(DE P MPO-carbon centered a dduct) were detected during oxi-dation of thiols by peroxynitrite (Figs. 2ba n d 7a). Evidence for

formation of -amino acid carbon-centered radicals from gluta-thionyl and other sulfur-centered radicals had been obtainedusing low-temperature (28) a nd fast-flow ESR spectroscopy(22). This mechanism involves an intramolecular abstraction ofa n -hydrogen atom of the gluta myl residue by the thiyl ra dicalcenter. A diagra mma tic representat ion of th is process is shownin Scheme 2.

It is possible tha t t he thiyl r adical, once formed, undergoes aconfor m at ional change w hich br ings t he t hiy l r ad ical cent ercloser to the -hydrogen atom. The equilibrium between GS

a n d G SH is dependent on the pH a nd is shifted to the right sideat higher pH (22, 23). H owever, a considerable fraction of G S

presumably exists as the -amino a cid radical a t physiologicalpH. A sim ilar r eact ion m echanism can also be pr oposed forformation of th e NAP-derived carbon-centered ra dical from

S-NAP . The -am ino ac id car bon-cent er ed r ad ical has anasymm etric carbon center and th e resulting DEP MPO-carbon-centered adduct will exist as diastereoisomers. Further ESRstructura l chara cterization of these DEP MPO-carbon-centeredadducts must await syntheses of carbon-13 labeled GSNO andS NAP.

Peroxynitrite-dependent Oxidation of FormateNo spin a d -ducts were observed from the reaction between peroxynitriteand formate in the absence of GSH. However, in the presence ofG S H , t h e f or m a t i on of C O

2

. was observed. Augusto et al . (9)r epor t ed sim ilar r esult s using DM P O a nd ind icat ed t hat for -

m at e w a s a c t ing a s a t ypical hyd r oxyl r ad ical scavenger . Theobser vat ion t ha t G S , generated from the photolysis of GSNO,is also able to abstract a hydrogen atom from formate to formC O

2

. indicates another mechanism whereby formate can affectspin adduct formation (30). Our results indicate that CO

2

. for-mat ion from formate cannot be used as a diagnostic indicat or ofhydroxyl radical in the presence of GSH.

M echani sm of Oxygen Act ivat ion by Sulfu r-centered Radi-

calsThe oxygen consumption observed during the oxidat ionof GSH by peroxynitrite can be explained by Equa tions 1115.G S reacts with O

2at a diffusion-controlled rate t o form G SOO

which then a bstra cts a hydrogen from G SH t o produce GS . Thereaction between GS a n d G S w ill r ead ily for m GS S G. whichcan reduce O

2t o O

2

. (31). Equations 11, 12, and 15 constitute achain reaction by which glutathione can be oxidized (Ref. 32and references therein).

k11 2 109

M1 s1

G S O2 L

|

; G S O O (Eq. 11)

k11 6.2 105 s1

G S O O G S H 3 GSOOH G S (Eq. 12)

k13 8 108

M1 s1

G S G SL|; G S S G. (Eq. 13)

k13 2.4 105 s1

G S S G2

.O2O

k14 1.6 108

M1 s1

G S S G O2

. (Eq. 14)

O2

. G S H HO

k15 103 104M1 s1

G S H 2O2

(Eq. 15)

NAP elicited a greater consumption of oxygen than GSH for thesame concentration of thiol and peroxynitrite. I t is likely thatt h e a d d i t ion a l s t er i c h i n dr a n ce a t t h e s u lf u r a t o m o f N APhind er s d im er izat ion of t he cor r espond ing t hiy l r ad ical (i.e.S-NAP). Whereas rapid dimerization of GS w il l r ed uce t heprobability of a reaction between GS and oxygen.

Oxygen consumption during the reaction between sulfite andperoxynitrite can be explained by the following reactions (27).S O

3

. for m ed fr om t he r eact ion bet w een per oxynit r i t e a ndN a H S O

3can react w ith oxygen to generat e a number of higher

oxidat ion species including OOSO3

a n d S O4

. . During this proc-ess, chain propagation reactions can occur as shown in Equa-tions 1820.

7 H. Karoui , N. Hogg, C . J . Frejavi l le , P . Tordo, a nd B. Kalya nara -man, unpublished data .

S C H EM E 2 . I ntramolecular transfor-mation of glutathionyl radical to thecorresponding -amino acid derivedcarbon-centered radical.



