Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting.

Abstract

Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals. The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3mm chromatographic paper was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried catechol-paper. A dark-brown band corresponding to PPO was visualized within 1 min and was further confirmed by a conventional Western blot using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with respect to its relevance for searching a natural PPO inhibitor is described and discussed.

Full text

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E-mail address: mao1010@ms7.hinet.net (S.J.T. Mao).
CHROMB 14764 1–6
CHROMB147641–6
Journal of Chromatography B, xxx (2006) xxx–xxx
Gel electrophoresis of polyphenol oxidase with instant
identification by in situ blotting?
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Tsai-Mu Chenga, Pei-Chen Huanga, Ju-Pin Panb,c, Kuan-Yu Lina, Simon J.T. Maoa,d,∗
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aCollege of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai Street, Hsinchu, Taiwan, ROC
bDivision of Cardiology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, ROC
cSchool of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC
dDepartment of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwan, ROC
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Received 1 June 2006; accepted 21 August 2006
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Abstract
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Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals.
The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present
study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3mm chromatographic paper
was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-
polyacrylamide gel electrophoresis (SDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried
catechol-paper. A dark-brown band corresponding to PPO was visualized within 1min and was further confirmed by a conventional Western blot
using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or
no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose
that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to
identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with
respect to its relevance for searching a natural PPO inhibitor is described and discussed.
© 2006 Published by Elsevier B.V.
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Keywords: Polyphenol oxidase; Tyrosinase; Catechol; Immobilized-paper; Browning reaction; Natural PPO inhibitor
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1. Introduction
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Plant polyphenol oxidase (PPO), also known as tyrosinase
in animals (EC 1.14.18.1), is an enzyme containing copper
that catalyzes two different reactions using molecular oxygen,
hydroxylation of monophenols to o-diphenols and oxidation of
the o-diphenols to o-quinones [1]. This enzyme, responsible for
melanization in animals and for browning in fruits and veg-
etables, is widely distributed in microorganisms, animals, and
plants.
LocalizationofPPOintheplantcellsdependsonthespecies,
age, and maturity [2–4]. In green leaves, a considerable part
of PPO activity is localized in the choloroplasts [5]. It is also
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?This paper is part of a special volume entitled “Analytical Tools for Pro-
teomics”, guest edited by Erich Heftmann.
∗Corresponding author. Tel.: +886 3 571 2121x56948; fax: +886 3 572 9288.
present in the soluble fraction in different fruits and vegeta-
bles [6–9]. Since colorless polyphenols are converted into dark
brown quinones, the browning reaction impairs the texture, fla-
vor, and nutritional values of fruits and vegetables. Prevention
ofundesirablebrowninghastraditionallybeenaccomplishedby
variouschemicals,includingtheantioxidantcompounds(ascor-
bic acid, citric acid, flavonoids, and sulfites) [10] and some PPO
inhibitors (tropolone, kojic acid, resorcinols and benzaldehyde)
[11–14].
Designing a PPO inhibitor to suppress its enzyme activity,
therefore, becomes an essential subject of challenge either in
preventing the browning reaction in fruits or in intervention of
darkening and aging processes for the cosmetic industry. In the
present study, we developed a rapid gel-electrophoretic blot-
ting technique to identify the possible molecular form of PPO
on a dried chromatographic paper that was immobilized with a
colorimetric substrate catechol. The method also allows us to
identify potentially potent inhibitors from natural products.
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1570-0232/$ – see front matter © 2006 Published by Elsevier B.V.
doi:10.1016/j.jchromb.2006.08.046
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Please cite this article as: Tsai-Mu Cheng et al., Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting, Journal
of Chromatography B (2006), doi:10.1016/j.jchromb.2006.08.046

