PREPARATM BIOCHEMISTRY, 2s(1&2), s7 -67 (1995)

PURIFICATION AND CIIARACTERIZATION OF AN

NAD(P)H:QUINONE OXIDOREDUCTASE FROM GLYCINE MAX

SEEDLINGS

A. Rescigno, F. Sollai, S. Masala, M. C. Porcu, E. Sanjust, A. C. RinaHif,

N. Curreli, D. Grifi and Augusto Rinaldi

Istituto di Chimica Biologica, Università di Cagliari, Via della Pineta7l - 09125

Cagliari, Italy-f

Dipartimento di Biochimica e Medicina sperimentale, università di Tor

Vergata, Roma

ABSTRACT

An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified

fromGlycine max seedlings by means of chromatographic procedures. After 1371-

fold purification, the enzyme showed a single band in IEF corresponding to an

isoeléctric point of 6.1. A single band was also found in native-PAGE both by

activity staining and Coomassie brilliant blue staining. The molecular mass

determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was

61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or

NADPH as the electron donor. Among the artificial quinones which are reduced

by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular

interest because of their neurotoxic effects.

INTRODUCTION

NAD(P)H:quinone oxidoreductase (EC 1.6-99.2) is a flavoprotein which

shows diaphorase activity catalyzing the two-electron reduction of quinonic

compounds to hydroquinones.

Copyright @ 1995 by Marcel Dekker, Inc

58 RESCIGNO ET AL.

The proposed scheme of the reaction is:

Q + NAD(P)H + H+ -r QHZ + NAD(P)+where Q is a quinone. This feature of the enzyme appears to be unique amongNADP(H)-oxidizing flavoproteins, which generally are one-electron transferringenzymesl, and therefore give rise to semiquinones that are readily autoxidizedwith the formation of the superoxide radical 02-. NAD(p)H:quinoneoxidoreductase (QR), reacting with both NADH and NADpH is widely spreadthroughout the animal kingdom, plants, bacteria and fungi, and has been isolatedfrom a number of sources including human tissues2 and plants3,+. In animal tissuesthe role of the enzyme is thought to constitute a cellular device controllingsemiquinone and superoxide radical formations. In fact, quinone reductase mightact in the cellular detoxification of quinones by shunting these substances awayfrom the redox cycling which is inherent to the one-electron reduction processes6.It has been proposed that in plants the quinone reductases may play a role in theenzymic mechanism for polymerization and depolymerization of lignin. In fact,

QR interacted with laccase and peroxidases, thereby increasing thedecarboxylation of the lignin model compound vanillic acid7. Moreover, theligninolytic basidiomycete Phanerochaete chrysosporium was seen to produceseveral quinone oxidoreductases8. other authors have proposed that eR might bea relevant membrane-peripheral redox system permitting an overproduction ofcarotenoids by regulating the redox state of quinonesa. on the other hand, thesignificance of the presence of QR in both roots and seedlings during the earlystages of germination still remains unclear. In this paper we report the purificationof QR from Glycine max seedlings and some properties are given. Crudehomogenate activity staining showed six bands, of which the largest was purified.

MATERIALS AND METHODS

Chemicals and instrumentation. Agarose Cibacron Blue-3GA, AH-Sepharose 48, NADH and NADPH were purchased from Sigma. 2,6-Dichlorophenol-indophenol was from Fluka. cM-cellulose was obtained fromwhatman International, and sephacryl s-200 HR from pharmacia. All otherreagents were of the highest grade available and used without further purification.SDS- and native-PAGE reagents, Mini-Protean ready gels and both molecularweight and isoelectric focusing standards were purchased from Bio-Rad. IEF was

NAD(P)H :QUINONE OXIDOREDUCTASE

carried out on a LKB-Multiphor tr electrophoresis unit. The molecular weight

determination was also carried out on a Beckman System Gold HPLC unit

equipped with a TSK-300 column, using standard gel-filtration from Bio-Rad.

Glycine max (L.) Merr seeds (1 Kg) were soaked in tap water for 3 hr.

