ARCHIVES

Vol.

OF BIOCHEMISTRY

AND

191, No. 1, November,

Coupled

BIOPHYSICS

pp. 351-357,1978

Oxidation

of NADPH DAVID

with Thiols

at Neutral

pH

P. BACCANARI

The Wellcome Research Laboratories, Received

Research Triangle May

Park, North Carolina 27709

19.1978

NADPH and NADH are rapidly oxidized in neutral imidazole chloride buffer at 3O’C in the presence of mercaptoethanol or dithiothreitol. The product of the NADPH reaction has been determined to be enzymically active NADP’. Oxidation of the pyridine nucleotides is coupled to the autooxidation of the thiol and is inhibited by ethylenediamine tetraacetic acid, stimulated by metal ions (FeSOJ, and requires oxygen. The rapid oxidation of thiols and NADPH at neutral pH was found to occur only in imidaxole and, to a lesser extent, in hi&dine buffer. Under the conditions employed, 300 pM dithiothreitol and 30 PM NADPH are oxidized in 30 min. Both NADPH and thiol oxidations are inhibited by catalase, whereas superoxide dismutase only inhibits the oxidation of NADPH. NADPH oxidation is also inhibited by the hydroxyl radical scavengers formate, mannitol, or benzoate. A reaction mechanism is proposed in which imidasole promotes the metal-catalyzed oxidation of thiols at neutral pH. The superoxide radical generated either by the thiol oxidation or directly oxidizes NADPH or forms hydrogen peroxide and hydroxyl radicals which can oxidize NADPH. Hydrogen peroxide is also involved in the autooxidation of the thiol.

Superoxide anion radicals (0~~) are produced upon the oxidation of reduced ferredoxins (l), reduced flavins and quinones (2), hemoglobin (3), epinephrine (4), phenylhydrazine (5), and thiols (6), and are capable of reducing ferricytochrome c and nitroblue tetrazolium and oxidizing epinephrine. The enzyme superoxide dismutase, which catalyzes the dismutation of superoxide to molecular oxygen and hydrogen peroxide, has been used as a tool to investigate the role of superoxide in these oxidations. It has also been shown that, in the presence of lactate dehydrogenase, superoxide can oxidize enzyme bound NADH (7). In a study by Misra (6) on the autooxidation of thiols at alkaline pH, it was demonstrated that the reaction was metal-catalyzed and produced superoxide, hydroxyl radicals (OH- ), and hydrogen peroxide. The reaction rate was pH-dependent, and the lack of oxidation below pH 8 (in phosphate buffer) indicated that the ionized form of the sulfhydryl was the required active species. In this report, I show that the metal-catalyzed oxidation of DTT’ can ’ Abbreviations

used: DTT,

dithiothreitol;

DTNB,

occur rapidly at pH values less than 8 in imidazole chloride buffer. Also, at neutral pH, NADPH can be readily oxidized by the superoxide, hydrogen peroxide, and hydroxyl radicals produced. The problems associated with using DTT-imidazole buffers in common laboratory procedures are also discussed. EXPERIMENTAL

PROCEDURES

Materials. NADPH was obtained from both P-L Biochemicals and Sigma. Imidazole was from Aldrich Chemical Co. and Sigma. 1-Methylimidasole and 2Methylimidaxole were from the Aldrich. Chelex-100 was from Bio-Rad and prepared according to the manufacturer’s instructions. All other reagents and enzymes were from Sigma. Assays. NADPH and DTT oxidations were routinely assayed at 30°C in unstoppered I3 x 10%mm Pyrex test tubes containing 1 ml of air-saturated 0.1 M imidasole cloride buffer. The standard concentrations of reactants were 60 pM NADPH and 12 mM mercaptoethanol or 2 mu DTT. The thiol was always added last, immediately before transferring the entire sample to a cuvette for a zero time absorbance reading. 5,5’-ditbiobis(2nitrobenxoic acid); droxyethyll-1-peperaxine-ethanesulfonic N-tris-(hydroxymethyl)methyl-2-aminoethane fonic acid.

