Chem -Biol. Interactions, 76 (1990) 3-- 18 3 Elsevmr Scmntlflc Pubhshers Ireland Ltd

SUPEROXIDE-DRIVEN NAD(P)H OXIDATION INDUCED BY EDTA-MANGANESE COMPLEX AND M E R C A P T O E T H A N O L

FRANCESCO PAOLETTI, ALESSANDRA MOCALI and DONATELLA ALDINUCCI

Ist~tuto d~ Patolog*a Generale, Unwerstta d~ Ftrenze, v~ale G B Morgagn~ 50, 5013~ F~renze rltatyj (Received August 15th, 1989) (Revmlon Received April 16th, 1990) (Accepted April 24th, 1990)

SUMMARY

A purely chemical system for NAD(P)H oxidation to biologically active NAD(P) ÷ has been developed and characterized. Suitable amounts of EDTA, manganous ions and mereaptoethanol, combined at physiological pH, induce nucleotide oxidation through a chain length also revolving molecular oxygen, which eventually undergoes quantitative reduction to hydrogen peroxide. Mn 2÷ is speclhcally required for activity, while both EDTA and mercaptoethanol can be replaced by analogs. Optimal molar ratios of chelator/metal ion (2 : 1) yield an active coordination compound which catalyzes thlol autoxidation to thiyl radical. The latter is further oxidized to disulfide by molecular oxygen whose one-electron reduction generates superoxide radical. Superoxide dlsmutase (SOD) inhibits both thlol oxidation and oxygen consumption as well as oxidation of NAD(P)H If present m the mixture A tentative scheme for the chain length occurring in the system is proposed according to stoichmmetry of reactions involved. Two steps appear of special importance m nucleotide oxidation: (a) the supposed transient formation of NAD(P)" from the reaction between NAD(P)H and thlyl radicals; (b) the oxidation of the reduced complex by superoxide to keep thlol oxidation cycling.

Key words" Superoxlde -- NAD(P)H oxidation -- Manganese-chelate -- EDTA-Mn - Thlol autoxldatlon -- Thiyl radical - Oxygen - Reductants

Abbreviations DTNB, 5,5'-dlthlo-bm-(-2-mtrobenzolc acid), DTT, dlthlothreltol, EDTA, ethylene dlammotetracetlc acid, EGTA, ethylenglycol-O,O'-bls(2-ammoethyl)-N,N,N',N'-tetracetlc acid, GSH, reduced glutathlone, HPLC, high pressure hqmd chromatography, MEMS, mercaptoethanol/EDTA/manganese system, SOD, superoxlde dmmutase, TDB, tnethanolamme- dlethanolamme-HCl buffer, TLC, thin-layer chromatography

0009-2797/90/$03 50 © 1990 Elsevmr Scmntlhc Pubhshers Ireland Ltd Printed and Pubhshed m Ireland

INTRODUCTION

The occurrence of non-enzymic reactions in a variety of NAD(P)H- oxidizing systems, related primarily to phagocytosis, has been reported [1- 4] and discussed in several reviews [5-9]. A common feature of such proposed non-enzymic reactions is the presence of reactive "oxygen species" which could be either the initiators or the propagators of a chain of reactions eventually leading to nucleotlde oxidation. Manganese ions [1,2,10,11] seem to be involved in these processes but their role in enhancing reduced nucleotide oxidation IS still unclear. Moreover, artificial chelators have been shown to affect the susceptibility of NAD(P)H to dehydrogenation and to induce radical formation [12,13]. According to recent views, chelators should exert their action through the formation of active complexes rather than by simply removing metal ions from the solution [14].

Our contribution to this topic derives from an accidental spectrophotometrIc observation on the rate of NAD(P)H oxidation in the presence of EDTA, manganese chloride and thlols, whose combination in aerated buffers yields superoxide radical The latter promotes a SOD- inhibitable nucleotide oxidation and that represents the principle of a new assay for superoxide dlsmutase recently developed in our laboratory [15] In the course of these studies we realized that, besides the SOD assay, the above system could also be used to investigate the mechanisms of NAD(P)H oxIdatmn. A more profound understanding of the reaction sequence might also provide useful information on the role of manganese in non-enzymic reactions linked to NAD(P)H-oxidase, on the generation of superoxlde radicals from thIol autoxidation, and on the interactions of transition metals and their chelates with biological molecules

This paper deals with the combined effects of EDTA, manganese and mercaptoethanol on the rate of NAD(P)H oxidation in aqueous media at physiological pH The report describes a sequence of purely chemical reactions for quantitative NAD(P)H oxidation, it demonstrates that the conversion of NAD(P)H to a biologically active NAD(P) is mediated by superoxide anions and It also proposes a posslble scheme for that conversion.

