Chem -Biol.Interactions, 76 (1990)3-- 18 Elsevmr ScmntlflcPubhshers IrelandLtd

3

S U P E R O X I D E - D R I V E N NAD(P)H O X I D A T I O N I N D U C E D BY EDTA-MANGANESE COMPLEX AND MERCAPTOETHANOL

F R A N C E S C O PAOLETTI, A L E S S A N D R A

M O C A L I and D O N A T E L L A

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, tnethanolammedlethanolamme-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)Hoxidizing systems, related primarily to phagocytosis, has been reported [14] and discussed in several reviews [5-9]. A c o m m o n 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 N A D ( 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 N A D ( P ) H oxidation in the presence of E D T A , manganese chloride and thlols, whose combination in aerated buffers yields superoxide radical The latter promotes a SODinhibitable 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 S O D assay, the above system could also be used to investigate the mechanisms of N A D ( 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 E D T A , manganese and mercaptoethanol on the rate of N A D ( P ) H oxidation in aqueous media at physiological p H The report describes a sequence of purely chemical reactions for quantitative N A D ( P ) H oxidation, it demonstrates that the conversion of N A D ( 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 t e m p e r a t u r e 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÷, Co2÷, 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 (2aminoethyl~N,N,N',N'-tetra-acetm acid (EGTA); 1,2- and 1,3-diammopropaneN,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 T r i s - H C 1 , 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. 3 Chemlcally induced N A D H oxldatlon can be reversed by the action of lactate dehydrogenase and lactate A mixture, as descmbed in Fig 1, is Incubated with 0 13 m M N A D H and reaction is allowed to reach completion at room temperature W h e n absorbance at 340 n m is steady the solution In the cuvette m diluted 1 : 1. with a buffer at p H 9 5. contamlng 0 4 M hydrazzne-sulfate, 1 M glycme. 1 M N a O H 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 N A D H corresponds exactly to that expected from the dllutlon

excess of M n 2÷ over E D T A kills the reactmn, but mhlbltlon can readily be reversed by restoring optlmal E D T A / M n 2÷ ratms W h e n the amount of (EDTA)2-Mn complex m the mlxture is kept constant (Fig 4B), an increase In mercaptoethanol up to 1 m M progressively stlmulates the reactmn, thereafter it begins to level off. N o such effect IS observed w h e n the amount of (EDTA)2-Mn IS varied m the presence of an excess of mercaptoethanol (Fig 4C). The rate of N A D ( 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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