Hepes, 4-(2-hyacid; TES, sul-

351 0903-9861/78/1911-0351$02.00/O Copyright

0 1978

by Academic

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in any form reserved.

352

DAVID

P. BACCANARI

The sample was returned to the test tube, and after 30 min, the procedure was repeated for a final absorbance reading. In this manner, up to 25 samples per hr could be monitored. The velocity of the reaction is expressed as change in concentration per 30 min, and each reported value is the average of at least four determinations. The concentrations of NADPH and oxidized DTT were calculated from molar extinction coefficients of 6.2 X lo3 at 340 nm (8) and 273 at 283 nm (9), respectively. The oxidation of DTT was assayed in the absence of NADPH. All glassware was washed with Impact detergent (Economics Laboratory, Inc.) and rinsed repeatedly with deionized water before use. NADPH oxidation was also measured in a superoxide and hydrogen peroxide generating system consisting of hypoxanthine and xanthine oxidase. Reaction mixtures contained 1.1 pmol hypoxanthine, 60 nmol NADPH, 0.1 mm01 imidaxole chloride buffer, pH 7.3, and 0.008 unit of xanthine oxidase in a hnal volume of 1.0 ml. Some assays also contained 8 pg/ml catalase. The oxidation of NADPH was measured at 30°C in a Gilford recording spectrophotometer by monitoring the change m absorbance at 340 nm over a 6-min interval. ’ Oxygen consumption was measured in air-saturated buffer (3 ml) using a Yellow Springs Instrument Co. model 53 biological oxygen monitor. High-pressure liquid chromatography was performed in a Varian LCS 1060 with the phosphate buffer system described by Zimmerman et al. (10). The reaction product was tested as a substrate for both 6-phosphogluconate dehydrogenase using the assay of Pontremoli and Graxi (11) and dihydrofolate reductase using the assay previously described (12). The ultraviolet difference of the reaction product compared to NADPH was determined in split cuvettes (Markson) in a Beckman ACTA III recording spectrophotometer. DTNB titrations were performed using the method of Habeeb (13). RESULTS

AND

DISCUSSION

Oxidation of NADPH. The coupled oxidation of thiols and NADPH was first observed while studying the kinetics of the enzyme dihydrofolate reductase. Reaction mixtures containing 0.1 M imidazole chloride (pH 7), 12 mu mercaptoethanol, and 60 PM NADPH showed a time-dependent decrease in absorbance at 340 mn in the absence of enzyme. No change in absorbance was detected without mercaptoethanol. The changes in spectral properties of the reaction mixture were further characterized by ultraviolet difference spectroscopy (Fig. 1). The difference spectra of the reaction mixture compared to NADPH showed a time-dependent loss of absorb-

II

240

I

280

320 WAVELENGTH

I

1

360

I

400

nm

FIG. 1. Ultraviolet difference spectra of the reaction product uersus NADPH. Spectra were measured in two-compartment cuvettes. The sample cuvette contained 12 rnru mercaptoethanol and 60 FM NADPH in 0.1 M imidazole chloride buffer, pH 7.3, in both compartments The reference cuvette contained 12 rnru mercaptoethanol in 0.1 M imidazole chloride (pH 7.3) in one compartment and 60 pM NADPH in 0.1 M imidaxole chloride (pH 7.3) in the other. The samples were incubated at 3O”C, and difference spectra were run at the times indicated.