10/11

H S O3ONOO 3 S O

3

.NO 2 H O

(Eq. 16)

S O3

.O2

O

k17 1.5 109

M1

s1

O3S OO (Eq. 17)

(Eq. 18)O3S OO H S O3

O

k18 3 106

M1 s1

O3SOOH S O3.

O3S OO S O323S O

4

. S O4

2 (Eq. 19)

S O4

. S O3

23S O42 S O

3

. (Eq. 20)

Superoxide is also formed as a minor product of reaction be-t w een S O

3

. a n d O2

(Equation 21):

S O3

.O2 3S O3 O2

. (Eq. 21)

Biological Impl icat ions of Peroxynitr i te-mediated Oxidat ion

of ThiolsIt ha s been suggested tha t nitric oxide-induced cy-totoxicity is am plified by thiol depletion (33). Nitric oxide-mediated neurotoxicity has been thought to be due to peroxyni-trite formation (34). Although the crit ical biological target oftoxicity for peroxynitrite is not known, it has been suggestedt hat t hiols a nd pr ot ein sulfhyd r yls a r e m ajor t ar get s for per -oxynit r i t e in neur ons (35). I t w a s r ecent ly show n t hat int ac tneurons are more susceptible tha n a strocytes to peroxynitrite-mediated toxicity. This differential sensitivity was attributedto higher concentra tions of GSH an d other an tioxidan ts/an ti-oxidant enzymes in astrocytes (35). Although the one-electronmechanism of oxidation of GSH by peroxynitrite probably oc-curs to a minor extent (36), the gluta thionyl radical can init iat eself-susta ining free ra dical chain reactions (Equa tions 1115)

limiting the effectiveness of GSH as an antioxidant (31, 32).Peroxynitrite, which is init ia lly formed by th e reaction betw eenN O a n d O

2

. , can r egener at e O2

. as a r esult of scavenging byGSH (Scheme 3). Thus, superoxide dismutase is required toprevent the chain oxidation of GSH (32). The formation of thiylr ad icals m ay cont r ibut e t o t he neur ot oxicit y associat ed w it hperoxynitrite a nd nitric oxide (Scheme 3). Thiyl ra dicals ha vebeen shown to init iate lipid peroxidation (37). Studies fromS t a d t m ans labor at or y (38) have show n t hat t hiy l r ad icals inbacterial systems a re detoxified by a thiol-specific antioxidantenzyme. However, it is not clear whether a similar enzymaticmachinery exists in mamm alia n cells to detoxify thiyl ra dicals.

I t h as a lso been reported that the reaction between peroxyni-t r i t e and GS H r esult s in low y ie ld s (1%) of G SN O (36, 39).The mechanism for this is as yet unclear but may involve the

interm ediacy of thiyl ra dicals (40). G SH clea rly protects the cellagainst oxid at ive d am age by a num ber of m echanism s. Ourd at a show t hat t he r eact ion bet w een per oxynit r i t e and GS Hcannot simply be regar ded as a 1:1 an nihilation of the oxidan tas ra dical species are generat ed that may propaga te the oxida-tive insult. This propagation is clearly of low efficiency and, atnormal GSH levels, can be easily controlled by the cell. How-ever, if GSH levels are compromised by peroxynitrite-depend-ent oxidation or by some independent route, the formation ofintracellular thiyl radicals may represent a mechanism for thepropagation of peroxynitrite-dependent oxidation.

P er oxyni t r i t e I nt er act i on w i t h S ul f i t e: B i ol ogi cal I m pl i ca-

tionsRecently it was reported that sulfites could counteractin a concent r at ion-d epend ent m anner t he abil i t y of N O t oinhibit platelet aggregation (41). This effect was attributed to afacile reaction between NO and sulf i t e anion. I n biologicalbuffers, sulfite a nion undergoes ra pid aut oxidat ion to producethe superoxide a nion (42, 43). I t is likely tha t peroxynitrite isformed during the addition of NO or NO-donors to solutionscont aining sulf i t e in t he absence of m et al- chelat or s such asDTPA. Since peroxynitrite has been shown to cause plateletaggregation (36), an alternative explanation for the effect ofsulf i t e and NO on plat e le t aggr egat ion could be d ue t o t he

oxidat ive cha in reaction tha t occurs a fter the rea ction betweenperoxynitrite a nd sulfite. Peroxynitrite-mediated forma tion ofsulfite anion radicals could also be involved in environmentalcarcinogenesis induced by polycyclic a romatic hydrocar bons(44).