Seedlings were grown for 7 days in the dark at25'C on wet vermiculite'

Purification. Soybean seedlings (630 g) were homogenized in 1500 ml of 50

mM Tris-HCl buffer (pH 7.5) for 3 min in a Waring Blender and the resulting

suspension was filtered through a nylon cloth. The filtered suspension was

centrifuged at26}00 x g for 40 min in a refrigerated centrifuge and the pellet then

discarded (Step l). The supernatant was applied to an Agarose Cibacron Blue-

3GA column (5x7 cm) previously equilibrated with 100 mM Tris-HCl buffer (pH

7.5). In these conditions QR was bound to the agarose. The resin was washed with

100 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl until the absorbance

at 280 nm was below 0.01. Elution of QR took place at a flow-rate of 1 ml min-l

with the same buffer containing 200 mM NaCl (Step 2). The enzyme solution

from step 2 was concentrated by ultrafiltration and dialyzed against l5 I of 30 mM

Tris-HCl buffer (pH 7 .5); it was then applied to an AH-Sepharose 48 column (1x5

cm) equilibrated with 30 mM Tris-HCl buffer (pH 7.5) and washed with the same

buffer until A2gg was < 0.01. The enzyme was eluted with 60 mM of the same

buffer (Step 3). The pooled fractions from step 3 were concentrated and twice

dialyzed against 15 I of 30 mM potassium phosphate buffer (pH 6.5). The next

step was performed on a CM-Cellulose column (2.5x5 cm) equilibrated with the

same buffer as above. In these conditions the enzyme did not bind to the resin and

was therefore recovered in the washing, which was collected until A2gg was <0.01

(Step 4). The solution from step 4 was concentrated by ultrafiltration and dialyzed

against 15 I of 50 mM Tris-HCl buffer (pH 7.5). Finally, gel-filtration

chromatography was carried out on a Sephacryl 5-200 HR column (2.5x80 cm)

equilibrared with 50 mM Tris-HCl buffer (pH 7.5) and elution was performed

with the same buffer. The fractions with the highest specific activity were pooled

and concentrated by ultrafiltration (Step 5). The purification procedure is

summarized in Table l. All purification steps were performed at 4". The overalì

purification achieved was 1371 fold with a recovery of about 5lvo.The purified

enzyme was yellow in solution like other flavoproteins.

59

RESCIGNO ET AL.60

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NAD(P)H : QUINONE OXIDOREDUCTASE

Enzyme assay. NAD(P)H:quinone reductase activity was measured with

NADH as the electron donor and 2,6-dichlorophenol-indophenol (DCPIP) as the

electron acceptor. The standard assay system contained 100 mM Tris-HCl buffer

(pH 7.5), 0.08% Triton X-100,0.5 mM NADH, and 40 pM DCPIP. The reaction

was started by the addition of 100 pl of a suitable diluted enzyme solution in a

final vol of 1 ml. The reduction of DCPIP was followed at l,6g6n- (e = 18 mM-l

"ttr-l; at 25'C. With the various other quinones used as acceptors, the enzymic

activity was followed by measuring the decrease of NADH at 340 nm. The effect

of temperature was analyzed by measuring the latter directly in the cuvette as soon

as the reaction was stopped. As for activity staining, after native-PAGE, the gel

was stained by immersing in 50 ml of 0.8 mM DCPIP dissolved in 100 mM Tris-

HCI buffer (pH 7.5) for 5 min. The excess of dye was removed and the gel was

washed twice with 100 ml of 100 mM Tris-HCl buffer (pH 1.5). A 2 mM NADH

solution was then applied to the stained gel. After 5-10 minutes, according to the

enzyme concentration, the QR activity showed up as bleached bands on a blue

background.

Protein analysis. Proteins were estimated by the Bradford method using

bovine serum albumin as the standarde. SDS- and native-PAGE were performed in

accordance with the Bio-Rad Mini-Protean tr ready gels Instruction Manual.

Isoelectric focusing was carried out as described by Righettir0.