ante at 340 nm, an increase in 259 nm absorbance, and an isosbestic point at 285 run. This is similar to the difference spectrum between NADP+ and NADPH. Sufficient material for product identification was obtained by incubating higher concentrations of NADPH and thiol. Samples containing 1 rn~ NADPH and 30 mu mercaptoethanol lost essentially all their 340 nm absorbance after 20 h at 30°C. The product was not a co-substrate with dihydrofolate for the NADPH-requiring enzyme dihydrofolate reductase but did act as a co-substrate with 6-phosphogluconate for the NADP’ utilizing enzyme 6-phosphogluconate dehydrogenase. Further evidence that the reaction product is NADP+ was obtained by high-pressure liquid chromatography. The reaction product (Fig. 2A) had the same retention time as authentic NADP’ (Fig. 2B). The ultraviolet-absorbing peak eluting at 9 min in the reaction product sample is mercaptoethanol. In addition, a mixture of the reaction product and NADP+ co-eluted as a single symmetrical peak (data not shown) which was distinct from NADPH (Fig. 2C). The heterogeneity in the chromatogram of NADPH is probably caused by formation of acid degradation products during chromatography in the pH 3.5 phosphate elution buffer. The rate of NADPH oxidation varied with the pH of imidazole chloride buffer

COUPLED

OXIDATION

OF NADPH

WITH

353

THIOLS

(Fig. 3), with an optimum at pH 7.3 where 15 ~~/30 min was oxidized. Therefore, all the following experiments were performed at pH 7.3. When the rate of NADPH oxidation was measured at different imidazole chloride concentrations at pH 7.3 (Fig. 4), little oxidation occurred until 40 mu imidA REACTION

PRODUCT

I

1

0

IO

20

RETENTION

I

30

40

TIME

( MINUTES)

50

60

FIG. 2. High-pressure liquid chromatography. The reaction product was obtained by incubating 1 mru NADPH and 30 mM mercaptoethanol in 0.1 M imidazole chloride, pH 7.3, for 20 h at 3O”C, and a 5-~1 sample was used for chromatography (A). In (B) and (C), 5~1 samples of 1 mu NADP+ and 1 mM NADPH, respectively, in 0.1 M imidazole chloride, pH 7.3, were chromatographed.

FIG. 3. Effect of pH on oxidation of NADPH. The reaction mixtures consisted of 1.0 ml 0.1 M imidasole chloride buffer of the appropriate pH, 60 PM NADPH, and 12 lll~ mercaptoethanol.

I 20

I 40 60 IMID*ZOLE

a ’ 80 100 120 CHLORIDE Cnlt.4)

%io

FIG. 4. Effect of imidazole chloride concentration on NADPH oxidation. The reaction mixtures contained 1.0 ml of pH 7.3 imidazole chloride buffer of the appropriate concentration, 60 pM NADPH, and 12 mM mercaptoethanol.

azole, then the rate of oxidation increased until the imidazole concentration reached 100 m. The rate did not change further even though the concentration of imidazole was increased lo-fold to 1 M. Therefore, the concentration of imidazole required for rapid oxidation of NADPH is at least severalfold higher than either reactant. Similar results were obtained with reagent-grade imidazole from several manufacturers, with sublimed imidazole, and with imidazole treated with Chelex-100. A variety of other buffers (0.1 M, pH 7.3) were tested in the reaction system. Sodium citrate, ethylene diamine, triethylamine, ethanolamine, HEPES, TES, potassium arsenate, N-ethylmorpholine, and Tris all showed only O-l.8 PM NADPH oxidized/30 min. In potassium phosphate buffer, the reaction was faster, 3-4 PM NADPH oxidized/30 min. However, since NADPH and NADH are known to interact with phosphate at neutral pH (14), the reaction in phosphate buffer was not studied further. The only other common buffer which effectively supported the oxidation of NADPH was histidine chloride, and its reaction rate was half that of imidazole. Therefore, the imidazolium ring structure appears to be essential for the mechanism of NADPH oxidation. Several thiol compounds were compared for their ability to promote the oxidation of NADPH. At 2 111~,cysteine and glutathione were ineffective, whereas mercaptoethanol and DTT resulted in 7 and 30 PM NADPH oxidized/30 min, respectively.