MATERIALS AND METHODS

All measurements were carried out at room tempera ture unless otherwise specified.

Spectrophotometrlc assays were performed with a Gllford apparatus, while spectra were obtained with a Kontron double beam spectrophotometer (Uvlkon 860) Oxygen consumption was estimated with the aid of a polarography (Gilson Medical Electronics) using a Clark-type oxygen electrode. The measurement of sulfhydryl groups was basically carried out according to Ellman [16]. Hydrogen peroxide was determined as the molecular oxygen generated after the addition of catalase in the polarographlc cell.

The separatmn of (EDTA)2-Mn complex was performed on a Sephadex G-10 (Pharmacia, Uppsala, Sweden) column (1 × 55 cm) equilibrated with 50 mM triethanolamine/diethanolamine-HCI buffer (TDB, pH 7.6). The complex was assayed by incubating 0.2 ml of each chromatographic fraction (2.5 ml) with 0.65 ml of an assay mixture which contains 0.3 mM NADH, 0.15 mM mercaptoethanol and 0.1 M TDB (pH 7.6) to reconstitute the complete nucleotide oxidation system. Activation of (EDTA)2-Mn was revealed by decrease in absorbance at 340 nm (AA340).

Reagents Oxidized and reduced E-forms of NAD(P) were purchased from Sigma

Chemmal Company (St. Louis, MO, U.S.A.}, which provided also cytocrome c, catalase (from bovine hver) and L-cysteme. Chloride salts of Zn 2÷, Mg 2÷, Fe 2÷ and Fe 8÷, Co 2÷, N12÷, Cu 2÷, Cd 2., and 2-mercaptoethanol were bought from Merck-Schuchard (Darmstadt, F R.G.), while ethylenediaminotetra-acetlc (EDTA) as both the acid and the dlsodmm salt, ethyleneglycol-O,O,'-bis (2- aminoethyl~N,N,N',N'-tetra-acetm acid (EGTA); 1,2- and 1,3-diammopropane- N,N,N',N'-tetra-acetm acids were from Fluka AG (Switzerland). Manganese chlorlde/4H20, lactate dehydrogenase (beef heart, cat. no. 106 984), D-lactate and pyruvate (monohthmm salts), were supplied by Boehrmger Mannheim GmbH (F.R.G.), together with 5,5'-dithm-bls-(-2-nItrobenzoic acid) (DTNB), dlthiothreitol (DTT), oxidized (GSSG) and reduced glutathione (GSH), coenzyme A and ascorblc acid Thmredoxln was a gift from Dr. A Holmgren (Karolinska Institute, Department of Medical Chemistry, Stockholm, Sweden) Superoxide dlsmutase (beef liver, spec. act 3300 units/rag) was prowded by Diagnostic Data Inc (Mountain View, CA, U.S.A.).

All other chemical were reagent grade.

RESULTS

Ev~ence /or NAD(PJH ox~at~on NAD(P)H is qmte stable in aqueous solutions at physiological pH and its

presence and/or amount can easily be detected spectrophotometrmally at 340 nm. However, the addition of EDTA/Mn 2÷ (2 • 1, on a molar ratio) and mercaptoethanol to nucleotide solutions causes a prompt NAD(P)H oxidation which is monitored by the decrease in absorbance (Fig 1) Coenzyme oxidation proceeds according to a sigmoidal kinetic reaction and reaches completion on a time scale of minutes The sequence of additions is not very relevant to the overall rate of reaction, on the contrary, the omission of any of the reactants from the mixture prevents nucleotlde oxidation The reaction has been carried out at physiologmal pH with trlethanolamine plus diethanolmame-HC1 buffer (TDB) (from 10 to 100 raM) but comparable concentrations of Tr i s -HC1, Na 1, Na2-phosphate and acetic acid/Na-acetic buffers are also suitable.