RESULTS AND DISCUSSION

Criteria of purity. Quinone reductase, obtained with the purification

procedure summarized in TABLE I, was checked for homogeneity by PAGE

under various conditions. Only one band was detected by activity staining of the

gel both on non-denaturing PAGE and isoelectric focusing. Similarly, one band

was detected by Coomassie brilliant blue staining. The isoelectric point of QR was

6.1. Only one band was noted in SDS-PAGE in the presence and absence of

mercaptoethanol.

Molecular weight determination. The MW determination was carried out by

SDS-PAGE and HPLC gel-filtration. The MW determined with SDS-PAGE was

21900, both in the presence and the absence of mercaptoethanol (fg. l). On HPLC

6 l

62 RESCIGNO ET AL.

FIGURE 1

SDS-polyacrylamide gel electrophoresis of purified NAD(P)H:quinoneoxidoreductase. The first lane indicates the molecular weight standard proteins;the second lane, the purified quinone reductase. Standard proteins in MWdecreasing order: phosphorylase b, bovine serum albumin, ovalbumin, bovinecarboanhydrase, soybean trypsin inhibitor, hen egg lysozyme.

gel-filtration chromatography a single peak, corresponding to a MW of 616fi) +

1000 SD, was eluted. The difference between these two determinations may be

due to the fact that the molecular mass determined by gel-filtration depends on the

shape of the protein and that the QR purified from Glycine rnuc may consist of

two identical monomers, as reported for most quinone reductases6. From the

flavine content, in accordance with the method described by Mahlerll, the

ininimum molecular weight was estimated to be about 20000.

NAD(P)H : QUINONE OXIDOREDUCTASE

Spectroscopic properties. In the UV-VIS region the purified QR, in its

oxidized form, exhibited the typical absorption spectrum of flavoproteins with

absorption maxima at270,365, and 443 nm (ftq.2).

Substrate specificity and inhibitors. Glycine max qtinone reductase is able

to reduce a number of compounds in the presence of NADH and NADPH as the

electron donor. Quinones, substituted naphthoquinones (including natural

occurring compounds as juglone and lawsone), dyes and other compounds were

all tested for their ability to act as an electron acceptor or as an inhibitor. TABLE

II shows the substrate specificity of the enzyme. Every tested compound showed a

lower relative activity in respect to DCPIP. It is noteworthy that QR is able to

catalyze the reduction of 6-hydroxydopa-quinone (6-OHDOPA-quinone) and 6-

hydroxydopamine-quinone (6-OHDA-quinone). Both these compounds have a

particular interest both because oftheir neurotoxicityl2'I3, and the discovery that 6-

OHDOPA-quinone is the cofactor at the active site of bovine serum amine

oxidasela. 6-OHDOPA-quinone and 6-OHDA-quinone were obtained by

autoxidation of the corresponding reduced forms. In fact, 6-hydroxydopa (6-

OHDOPA) and 6-hydroxydopamine (6-OHDA) autoxidized in 100 mM Tris-HCl

buffer (pH 7.5) when in the presence of molecular oxygen. Autoxidation of these

compounds was followed at 485 nm (maximum absorption of the oxidized form)

and was complete after 10 min. Quinone reductase (0.2 pg rn1-l; and 0.5 mM

NADH were incubated in 100 mM Tris-HCI buffer (pH 7.5) containing 40 pM 6-

OHDOPA- or 6-OHDA-quinone in 1 ml volume. The relative activity of these

compounds was not referred to DCPIP activity since, during the reaction, an

equilibrium is established between 6-OHDOPA-quinone (or 6-OHDA-quinone),

which undergoes reduction, and 6-OH-DOPA (or 6-OH-dopamine) which is

rapidly oxidized by molecular oxygen. As a consequence of the above, NADH

consumption was greater than the stoichiometric amount needed to reduce 40 pM

quinone solution. 6-OHDA-quinone's relative activity was then calculated to be

30Vo of the value found for 6-OHDOPA-quinone.

