ANALYTICAL

BIOCHEMISTRY

188,101-104

(1990)

Electrophoretic Detection of Ascorbate Oxidase Activity by Photoreduction of Nitroblue Tetrazolium’ Mauro Maccarrone, Antonello Rossi,* Gabriele D’Andrea, Gianfranco Amicosante, and Luciana Avigliano Department of Biomedical Sciencesand Technologiesand Biometrics, University of L’Aquila, I-67100 L’Aquila, Italy, and *Department of Experimental Medicine and BiochemicalSciences,University of Rome“Tor Vergata, “I-001 73 Rome,Italy Received

December

13,1989

A method for the detection of ascorbate oxidase in electrophoretic gels is described. This method relies on the ability of the enzyme to prevent the photoreduction of nitroblue tetrazolium (NBT). The method is based on that described by C. Beauchamp and I. Fridovich (1971, Anal. Biochem. 44,276-287) for the superoxide dismutase and was made specific for ascorbate oxidase detection by treating the gel with 0.1 M hydrogen peroxide. Ascorbate (25 FM) or riboflavin (500 jiM) was used as the electron donor. The possible reaction mechanism in the presence of ascorbate has been investigated. Western and Northern blot analyses confirmed the results obtained from the NBT staining procedure. 0 1990 Academic Press, Inc.

Ascorbic acid oxidase (AAO’; EC 1.10.3.3) is a copper enzyme widely distributed in the plant kingdom, as determined by activity measurements based on oxygen uptake (1) and by immunohistochemical localization (2). It catalyzes the following reaction: 2 L-ascorbate + O2 + 2H+ + 2 L-dehydroascorbate + 2Hz0 A role of this enzyme in a redox system, alternative to the mitochondrial chain, in growth promotion or in susceptibility to diseases,has been postulated (3). Several methods allowing the detection of AA0 activity in crude samples have been described, but no one method can be directly applied to electrophoretically run 1 This investigation was supported by the E.E.C. Contract SCl0197-C (GDF). ’ Abbreviations used: AAO, ascorbic acid oxidase; NBT, nitroblue tetrazolium; TEMED, N,N,N’,N’-tetramethylethylenediamine; DAB, 3,3’-diaminobenzidine; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; NC, nitrocellulose; SOD, superoxide dismutase. 0003~2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

gels. Thus a new method based on the chromophore formation upon reduction of nitroblue tetrazolium (NBT) has been developed. This method was applied to investigate the presence of AA0 both in different plants and at various growth stages in the same plant, comparing the resulting patterns with those obtained from immunoblot and from determinations of AAO-coding mRNA. MATERIALS

AND

METHODS

Chemicals. Chemicals were of the purest analytical grade. T7 RNA polymerase was purchased from Boehringer, [w~‘P]UTP from Amersham, and nitroblue tetrazolium from Sigma. Enzyme purification. Green zucchini (Cucurbita pepo medullosa var. Milano) and red pepper (Capsicum annuum) were from commercial sources, and zucchini shoots were grown at 25°C in a greenhouse and harvested 15 days after seeding. AA0 from zucchini peelings was purified to homogeneity as described by Avigliano et al. (4), while all the homogenates used in the experiments were prepared following the procedure reported by D’Andrea et al. (5). Antibodies. Antibodies against green zucchini AA0 were elicited in rabbits and purified as described elsewhere (5). Electrophoresis. The following samples were subjected to polyacrylamide gel electrophoresis under nondenaturing, nonreducing conditions, using a polyacrylamide gradient 5-15%: purified AA0 (10 pug),green zucchini peelings homogenate (100 pg), green zucchini shoots homogenate (100 pg), and red pepper homogenate (100 pg). After the electrophoretic run the gelswere subjected to either the NBT reaction or the immunoblotting analysis. Coomassie blue. R-250 0.2% (w/v) was used for gel proteins staining. AA0 activity detection in gels. The method originally described by Beauchamp and Fridovich for the detection 101