Quantitative and quahtat~ve analys~s of pymd~ne nucleot~des The mercaptoethanol/EDTA/Mn 2÷ system (MEMS) has been challenged

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Fig 1 Oxidation of NADH The incubation mixture (1 ml) contains 0 13 mM NADH, 6 mM EDTA and 3 mM MnCl 2 in 0 1 M TDB (pH 7 4) Nucleotide oxidation is started by the additmn of mercaptoethanol (3 mM final concentration) and followed at 340 nm Under these conditions the maximal rate of nucleotide oxidation, calculated m the linear portion of kinetics, approaches 20 nmol NADH transformed per min at room temperature AAs4 o value recorded within 30 mm after the addition of mercaptoethanol IS about 0 8 No oxidatmn occurs w~thout mercaptoethanol

with mcreasmg amounts of N A D H and N A D P H (Ftg 2). A hnear correlation be tween AA340 nm and nucleot lde concentrat ions up to 100/~M is obtained and both the coenzymes m the sys tem are quantttat lvely oxtdized (see sertal absorbance spectra m the inset) wtth the same specff |city NAD(P)H oxidatmn has also been confirmed by the HPLC analysts of the reachon products among whmh we detected a single peak of nucleottde e lutmg at the same posttlon of authent ic NAD(P) ÷ in its monomerlc state (results not shown) In additton, the product of N A D H oxtdation is biologically active and can quanti tat ively be reduced back in the presence of lactate dehydrogenase and lactate (Fig. 3)

Charactemst:cs of the reactwn and opt:mal cond~t:ons for NADfP)H ox:datwn

The reaction has a specific requirement for Mn 2÷ Under the same conditions none of the fol lowing cations tested, namely Ca 2÷, Cu 2÷, Fe 2+, Fe 3+, Mg 2÷, Co 2÷, Ni 2÷, Cd 2÷ and Zn 2÷, is capable of promoting nucleotlde oxidation. However , to be effective, Mn ~÷ must be completely complexed with EDTA as revealed by the rate of NAD(P)H oxidation at increasing concentrat ions of the metal 1on, m the presence of fixed amounts of EDTA and mercaptoethanol (Fig. 4A) Maximal rates of NAD(P)H/NAD(P) ÷ conversion are obtained when EDTA/Mn 2÷ molar ratios are about 2 : 1, while at eqmmolecular concentrations, the reaction ts barely detectable A shght

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NAD(P)H (mM) Fig 2 Quantitative NADP(H) oxidation. Increasing amounts of ei ther NADH (o) or NADPH (O), in a range of 0--100/~M have been oxidized as descmbed m the legend to Fig 1 Reaction m allowed to reach completion and AA3~ 0 values determined thereaf ter Each assay m carrmd out in tmphcate Inset. absorptmn spectra of the reaction mlxture containing NADH are sequentmlly recorded within 0 (curve 1) and 20 mln (curve 5) after mercaptoethanol addition

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Fig. 3 Chemlcally induced NADH oxldatlon can be reversed by the action of lactate dehydrogenase and lactate A mixture, as descmbed in Fig 1, is Incubated with 0 13 mM NADH and reaction is allowed to reach completion at room temperature When absorbance at 340 nm is steady the solution In the cuvette m diluted 1 : 1. with a buffer at pH 9 5. contamlng 0 4 M hydrazzne-sulfate, 1 M glycme. 1 M NaOH and 0 1 M lactate Increase m absorbance takes place on addition of 2 ~l of crystalhne lactate dehydrogenase (LDH, from rabbit muscle, Boehrlnger MannheIm. 1 mg/ml of ammonium sulfate suspenslon) The amount of enzymatzcally active nucleotlde converted to NADH corresponds exactly to that expected from the dllutlon

excess of Mn 2÷ over EDTA kills the reactmn, but mhlbltlon can readily be

reversed by restoring optlmal EDTA/Mn 2÷ ratms When the amount of (EDTA)2-Mn complex m the mlxture is kept constant (Fig 4B), an increase In mercaptoethanol up to 1 mM progressively stlmulates the reactmn, thereafter it begins to level off. No such effect IS observed when the amount of (EDTA)2-Mn IS varied m the presence of an excess of mercaptoethanol (Fig 4C). The rate of NAD(P)H oxldatlon seems dlrectly dependent on the

amount of the complex, wlthout reaching saturatmn.