354

DAVID

P. BACCANARI

When the rate of NADPH oxidation was measured at various DTT concentrations, the pattern illustrated in Fig. 5 was observed. The reaction rate increased with DTT concentration until 0.7 mu, then the rate was progressively inhibited by higher thiol concentrations. A similar pattern was observed with mercaptoethanol, except maximal NADPH oxidation (15 p~/30 min) occurred at 12 mu mercaptoethanol. Therefore, of the thiol compounds tested, DTT was effective at low concentrations and showed the fastest rate of NADPH oxidation. The oxidation of a pyridine nucleotide by DTT or mercaptoethanol was not limited to NADPH; NADH (60 FM) was oxidized at half the rate of NADPH by either thiol. A possible mechanism of NADPH oxidation in this system could be an oxidized DTT contaminant in the reduced DTT resulting in the reaction NADPH + oxidized DTT + H’ + NADP+ + reduced DTT. This was tested by incubating NADPH (60 PM) with 2m~ oxidized DTT. No oxidation of NADPH occurred. Also, NADPH oxidation was not observed in an anaerobic consisting of reduced DTT, system NADPH, and degassed imidazole buffer under a nitrogen atmosphere. Therefore, imidazole, a reduced thiol, and oxygen are required for the oxidation of NADPH. Oxidation of DTT. Misra (6) showed that thiols auto-oxidized at alkaline pH and produced superoxide radicals. Since this oxidation was pH dependent, the ionized thiol was implicated as the reactive species.

‘\_ 2

4 DITHIOTHREITOL

6

8 (mM)

FIG. 5. Effect of DTT concentration on NADPH oxidation. The reaction mixture contained 0.1 M imidazole chloride pH 7.3 (1.0 ml), 60 pM NADPH, and the appropriate concentration of DTT.

There was little thiol oxidation at pH 7.8 in phosphate buffer, but the rate increased as the pH increased, and in most cases, measurements were made at pH 10.2 (6). Because of the acid-base properties of imidazole, and the ability of imidazole, metals, and sulfhydryls to form complexes, I felt that thiols may be ionized in imidazole buffer at neutral pH then auto-oxidize via a mechanism similar to that reported by Misra. The resulting superoxide, hydrogen peroxide, or hydroxyl radicals could then oxidize NADPH. Thiol oxidation was studied by several different assays. DTNB titrations showed that 2 mu DTT in 0.1 M imidazole chloride, pH 7.3, was completely oxidized during a 20-h incubation at 30°C in the presence or absence of 1 mu NADPH. If the oxidation of thiols and NADPH are linked, then the lack of NADPH oxidation in other buffers implies thiol oxidation does not readily occur in these buffers. When the oxidation of DTT (2 111~) was monitored by absorbance at 263 mu, 330 ELM DTT was oxidized/30 TABLE FACTORS

AFFECTING

ComponentS

I

THE OXIDATION OF DTT AND NADPH” DTT oxiNADPH dized oxidized

a) complete b) Gnidazole buffer + phosphate or Tris buffer c) +1 PM EDTA d) +20 /.tM EDTA e) +50 pM FeS04 fJ +5 pg/mI superoxide dismutase g) +l pg/ml cat&se h) +20 mM formate or +20 mM benzoate or +20 mM mannitol

PM

%

)hM

%

330 70

100 21

30 2

100 7

260 50 550 320

79 15 167 96

4 1 38 19

14 3 127 63

180 320

55 97

5 28

17 93

’ The complete system consisted of 0.1 M imidazole chloride, pH 7.3,2 mM DTT, f60 pM NADPH. (DTT oxidation was measured in reaction systems which did not contain NADPH). The concentrations of DTT and NADPH oxidized in these systems were normalized to 100%. In b), 0.1 M potassium phosphate or Trischloride, pH 7.3, was substituted for the imidazole buffer. In all cases, DTT and NADPH oxidations were measured by change in absorbance at 283 and 340 nm, respectively, in 30 min.