102

MACCARRONE

of superoxide dismutase (6) was used except that the gel was treated with 0.1 M HzOz, before staining, and ascorbate or riboflavin in the presence of TEMED and light was used as the electron donor. Different concentrations of the reducing agents were used, the most effective ones being 500 pM for riboflavin and 25 pM for ascorbate. A blue color is formed after NBT reduction, due to the accumulation of formazan. The presence of AA0 prevents this reduction and, therefore, the enzyme can be detected as an achromatic band in a blue background. After the electrophoretic run the gel (1.5 mm thick) was dipped in 0.1 M HzOz aqueous solution for 20 min, and then it was washed five times (5 min each) with 36 mM K-PO4 buffer; afterward it was soaked in 2.45 mM NBT aqueous solution for 30 min and in 36 mM K-POJ 28 mM TEMED/25 pM ascorbate (or 500 pM riboflavin) buffer for a further 30 min in the dark. The pH of the buffer solutions was set to 7.8 for ascorbate and to 6.0 for riboflavin. The gel was finally put at a 15-cm distance from a 250-W HQl-TS Osram lamp, at a luminous flux of 7.5 f 0.8 mW cmB2 min-’ in the interval 400-700 nm, while keeping the gel temperature at 10 + 1°C. After development of the blue color the reaction was stopped by washing the gel with deionized water. Control experiments. Control experiments were performed in order to quantitate the AA0 ability to inhibit the formazan production and to understand the mechanism of this inhibition. The NBT reduction was followed at the isobestic point of formazan formation (585 nm), using a Uvikon 860 spectrophotometer (Kontron Instruments). Samples each containing 1 ml of 36 mM K-Pod/28 mM TEMED/2.45 X lop5 M NBT/25 pM ascorbate (or 500 PM riboflavin) and different amounts of pure AA0 were buffered at pH 7.8 (or 6.0) and equilibrated with air or nitrogen, by bubbling the gas for at least 10 min before illumination. Then they were illuminated for 2 (or 10) min with the same Osram lamp as above and the formation of formazan was followed for the first 3 min. Polurographic measurements. Oxygen consumption was evaluated in samples containing 3 ml of 36 mM KPOJ50 pM ascorbate/O.25 pg AA0 buffered at pH 7.8, in the presence or absence of both 50 mM TEMED and light. In these experiments, performed by means of a YSI Model 5300 biological oxygen monitor, the light energy was 10 times weaker than that coming from the Osram lamp. Immurwblotting analysis. After the electrophoretic run the proteins were transferred from the gel to a nitrocellulose (NC) sheet following the procedure reported by Towbin et al. (7). Briefly, the NC sheet was dipped for 2 h at room temperature in a rabbit anti-AA0 IgG solution and was then extensively washed in 10 mM Tris/HCl buffer, pH 7.4, containing 0.9% NaCl (w/v); afterward it was plunged for a further 2 h in a goat anti-rabbit IgG solution, diluted 1:200 in the washing buffer, and was then extensively washed again. The gel was finally

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0

IO

20

30

40 AAO.

50 cB

FIG. 1. The AA0 ability to inhibit the NBT reduction was tested as described under Materials and Methods. 25 pM ascorbate, in air (0) or under nitrogen (0); 500 pM riboflavin, in air (A) or under nitrogen (A).

dipped for 2 h in a “peroxidase-anti-peroxidase complex” solution, which was diluted 1:500 in the Tris buffer mentioned above, and was then extensively washed for the last time. The AA0 detection was based on the oxidation of DAB (25 mg of DAB plus 10 ~1 of 30% H202 in 100 ml of 10 mM Tris/HCl buffer, pH 7.4). RNA extraction and Northern blot analysis. Total RNA was isolated from zucchini and Capsicum fruits as reported by Logemann et al. (8). Poly(A)+ RNA was selected by oligo(dT) cellulose column chromatography. RNA was electrophoresed on a 1% agarose gel containing 6.6% formaldehyde and then transferred to a NC membrane. The probe in the Northern blot analysis was a 32Plabeled RNA (sp act 5 X lo* cpm/pg) synthesized by T7 RNA polymerase runoff transcription of a linearized plasmid containing a DNA fragment coding for the C-terminal part (about 180 amino acids) of zucchini AA0 (9). Hybridizations were performed at 42°C in the presence of 50% formamide (v/v), 50 mM Na-PO4 pH 6.5,50 mM NaCl, 50 mM Na-citrate, 0.02% BSA (w/v), 0.02% Ficoll (w/v), 250 pg/ml sonicated calf thymus-denatured DNA, 0.02% polyvinylpyrrolidone (w/v), 0.1% SDS (w/ v). The filters were then washed four times with 50 mM NaCl plus 50 mM Na-citrate, pH 7.0, and 0.1% SDS (w/ v) at the same temperature. RESULTS