Effects of temperature and pH The increase m temperature within 15°C and 50°C speeds up the reactmn

according to an exponential curve The pH at whmh the reactmn is performed greatly affects the rate of NAD(P)H oxldatmn (Fig. 5) Maximal activity is observed at near physmlogical pH (7.4-7 6) On the mght and on the left of that value, coenzyme oxidation decreases and it ~s completely inh~bited around pH 9.5.

Ox~datwn of mercaptoethanol A decrease m SH-groups always occurs in the system during incubation

thus suggesting that mercaptoethanol undergoes oxidation. However, there are differences in the rate and extent of SH-group disappearance depending on the presence of nucleotldes and of oxygen m the mcubatmn m~xture Experiments carried out with and without nucleotides (F~g. 6) have shown that the addltmn of NADH to the system causes an mltml increase followed by a slow and progressive dimmutmn m the concentratmn of free thmls The omission of NADH, on the contrary, allows a prompt and extensive

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sotuhon (mL)

Fig 4 Effects of Mn 2÷, mercaptoethanol and (EDTA)~-Mn complex on maximal rates of NADH oxidation Reactions are carmed out m the presence of 0 1 mM NADH and 0 1 M TDB (pH 7 6) Incubation mixtures contain also (A) 20 mM EDTA plus 2 mM mercaptoethanol, reaction s tar ted by increasing concentrations of MnC12 ( 0 - 4 0 raM), (B) 20 mM EDTA plus 10 mM MnC12, reaction s tar ted by increasing concentrations of mercaptoethanol (0--4 raM), (C) 2 mM mercaptoethanol, reaction s tar ted by increasing ahquots ( 0 - 0 2 ml) of (EDTA)2-Mn solution The complex solution contains 66 mM EDTA and 33 mM MnCI 2

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Fig 5 Effect of pH on NADH oxidation Maximal rates of nucleotlde oxidation at ddferent pH (6 -9 ) are reeorded m a complete system (1 ml) containing 0 1 M TDB, 0 25 mM NADH, 1 mM mercaptoethanol, 6 mM EDTA and 3 mM MnCI 2 Values are reported as nanomoles of NADH oxl&zed per m m at room temperature and are the mean of two separate determinat ions

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Fzg 6 Effect of NADH on mercaptoethanol oxidation Changes m free thlol levels m the system (MEMS) are determlhed [16] during the incubation with and without 0 5 mM NADH The incubation mixture (1 05 ml, 5hal volume) contains also 0 1 mM TDB (pH 7 4), 6 mM EDTA, 3 mM MnCl~-Mn and 0 5 mM mercaptoethanol At the t imes estabhshed samples (50 ~1) are taken and dehvered to tes t tubes containing an equal volume of 0 1 M MnC12 to stop the reaction before adding the DTNB reagent Values are the mean of two separate determinations

10

mercaptoethanol oxidation which accounts for a decrease of approximately 30% m total sulfhydryl groups within 15 ram. From these data ]t might reasonably be assumed that reduced nucleotides m MEMS exert an m~tml protectmn against mercaptoethanol oxidation To avoid these partml interferences further expemments on mercaptoethanol oxldatmn have been performed om]tting nucleotldes from the system (F~g. 7). The kmetms of SH- disappearance m reaction mixtures containing either de-aerated, control or thoroughly aerated solutmns clearly mdmate that molecular oxygen ~s revolved m the process, as also reported for the spontaneous autox~datmn of other thmls [17--19]. The extent of mercaptoethanol oxldatmn under a continuous air stream approaches almost 90% of SH-groups w~thm a 30-mm mcubatmn.