ConclusionWe have shown using a novel phosphorylatedspin t r ap t hat per oxynit r i t e oxid izes a var ie t y of t hiols andsulfite forming the corresponding thiyl and sulfite anion radi-cals. These reactions are not mediated by h ydroxyl radical. Incontra st to DMP O, DEP MPO does not undergo direct oxidationby peroxynitrite to form the corresponding hydroxyl adduct.P eroxynitr ite-dependent oxidat ion of forma te in th e presence ofthiols is mediated by thiyl radicals. The oxidation of thiols byper oxynit r i t e is accom panied by oxyr ad ical for m at ion. I t ispossible that self-susta ining free ra dical reactions triggered by

peroxynitrite contribute to cellular t oxicity an d limit t he a nti-oxid ant pot ent ial of G S H.

R E F E R E N C E S

1. Huie, R. E. , and Padmaja, S. (1993) Free Rad. Res. Comms. 18, 1951992. Goldstein, S. , and Czapski , G . (1995) Free Rad. Bi ol . Med. 19, 5055103. Roya ll, J . A., Kooy, W. W., and Beckma n, J . S. (1995)New Hori zons3, 1131224. Da rley-Usmar , V. M., Hogg, N., OLeary, V. J . , Wilson, M. T., and Monca da , S.

(1992) Free Rad. Res. Comms. 17, 9205. Beckman, J . S. (1991) J . Dev. Physi ol . 15, 53596. Ra di, R., Beckman, J . S., Bush , K. M., an d Freema n, B. A. (1991) J. Bi ol . Chem.

266, 424442507. R adi , R . , B e ckm an , J . S . , B u s h , K . M . , an d F r e em a n , B . A . (1991) A r c h .

Biochem. Biophys. 288, 4814878. King, P . A., J am ison, E., Stra hs, D., Anderson, V. E., and B renowitz , M. (1993)

Nucleic Acids Res. 21, 247324789. Augusto, O. , Gatt i , R. M. , and Radi , R. (1994) Ar ch. Bi ochem. Bi ophys. 310,

11812510. H ogg, N. , J oseph, J . , and Kalya nara man, B. (1994) Ar ch. Bi ochem. Bi ophys.

314, 15315811. Pou, S., Ngu yen, S. Y., Gla dw ell, T., and Rosen, G . M. (1995)B i ochi m. Bi ophys.

Acta1244, 626812. Lemercier , J -N. , Sq uadri to, G. L. , a nd Pryor , W. A. (1995) Arch. Bi ochem.

Biophys. 321, 315913. Shi, X., Lenha rt, A., and Ma o, Y. (1994)Biochem. B iophys. Res. Commu n. 203,

1515152114. Koppenol, W. H., Moreno, J . J ., P ryor, W. A., Ischiropoulos, H., a nd B eckman,

J . S. (1992) Chem. R es. Toxicol. 5, 83484215. Beckman, J . S., Beckman, J . W., Chen, J . , Marshall, P. A., and Freeman, B. A.

(1990) Proc. N at l . Acad. Sci . U. S. A. 27, 1620162416. Pr yor, W. A., J in, X., and S qua drito, G . L. (1994)Proc. Nat l . Acad. Sci . U . S. A.

91, 111731117717. Ka roui, H . (1994) Ph .D thesis, U niversite de S aint -J er ome, Ma rseille, Fra nce18. Freja ville, C., Kar oui, H., Le Moigne, F., Cu lcasi, M., Piet ri, S., La uricella, R.,

Tuccio, B., a nd Tordo, P . (1995) J. M ed. Chem. 38, 25826519. Freja ville, C., Kar oui, H., Le Moigne, F., Cu lcasi, M., Piet ri, S., La uricella, R.,

Tuccio, B., a nd Tordo, P . (1994) J. Chem. Soc. Chem. Comm. 179320. Field, L. , Di l ts , R. V. , Ravichandran, R. , Lenhert , P . G. , and Carnahan, G. F.