AA0 was able to inhibit the accumulation of the formazan due to the photo-induced reduction of NBT by TEMED and ascorbate or riboflavin in the presence of air (Fig. 1). The same figure shows that NBT reduction can occur also under nitrogen. In this case however AA0 is unable to prevent the formation of formazan. From Fig. 1 it can be observed that a unit of enzyme activity (i.e., the amount of AA0 which inhibits the reaction by

ELECTROPHORETIC

DETECTION

OF

ASCORBATE

OXIDASE A

0

I

I

I

1

I

I

2

4

6

8

IO

I2

ACTIVITY B

c

IN D

PLANT A

103

TISSUES B

CD

J

time, min

FIG. 2. Polarographic tracks obtained by the oxidation of 50 pM ascorbate in 36 mM K-PO4 buffer, pH 7.8. The continuous line shows the effect of the addition of 50 mM TEMED to the reaction system in the presence of light: light alone has no effect on the oxygen uptake, while in the presence of TEMED the oxygen consumption starts again. The dashed line shows that the oxygen uptake exceeds the stoichiometric value, when light and 50 mM TEMED are present in the mixture from the beginning.

50%) varies between 8 and 13 pg, depending on which electron donor is used. Polarographic measurements of the effect of TEMED and light on the oxidation of ascorbate in the presence of AA0 were performed. Figure 2 shows that, in the presence of light, the addition of TEMED increased the amount of O2 consumed. In fact, in the presence of 50 mM TEMED and light 150 nmol of ascorbate is oxidized with the consumption of 96 nmol of oxygen, while without TEMED the Oe uptake leveled off at the expected value of 75 nmol. Moreover, the addition of 50 mM TEMED and light at the end of the reaction allows the oxidation to start again.

FIG. 4. Immunoblot (left) and the same samples as in Fig. 3.

Coomassie

blue

staining

(right)

of

The reaction including NET, TEMED, and ascorbate was used to detect AA0 on electrophoretic slabs (Fig. 3). This figure shows that pure AA0 gives an achromatic band (lane A) when the gel is treated as reported under Materials and Methods. The figure also shows that AA0 activity can be detected in zucchini peels and shoots (lanes B and C), while homogenates of red pepper do not display any achromatic band in the molecular weight region corresponding to native AA0 (lane D). These results were confirmed by immunoblotting analysis, showing a band positive to the reaction with anti-AA0 antibodies in all the homogenates, pepper included. The relative intensity of this band is higher in zucchini peels than in shoots or pepper homogenates (Fig. 4). The presence of RNA coding for AA0 in plant homogenates was checked too. Figure 5 shows the Northern blot of zucchini peel homogenate with a strong positive band, while a greater amount of pepper homogenate did

ABCDABCD

FIG. 3. Staining of electrophoretic slabs with NBT (left), performed as described under Materials and Methods. The same patterns were obtained, using either 25 @M ascorbate or 500 pM riboflavin as the electron donor. On the right the proteins present in the same gels are represented, as revealed by the Coomassie blue staining procedure. Lanes: A, purified AA0 (10 pg); B, green zucchini peeling homogenate (100 pg); C, green zucchini shoot homogenate (100 pg); D, red pepper homogenate (100 pg).

FIG. 5. Northern blot of 5 pg of total RNA from zucchini A) and of 45 fig of poly(A)+ RNA from red pepper (lane show the position of the ribosomal RNAs (18s and 28s).

peels (lane B). Arrows

104 not show any RNA band cross-reacting chini AA0 RNA probe.