Oxygen consumptwn and hydrogen peroxide productwn Polarographm measurements have shown that concomstant to SH-group

disappearance from the incubatxon mxxture, oxygen levels decrease at rates whmh are d]rectly dependent on mercaptoethanol concentration m the system. To determine the stoichmmetry of the reaction we have carrmd out expemments by whmh thml levels, oxygen consumptmn and hydrogen peroxide productmn m the system were estimated at given times of mcubatmn. Results of Table I mdmate that about two moles of thmls are

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Fig 7 Air-dependence of mercaptoethanol ox]datnon reduced by the (EDTA)2-Mn complex A m]xture (2 ml, final volume) containing 0 1 M TDB (pH 7 4), 1 mM mercaptoethanol, 6 mM EDTA and 3 mM MnC12 is incubated either under vacuum (deaerated) or kept under a mild anr stream (thoroughly aerated) An untreated mixture is used as control The mixtures are pretreated for 15 mm before adding the (EDTA)2-Mn complex to start the reactxon Incubation is carrmd out at room temperature for 30 mm At given intervals samples (50 pl) are taken and mixed with an equal volume of 0 1 M MnC12 before assaying for SH-groups by the DTNB method [16] Each assay is earrmd out m duphcate Free thml levels m an aerated mixture wxthout the (EDTA)~-Mn complex do not change within the time of mcubatmn

11

TABLE I

CHANGES IN OXYGEN, THIOL AND HYDROGEN PEROXIDE LEVELS IN THE SYSTEM (MEMS), WITH AND WITHOUT NADH

Values, expressed m i~nol/ml, are the mean of two separate experiments _+ S E M Experiments are carried out in a polarographm cell at 25°C, with a mixture (1 6 ml hnal volume) containing 5 mM mereaptoethanol, 6 mM EDTA, 3 mM MnC½ and when present 5 mM NADH, m 50 mM TDB (pH 7 6) The reaction is s tar ted by the ad&tlon of (EDTA)2-Mn complex

AOxygen ARSH • AH202 b (consumed) (consumed) (produced)

MEMS - 0 2 5 ± 002 - 0 5 5 ± 004 + 0 2 4 ± 002 MEMS + NADH - 0 2 5 ± 002 - 0 5 5 ± 003 + 0 2 5 ± 001

• Free thlols m the mixture are assayed by the DTNB method [16] using 10 and 20 ~1 of sample b Hydrogen peroxide levels are determined at the end of the incubation by adding catalase (2 ~1

of enzyme suspensmn) to the polarographm cell and measuring the amount of oxygen released

oxidized for each molecular oxygen consumed. In turn, oxygen is almost quantitatively reduced to hydrogen peroxide whmh is eventually determined in the presence of catalase. The addition of NADH to the reaction mixture slows down the rates of both oxygen and SH-groups decrease in a NADH concentration-dependent fashion (as also suggested by results of Fig. 6). However, hnal values of thiol and oxygen consumption and of hydrogen peroxide production do not significantly vary with and without nucleotides.

Evutences for superoxzde formation. Effects of SOD, catalase and hydrogen peroxide on the rate of NAD(P)H oxutatzon

The first product of 02 reduction has been Identified as the superoxide anion by experiments with superoxide dismutase (SOD} (Fig. 8). Tiny amounts of this enzyme added to the system cause a prompt inhibition of the ongoing nucleotide oxidatlon and prevent the reaction from starting when pre-incubated with the mixture. Heat-inactivated SOD has no inhibitory effect on NAD(P)H oxidation and neither has active catalase. Moreover, H202 acts as an inhibitor of nucleotlde oxidation and the inhibition can be released by eatalase. Further proof of the formation of superomde in MEMS has been provided by the characteristic reduction of ox-eytochrome c followed at 550 nm, whmh occurs either m the absence or in the presence of nucletoldes (results not shown).

The posslbihty that free radicals other than superomde, mainly OH', might play some role m our system has been ruled out by experiments with manmtol, formate and T m s - H C l (0.1 M each) and ethanol (1.5 M). None of these known OH" scavengers is able to prevent nucleotlde oxidation.