We have noted that QR showed an action opposite to that of peroxidases

and laccases, the two enzymes involved in the metabolism of lignin. For example,

horseradish peroxidase catalyzes oxidation of synthetic compounds like

syringaldazine and o-dianisidine to the corresponding quinone derivatives in the

presence oî H2O2. In the same way, laccasesls and peroxidasesl6 use 2,4,5-

63

64 RESCIGNO ET AL.

300 nm 400 nm 500 nm ó00 nnr

rv{velengt h

FIGURE 2

Absorption spectrum of purified NAD(P)H:quinone oxidoreductase. Theabsorption spectrum was recorded between 600 and 24O nm.

F

5 l o r o E- - _ - o

NAD(P)H:QUINONE OXIDOREDUCTASE

TABLE II

Relative activity of the Glycine nax NAD(P)H:quinone oxidoreductase (QR) with

different electron acceptors. Activities are reported in relation to the standard QRassay with DCPIP. Quinone reductase was 0.04 mg ml-I. Acceptors concentration

was 40 pM, NADH was 0.5 mM' Acceptors reduction was measured as NADH

oxidation at 340 nm. N.D., not detectable.

65

Electron acceptor

DCPIPJugloneLawsonePotassium ferricyanide5,8-Dihydroxynaphthoquinone6-OHDOPA-quinone (*)6-OHDA-quinone (*)

Relativeactivity

(7o)

10058

N.D.44t9

10030

(*) Details about relative activities of 6-hydroxydopa-quinone and 6-

hydroxydopamine-quinone are given in the Results and Discussion section.

trihydroxyphenethylamine (6-OHDA) and 2,4,5ìrihydroxyphenylalanine (6-

OHDOPA) as substrates. Glycine max QR reduces the quinones generated by

peroxidase and laccase to the corresponding hydroquinones using NADH and/or

NADPH as a substrate. In the presence of DCPIP, QR showed a K- value for

NADH 341 pM, while Kn' for NADPH was 190 pM.

We have tested the inhibitory effect of a number of compounds on the QR

activity. TABLE III shows the more significant results. The enzyme was totally

inhibited by a0 pM dicumarol, a typical inhibitor of quinone reductases.

Effect of pH and temperature. Effect of pH on QR activity was tested in 100

mM Tris-HCl buffer using DCPIP as the electron acceptor. The pH curve showed

an optimum at 7.5. Over 6O7o of activity still remained at pH 9.

The effect of temperature on the rate of the reaction was studied over the

range 7 -57"C under standard assay conditions. Over the tange 7 -3O" a straight line

was obtained in the Arrhenius plot with an activation energy of 6 kcal mol-l.

66 RESCIGNO ET AL.

TABLE III

Relative inhibition of the G.max NAD(P)H:quinone oxidoreductase. Inhibition isgiven in relation to the standard assay with DCPIP taken as l00%o of activify. Allinhibitors and DCPIP were present in the concentration of 40 pM; NADH was 0.5mM. The inhibiting effect was measured as NADH oxidation at 340 nm.

Inhibitor Relative inhibition ( 7o )

DicumarolCibacron Blue F3G-AProcion Blue MX-RAlizarin Red S

Between 30' and 43'C, the enzyme activity was unaffected by the

temperature, the graph showing a plateau; over 43oC it began to decrease. This

behaviour could not be compared with other quinone reductases because of the

general lack of data in this regard.

CONCLUSIONS

We have reported the purification of QR ftom Glycine max seedlings.

The yield obtained and the high specific enzymic activity achieved make

this purification procedure useful for obtaining good amounts of QR, sufficient for

further studies. In fact, QR is not yet commercially available and, therefore,

Glycine max seedlings seem to constitute an inexpensive and easily accessible

source.

As for the role this enzyme plays in lignin metabolism of plants, it is

interesting to point out its behaviour opposite to that of laccase and peroxidase

activities. On the basis of our present knowledge, it is not yet possible clearly to

understand to what extent QR is involved in the early stages of seed germination.

Finally, the purified enzyme can be seen to be a useful tool in the study of

the relationship between the brain's NAD(P)H:quinone oxidoreductase and the

neurotoxic quinone derivatives of cathecholamines. In this regard, further studies

are in progress in our laboratories.

100t )

7538

NAD(P)H : QUINONE OXIDOREDUCTASE

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