MACCARRONE

with

the zuc-

DISCUSSION

The reduction of NBT to a poorly soluble, highly colored derivative has been largely used in both biochemistry and histochemistry in various analytical techniques. One of the applications is based on the ability of a mixture of TEMED (or EDTA) and riboflavin to give a photoinduced reduction of NBT. The species directly responsible for NBT reduction has been thought to be O2 (6). In this paper we have shown that ascorbate may substitute for riboflavin in the reduction of NBT by TEMED. NBT can be reduced directly by ascorbate, provided that the stoichiometry ratio is above 4O:l in favor of ascorbate at pH 7.8. In the presence of TEMED and light much lower concentrations of ascorbate are able to reduce NBT. This indicates that dehydroascorbate is reduced back to ascorbate by TEMED, as demonstrated by the finding of an oxygen uptake greater than stoichiometric with respect to ascorbate when TEMED was present in the incubation mixture (Fig. 2). The reduction of NBT occurs both in the presence and in the absence of oxygen, and the addition of AA0 is able to prevent the formation of formazan only in the presence of oxygen. We took advantage of this activity of AA0 in order to detect the enzyme on gel slabs (Fig. 3). This assay is rather similar to the well-known colorimetric method of detecting superoxide dismutase (SOD) (6) but it was made specific for AA0 by treating the gel with 0.1 M hydrogen peroxide as a preliminary step of the procedure. HzOz irreversibly inactivates superoxide dismutase at low H202/SOD molar ratios (10, 11) and indeed when the hydrogen peroxide treatment was omitted the NBT staining revealed in the electrophoretically run homogenates an achromatic band corresponding to AA0 and another one, fast moving, attributable to superoxide dismutase (12). Moreover samples identical to those subjected to the NBT staining retained up to 50% of their ascorbate oxidase activity after 20 min of incubation with 0.1 M H202 at room temperature, as measured by the spectrophotometric assay of Oberbacher and Vines (13). This finding supports the evidence that the hydrogen peroxide treatment of the gel does not prevent AA0 from being detected by the NBT reaction. As far as the NBT staining mechanism is concerned, AAO, at variance with SOD, does not appear to act through an interaction of O2 radicals, although it is able to react with them at a rate about l/100 of that of SOD (I. Mavelli, personal communication). AA0 was also found able to reoxidize reduced riboflavin at a rate that is at least 10 times higher than that of oxygen alone. On the other hand it is well known that AA0 catalyzes the reduction of 0, to water, through the intermediacy of partly reduced species. Thus the inhibition of formazan reduction by AA0 may be attributed to the catalytic re-

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moval of ascorbate and reduced riboflavin with no concomitant production of superoxide. The good agreement between the achromatic bands in NBT staining and the immunoblot patterns confirms that these bands correspond to AAO. Furthermore, the quantitation of mRNA is in line with these findings, showing the presence of AAO-coding mRNAs in the same samples which are active in the gel slabs and able to bind the anti-AA0 antibodies. Some discrepancies arise regarding the red pepper homogenates. In fact on the basis of NBT analysis and poly(A)+ RNA detection one could argue that no AA0 was present in this plant. On the contrary the immunochemical analysis shows a band having the same mobility as AAO. However, one should keep in mind that polyclonal antibodies used to perform immunoblotting might have recognized epitopes in red pepper homogenate different from those of AAO. In conclusion, the new method seems to be rapid and reliable, allowing the AA0 detection in gels in a couple of hours instead of a couple of days, as required for instance by immunoblotting. Moreover, unlike the spectrophotometric and polarographic determinations of AA0 activity in rough homogenates, this procedure allows a direct detection of AA0 activity, avoiding false positive results. ACKNOWLEDGMENTS The authors thank Professor A. Finazzi-Agro versity of Rome, Italy) and Dr. A. Oratore Italy) for their helpful advice.

(“Tor (University

Vergata,” Uniof L’Aquila,

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Electrophoretic detection of ascorbate oxidase activity by photoreduction of nitroblue tetrazolium.

A method for the detection of ascorbate oxidase in electrophoretic gels is described. This method relies on the ability of the enzyme to prevent the p...
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