Catalytw role of (EDTA)~-Mn complex The fate of (EDTA)2-Mn in MEMS has been investigated by comparing the

activity of the complex before and after reaction with mercaptoethanol. This

12

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12 - M E M system

8

4

0 J J 0 10

Premcubated wi th SOD

. ' ~ " SOD

"?_ A T A L A S E

I I I I I 20 30

I N C U B A T I O N (mm)

Fig 8 Effects of SOD, H202, and catalase on the ra te of NADH oxldatmn by MEMS Nucleot~de omdatmn is carrmd out under the cond~tmns descmbed m Fig 1 SOD (0 5 ~g) m added d~rectly to the cuvette e~ther during the pre-mcubatmn or the mcubatmn time Catalase (2 ~l of ammomum sulfate suspensmn, 250 mg/ml) and HzO 2 (1% final concentratmn) are added along with nucleotlde omdatmn

expemment was aimed at ascertaining whether the complex was consumed or reactivated along w~th thml oxidatmn. A reactmn m~xture containing 40 mM EDTA, 20 mM MnCl 2 m 50 mM TDB (pH 7.6) was incubated m the absence {mixture A) and m the presence of 20 mM mercaptoethanol (mixture B) for about 8 h under stirring. At the end of incubation free thlol groups m mixture B decreased by 80%. An aliquot (1 ml) of each mixture has been further separated on a Sephadex G-10 column and the collected fractmns (2 5 ml) were assayed for the presence of active (EDTA)~-Mn (see Materials and Methods). The (EDTA)2-Mn complex separated from mixtures A and B was found to elute at the same position and w~th ~dentmal activity despite the large oxldatmn of mercaptoethanol occurring m mixture B. Based on these data it can be assumed that (EDTA)2-Mn is not consumed along with mercaptoethanol oxldatmn and it might reasonably be regarded as the catalyst of the reactmn sequence.

Effects of other chelators and reductants Whereas Mn 2÷ is speclhcally reqmred for MEMS, the other components

can be succesfully replaced by analogs {Table II). Chelators like 1,2- and 1,3- diammo-propanol are good substitutes for EDTA, while EGTA is completely ineffective. As far as reductants are concerned, SH-groups appear essential for the reaction. Moreover, there are marked differences m the reactivity among thlols, conceivably reflecting the relative susceptlblhty of each

13

compound to oxidation. L-Cystelne yields the highest rate of nucleotlde oxidation by far, but the reaction does not reach completion. Reduced glutathione {GSH) and coenzyme A, on the other hand, react very slowly but allow a quantitative nucleotide conversion as does mercaptoethanol. Conversely, no detectable oxidatlon is observed with dithiothtreltol (DTT), ascorbic acid and thioredoxm-SH 2. The fate of thlols m MEMS, following univalent 0 3 reduction, was investigated using L-cystelne as reductant. Preliminary thin-layer chromatography (TLC) analyses indicate that L- cysteine rather than L-cysteic acld is the ultlmate product of L-cysteme oxidation.

DISCUSSION

Our results indicate that the oxidation of reduced nucleotldes can be achieved by purely chemical reactions involving (EDTA)2-Mn complex, mercaptoethanol and molecular oxygen. This system differs from others reported in the literature, such as vanadate [20], molybdate [21] and horseradish peroxidase [10] which indeed deal with non-enzymic reactions and nucleotide oxidation but also involve either enzymes or membrane preparations as sources of free radicals.

TABLE II

DIFFERENT ABILITIES OF SEVERAL CHELATORS AND REDUCTANTS TO PROMOTE NADH OXIDATION IN THE PRESENCE OF MnCl 2

The reactlon m carrled out at room temperature, and NADH oxldatlon is monitored by decrease m absorbance at 340 nm The extent and maximal rate of nucleotlde oxldatlon are evaluated m the presence of 50 ~M NADH, 5 mM MnCl 2 and 0 1 M TDB (pH 7 4) Final concentrations of che- lators and reductants m the mixture are 15 mM and 1 mM, respectlvely Relative rates of

NADH oxldatlon are expressed as percentages of the rate obtained wlth the mercaptoethanol/ EDTA/Mn system (MEMS)

Oxldatlon Relatlve rate (%)

Chelators EDTA Complete 100 EGTA Undetectable 0 1,2-Diammo-propanol Complete 90 1,3-Dlammo-propanol Complete 130