COUPLED

OXIDATION

min in 0.1 M imidazole chloride, pH 7.3 (Table I, a). Buffers made from the imidazole analogs l-methyl imidazole or 2methyl imidazole also showed rapid oxidation of DTT at 480 and 160 ~J.M/~O min, respectively. However, in 0.1 M potassium phosphate or Tris-chloride buffer, pH 7.3, only 70 PM DTT was oxidized/30 min (Table I, b). Since oxygen is required for thiol oxidation, oxygen consumption should also be buffer dependent. Oxygen uptake was measured in a 3-ml system containing air-saturated 0.1 M imidazole chloride or 0.1 M potassium phosphate buffer, pH 7.3. When mercaptoethanol (final concentration 23 mu) was added, there was an immediate oxygen consumption in each case. Although the rate of oxygen consumption slowly decreased with time, initially 34 PM oxygen/2 min was utilized with imidazole and only 4 PM/~ min was utilized with phosphate. From all these experiments, it is concluded that NADPH is oxidized only under those conditions where thiols are oxidized. Inhibition of oxidation. According to the mechanism proposed by Misra (6) for thiol oxidation at alkaline pH, oxygen and trace metal ions are required, and superoxide, hydrogen peroxide, and hydroxyl radicals are generated. These factors were examined in the neutral imidazole chloride buffer system. The oxidations of both DTT and NADPH were inhibited by EDTA, but as shown in Table I (c and d), NADPH oxidation was more sensitive than DTT oxidation. At 1 PM EDTA, NADPH oxidation was inhibited 86%, whereas DTT oxidation was only inhibited 21%. A concentration of 20 pM EDTA was required to inhibit DTT oxidation 85%. These data indicate that either metal ions may be required at different concentrations for both NADPH and DTT oxidation or that a slight inhibition of DTT oxidation (21%) can lead to a larger inhibition of NADPH oxidation (86%). Although metal ions are known to complex with and oxidize NADPH (15), this alone does not appear to be the case in the imidazole system since oxidation does not occur without a thiol. The stimulation of DTT and NADPH oxidations by 50 PM ferrous sulfate (Table I, e) also indicate there is a

OF

NADPH

WITH

355

THIOLS

role for trace metal contaminants in the reaction mixture. EDTA (50 PM) inhibited these ferrous sulfate-stimulated NADPH and DTT oxidations greater than 90%. The relative importance of superoxide, hydrogen peroxide, and hydroxyl radicals in the oxidation of DTT and NADPH can be assessed by measuring the effects of superoxide dismutase, catalase and formate, respectively, on the reaction rate. These data are also shown in Table I, and in each case, the percentage of inhibition recorded was the maximum obtainable by each particular agent. Superoxide dismutase had little effect on DTT oxidation yet inhibited NADPH oxidation almost 40% (Table I, f) , showing superoxide or a derived radical-oxidized NADPH. Catalase inhibited NADPH and DTT oxidations 83 and 45%, respectively (Table I, g). Although a portion of the effect on NADPH is undoubtedly due to inhibition of DTT oxidation, these data indicate hydrogen peroxide (probably formed by the spontaneous dismutation of superoxide) is capable of oxidizing NADPH. Supporting evidence was obtained by measuring the direct oxidation of NADPH in dilute hydrogen peroxide in the absence of thiols. Oxidation of NADPH by 5 x 10m4 M hydrogen peroxide proceeds at half the rate of the standard mercaptoethanol:NADPH:imidazole chloride reaction. The hydroxyl radical scavengers, formate, benzoate, and mannitol inhibited NADPH oxidation slightly but had little or no effect on DTT oxidation (Table I, h). Mechanism. The following reaction mechanism for the oxidation of DTT and NADPH is consistent with the experimental evidence obtained in this study. HS-R-SH HS-R-S

+ imidazole + imidazole

HS-R-S-

+ Fe’3 4 HS-R-S.

HS-R-S.

+Oz+R

Fe+2 + 02 +

Fe +3 + o*-

Op- + Op- +

2H+ + Hz02

H202+02-‘OH. HS-R-S.