(1978) J. Chem. Soc. Chem. Commu n. 249250

S C H EM E 3 . Hypothetical scheme showing the sustained pro-duction of peroxynitrite in the neurotoxicity associated withnitric oxide. Both NO and O

2

. can be generated enzymatically duringinflamma tion. The rea ction betw een ONOO and G SH can ampli fy theoxidat ive damage via formation of O

2

. and may l imit the ant ioxidantpotential of GS H.



11/11

21. Riley, D., and Weiss, R. H. (1994) J. Am. Chem. Soc. 116, 38738822. Grierson, L. , Hilderbrand, K. , and Bothe, E. (1992) I n t . J . R a d . B i o l . 62,

26527723. Zhao, R., L ind, J . , Mereny i, G., a nd E ricksen, T. E. (1994)J. Am. Ch em. Soc.

116, 120101201524. F inkelstein, E. , Rosen, G. M. , a nd Ra uckman, E . J . (1980)J. Am. Chem. Soc.

102, 4994499925. Harman, L. S. , Carver , D. K. , Schreiber , J . , and Mason, R. P . (1986) J . B i o l .

Chem. 261, 1642164826. Adams, G. E., and Wilson, R. L. (1969) Tr ans. Faraday Soc.65, 2981298727. Mott ley, C. , a nd Ma son, R. P . (1988) Arch. Biochem. Biophys. 267, 68168928. S evi lla, M. D. , B ecker, D. , a nd Ya n, M. (1990) I n t . J . R a d . B i ol . 57, 6581

29. Har m an , L . S . , M o t t l e y , C . , an d M as o n , R . P . ( 1984) J . B i o l . C h e m . 259,56065611

30. Forni, L. G., and Wilson, R. C. (1986) B i ochem. J . 240, 89790331. Winterbourn, C. C. (1993) Fr ee Rad. Bi ol . M ed. 14, 859032. Winterbourn, C . C. , and Metodiewa, D. (1994) Ar ch. Bi ochem. Bi ophys. 314,

28429033. Walker, M. W., Kinter, M. T., Roberts, R. J . , and Spitz, D. R. (1995) Pedi atr .

Res. 37, 414934. Lipton, S. A., Choi , Y-B. , Pa n, Z-H. , Lei , S . Z. , Chen, H -S. V., S ucher, N. J .,

Loscalzo, J . , Singhel, D. J . , and Stamler, J . S. (1993) N a t u r e 364, 626632

35. B olanos , J . P . , Heales , S . J . R. , a nd Clark, J . B . (1995) J. Neurochem. 64,19651972

36. Moro, M. A., Darley-Usmar, V. M., Goodwin, D. A., Read, N. G., Zamora-Pino,R., Feelisch, M., Radomski, M. W., and Moncad a, S . (1994)Proc. Nat l . Acad.Sci . U. S. A. 91, 67026706

37. Schoneich, C. , Asmus, K-D. , Dil l inger , U . , a nd Bruchhausen, F. V. (1989)Biochem. Biophys. Res. Commun. 161, 113120

38. Yim, M. B. , Cha e, H. Z. , Rhee, S. G . , Chock, P . B . , and S tadt man, E . R. (1994)J. Bi ol . Chem. 269, 16211626

39. Wu, M., Pritcha rd, K. A., J r. , Kam insky, P. M., Fa yngersh, R. P ., Hintze, T. H.,an d Wolin, M. S. (1994)Am. J. Physi ol . 35, 5, H2108-H2113

40. Pryor , W. A. , Church, D. F. , Govindan, C. K. , and Crank, G. (1982) J . O r g .Chem. 47, 156159

41. Har ve y , S . B . , a n d N e ls e st u e n , G. L . ( 1995) B i ochi m. Bi ophys. Acta 1267,4144

42. Fridovich, I . , and H andler , P . (1961) J. Bi ol . Chem. 236, 1836184043. McCord, J . M., and Fridovich, I. (1968) J. Bi ol . Chem. 244, 6056606344. Reed, G . A. , Curtis , J . F. , Mott ley, C. , E l ing, T. E. , and Mason, R. P . (1986)

Proc. Nat l . Acad. Sci . U. S. A.83, 7499750245. Tuccio, B., La uricella, R., F reja ville, C., B outeiller, J . C., an d Tordo, P. (1995)

J. Ch em. Soc. Perki n T rans. 2, 295298


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