Reductants 2-Meraptoethanol Complete 100 L-Cystelne Incomplete 219 GSH Complete 33 Dltlothreltol Undetectable 0 Ascorblc acid Undetectable 0 Coenzyme A Not determined 20 Tbloredoxln-SH 2 Undetectable 0

14

We have shown that changes m the rate of nucleohde ox~datlon closely depend on relahve reactant concentratmns by which the system can be easily controlled and modulated. Such a flexibility might recall that of bmlogmal reactmns partmularly because NAD(P)H oxldatmn occurs at physmlogical pH and yields an enzymmally active nucleohde For a better understanding of the mechanisms by whmh nucleotldes are eventually oxidized we decided to dmcuss separately the sequence of reactmns occurring without and with NAD(P)H.

The role of the EDTA-Mn complex ~s essentmlly that of catalysmg mercaptoethanol autoxldatmn Moreover, the fact that chelator/metal ophmal ratms for achvlty are about 2 : 1 would suggest that the real catalyst might be (EDTA)2-Mn, L e a complex where two EDTA molecules coordinate with a single manganese mn The metal mn Involved might be Mn(IV) whmh was reported to form a complex with desferal [22] or Mn(III) as proposed by Thomas et al [23] So far, however, owing to the lack of mformatmn, the oxldatmn state of Mn m reactmn (1) (see below) has been mdmated with n Ewdence has been provided that oxygen undergoes umvalent reductmn and ~s converted to superoxlde anion. Accordingly, the hrst set of reactmns might be as follows:

(EDTA)~-Mn (') + RS---* (EDTA)2-Mn(~-I) + RS" RS" + RS- ~ RSSR-" RSSR-" + 02 ~ RSSR + 02-" (EDTA)~-Mn (n-i~ + 0 z + 2H ÷ ~ (EDTA)2-Mn(.) + H202

(1) (2) (3) (4)

sum: 2RS- + 02-" + 2H ÷~ RSSR + H202

Reaction (1) represents the imtlal event m thlyl radmal formation as proposed for the so-called spontaneous autoxldatmn of thmls catalyzed by metal ions and metal ion complexes [17--19]. The propagation (reachons 2 and 3) leads to thml ox~datmn and contemporary superoxlde radical formahon. The latter might be reduced to hydrogen peroxide by reacting with (EDTA)2-Mn(n-'I to ymld back the oxidized complex and close the catalytm cycle. The rate of the reachon, as measured by the oxidation of thiols, is dependent on oxygen availablhty and also strikingly inhibited by SOD This suggests that superoxlde is heavily involved in the propagation and is not just an end-product of the all sequence

The stolchlometry of reachons (1)--(4), mainly based on results of Table I, mdmates that two thlol groups are oxidized by a single oxygen molecule, generating one molecule of disulfide and of hydrogen peroxide However, s~de-reactmns are hkely to occur m the system besides those already outhned For instance, the termination between two RS" and m the presesnce of adventltmus iron, a partial contmbutlon of the Haber-Welss reaction might also take place.

The addltmn of excess of Mn e÷ to the ongoing reaction causes a prompt mhlbltmn of thml oxidatmn and both oxygen consumptmn and superoxlde

15

production (data not shown). In analogy with the inhibitory effect of SOD, Mn 2÷ is likely to act here as superoxlde scavenger [24] and inhibitor of complex re-omdation. Moreover, an increase in Mn 2÷ concentratmn over EDTA, would decrease the amount of "active" complex and shut down the source of thlyl radical formation (reactmn 1). This, m turn, would prevent reactions (2) and (3) from occurring and stop the ent,re sequence of recorded events.

As regards NAD(P)H oxidation mechamsms it must be mentmned that reduced nucleotides are not sigmhcantly oxidized by superoxlde We also ruled out this possibility by submitting the nucleotlde solution, m the presence of either (EDTA)2-Mn or mercaptoethanol, to superoxlde generated by means of the xantme-xantme oxidase sytem. One poss,ble mechamsm to explain the oxidation of nucleotides by MEMS ~s the mteractmn between NAD(P)H and the supposed EDTA-Mn(III) complex; the latter mlmmkmg the action of lactate dehydrogenase in the superox,de-dependent NADH oxldatmn system proposed by Bielsky and Chan [25,26]. Accord,ngly, the resulting NAD(P)" would be further converted to NAD(P) ÷ by reacting with molecular oxygen. This lmphes that NAD(P)H added to the mixture should increase oxygen withdrawal from the polarographm cell as compared to controls without nucleotldes. Our results are against th~s hypothesis and showed that both oxygen consumpt,on and SH-group ox~datmn occur at lower rates in the presence than m the absence of reduced nucleot,des Therefore, in analogy to the reaction reported by Wfllson [26] between GS" and NADH, we propose that the first step m nucleotlde oxidatmn in MEMS is the reaction between NAD(P)H and the th~yl radmal as follows:

RS" + NAD(P)H-* RSH + NAD(P)" (5)

The partial protection exerted by NADH on thlol oxidat,on shown m Fig. 6 would support this belief. NAD(P)" might react further with either thiyl radmals and molecular oxygen or terminate with another nucleot~de radmal, being eventually converted into the oxidized form.

These results, with and without nucleotldes, seems to point to the re- omdation of the complex (reaction 4) as the crucial mhlbitable step of the entire sequence. The removal of superoxlde prevents (EDTA)2-MnIn-1~ from oxidatmg and consequently all the following steps will result lmpa~red. Any superoxlde scavenger actually present in the system, namely, SOD and Mn e÷ will compete for superoxlde anmns thus modulating the rate of the chain length and that is probably the principle on which our assay for SOD activity determination [15] is based.

In the presence of adventitious ~ron, hydrogen peroxide may also mteract with superoxlde through a Fenton-type reaction or with thlyl and dlsulhde amon radicals. According to the scheme proposed (Fig 9) the removal of either superox~de or thlyl and dlsulhde anmn radicals from the solutmn would decrease the rate of NAD(P)" formatmn and eventually mh~b~t nucleotlde oxidation. We have also considered the possibility that SOD

16

RSSR- 1

NAD + H+~:~ 2#, NADH 02~ ~ 02-

NADH NAB NA D + \__ , %

RS- RS - - RS-

RSSR- (EDTA)2-Mn .RSSR (EDT^~_~,~., " I, ,I 'qC_' ~ - o ~ - ~ ~ o ~ o ~

I H20 ~ 2H +

Fig 9 Proposed mechamsms for nucleotlde oxldatmn by the mercaptoethanol/EDTA/manganese system (MEMS)

inhibition might occur through the production of hydrogen peroxide rather than by superoxlde removal. However, there is evidences against this hypothesis: (a) H202 is always formed m the system by spontaneous superoxlde dismutation and that does not affect nucleot~de oxidation, (b) experiments carrmd out m the presence of SOD and of SOD plus catalase have shown that catalase does not abohsh the mh~b~hon of SOD, on nucleotide oxidation (unpubhshed results).

Differences in reductant react lwty closely reflect mdlwdual susceptlblhty to autoxldation as widely reported in studms on thlol chemistry Moreover, it is worth mentmmng that among the compounds hsted there are monothlols, d~thiols and ascorblc acid. Only monothlols react m the system, conceivably because they generate a relatively stable thlyl radical, whilst thlyl radmals from dithlols might be more prone to termmatmn by the formatmn of an mtramolecular disulfide bridge before they collide with NAD(P)H.

Our results concern the combined effects of EDTA, manganese ions and mercaptoethanol on the rate of NAD(P)H oxidation m aqueous media at physmloglcal pH. The scheme proposed m Fig. 9 is mostly based on experimental observatmns and is the sum of all cons~deratmns mentmned m the Dlscussmn. We are aware that further studies will be necessary to prove some of the assumptmns, such as the oxidatmn state of manganese m the (EDTA)2-Mn active complex and the ulhmate electron acceptor for NAD(P)" Nevertheless, this work represents a step m the descrlptmn of a purely chemical reactmn sequence for nucleotlde oxldatmn. We are confident that our system might add new mformatmn on the bmchemlstry of manganese and be of import m further studms on bmlogmal dehydrogenatmn mechanisms

ACKNOWLEDGEMENTS

This work was supported by research grants from the Mmlstero della Pubbllca Istrumone (60 and 40%) and by A.I R.C (Assomamone Itahana per la Rmerca contro 11 Cancro)

17

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