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immediatelyblottedinsitu(atroomtemperature)forPPOactiv-
ity by pressing it onto the top of a dried catechol-paper without
addingbufferedsolution(seeSection3).QuantumOneImaging
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T.-M. Cheng et al. / J. Chromatogr. B xxx (2006) xxx–xxx
2. Experimental
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2.1. Fruits and vegetables
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Fresh fruits and vegetables at commercial maturity were pur-
chased from a local market. They were then placed in a 4◦C
ice-packed box and immediately used for the study.
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2.2. Preparation of extract from fruits and vegetables
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Approximately150–200mL(at4◦C)ofaphosphatebuffered
solutioncontaining0.02Msodiumphosphate,pH7.4(PB),were
added to each 100g of chopped fruit or vegetable to make a
final volume of 200mL or 50% (w/v) solution. The mixture was
homogenizedthoroughlybyablenderatamaximalspeedovera
period of 5min with 5 stops. The homogenate was sonicated for
15s and centrifuged at 3000×g for 30min at 4◦C to remove
the pellets. The supernatant was then passed through a 3mm
filter paper. The pass-through extract stored at 4◦C for less than
12hwasusedforconventionalPPOenzymeassayandforSDS-
PAGE blot.
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2.3. Determination of PPO activity in solution
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To monitor the PPO activity, 200?L of 4mM catechol sub-
strate in PB were added to 10?L of the plant extract in a
microtiterwell[15].Thereactionmixturewasincubatedatroom
temperature(∼24◦C)overtime.Theenzymeactivitywasmoni-
toredbyreadingincreasedabsorbanceat415nmoveraperiodof
30min using a plate reader equipped with an automatic printer.
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2.4. Preparation of catechol-immobilized paper
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Unless otherwise mentioned, 3mm chromatographic papers
(Whatman® 3mm Chr, Cat no. 3030-917, Maidstone, England)
were briefly soaked into a 0.5% (w/v) catechol solution. After
a brief blot on an absorption paper, the “catechol-paper” was
dried at 37◦C for 5min. The dried paper is stable for at least
60 days at room temperature when kept in a dark environment.
SincetheKmofPPO(mushroom,forexample)isapproximately
0.1–1mM [16] and the concentration of catechol immobilized
was in excess at 40mM (or 0.5%), the maximal rate of PPO of
tested extract could be achieved on the catechol-paper.
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2.5. Detection of PPO activity following SDS-PAGE and
densitometry
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Electrophoresis was conducted in a vertical slab gel unit
(Mini Protein®3 Cell, Bio-Rad, Hercules, CA) equipped with
a PAC 300 power supply (Bio-Rad, Hercules, CA). Sample
extract 10–15?L equilibrated in 10mM Tris–HCl and 0.1%
SDS, pH 6.8, without heat was loaded onto a 10% SDS-PAGE
gel according to procedures described previously [17,18]. Pre-
stained protein markers were used for the molecular weight
index. Gel run at a constant current of 20mA for 60min was
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Software (Bio-Rad) was used to measure the image density on
the PAGE-blot.
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2.6. Preparation of mushroom PPO
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A gel-filtration of high performance liquid chromatography
(HPLC) column Sephadex 200 was used for further purification
of mushroom PPO purchased from Sigma–Aldrich (St. Louis,
MO). HPLC was conducted at a flow rate of 0.5mL/min and
monitored at 280nm when using PB as a mobile phase [19].
Tubes corresponding to PPO activity were pooled and stored at
−20◦C prior to use.
2.7. Test of endogenous activity of PPO inhibitor
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To0.5mLofplantextract(describedinSection2.2),5mLof
98% alcohol were added and vortexed for 5min. The reaction
mixture was then centrifuged at 3000×g for 10min to remove
thepellets.Thesupernatantwasdriedundernitrogenandrecon-
stituted to 0.5mL of PB. To test the inhibitory activity for PPO,
various amounts of the reconstituted extract were aliquoted into
the standard PPO assay as described above (Section 2.3.).
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2.8. Preparation of antibody against PPO
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Female Balb/c mice (5–7 weeks of age) were immunized
according to the method previously described [20]. In brief,
200–300?g of purified mushroom PPO in 0.5mL of a ster-
ilized buffered solution containing 0.12M NaCl and 0.02M
phosphate, pH 7.4 (PBS), was mixed and homogenized with
0.5mL of Complete Freund’s Adjuvant (Sigma–Aldrich) by a
three-waystopcock.Eachmousewasinitiallygivenatotalemul-
sion of 0.5mL with six subcutaneous injections onto the back
and one intraperitoneal injection. At day 7, a same dose with
complete adjuvant was given intraperitoneally, followed by two
intramusclar injections without adjuvant at day 14. At day 21,
blood was collected in 0.1% (w/v) EDTA to obtain plasma.
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2.9. Western blot
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Western blot analysis was performed according to the pro-
cedures described previously [21]. In brief, protein (typically
10?g) or plant extract resolved by SDS-PAGE was transferred
onto a nitrocellulose membrane and blocked by a 5% (v/v)
skim-milk in PBS. Following washes, 10mL of mouse PPO
antiserum (1:2000 dilutions) were added, incubated for 1h, and
washed. A commercially available secondary antibody (goat
anti-mouse IgG conjugated with HRP) (Sigma–Aldrich) was
then added, incubated for 1h, washed, and developed by 3,3-
diaminbenzidine containing 0.01% peroxide.
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3. Results and discussion
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3.1. Enzymatic reaction of PPO using catechol as a
substrate
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Oxidation using catechol as a polyphenol substrate for PPO
activity and formation of o-benzoquinone product is given
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Please cite this article as: Tsai-Mu Cheng et al., Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting, Journal
of Chromatography B (2006), doi:10.1016/j.jchromb.2006.08.046

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removing, washing, transferring, electroblotting, or using sol-
uble reagents were not required in identifying the PPO. One
single band corresponding to PPO of mushroom extract was
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below:
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2catechol + O2→ 2o-benzoquinone + 2H2O
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Because of the chromogenic property of o-benzoquinone
(dark brown),catechol[22]
dihydroxyphenylalanine (l-Dopa) [23] has been conventionally
used for the PPO activity assay.
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or itsderivativel-3,4-
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3.2. Rationale for the use of SDS gel electrophoresis in
identification of PPO
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PPO is widely distributed in all the plants. The molecular
weight of PPO, however, is varied among the species [2–4].
IdentificationofthemolecularformofanunknownPPOinplant
via a purification procedure is time consuming. An alternative
method is to use Western blot analysis, but the procedure is also
tedious and the antibody employed must recognize or crossly
react with the PPO of an unknown species. For this reason, we
sought a new method for rapid identification of the plant PPO.
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3.3. Evaluation of PPO activity on catechol-immobilized
paper
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Catecholisaspecificsubstratebeingwidelyusedformeasur-
ing the PPO activity of a given plant species. We tested whether
catechol could be immobilized on a paper for directly detect-
ing the PPO activity. First, 3mm chromatographic paper was
immersed into a catechol solution as described in the Section 2.
After brief blotting and drying at 24, 37, or 60◦C, mushroom
PPO was then spotted on the “catechol-paper” to develop the
chromogenic product o-benzoquinone (if any). A dark brown
spot was clearly observed. There was no difference in PPO
induced chromogeneity among the papers dried at 24–60◦C,
although the shape of the paper was somewhat distorted when
dried at 60◦C (data not shown). The catechol-paper dried at
37◦C for 5min was, therefore, used for the subsequent studies
and was found to be stable for at least 60 days at room tempera-
ture. The optimal concentrations of catechol immobilized were
found to be between 0.5 and 1% (data not shown).
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3.4. Determination of mushroom PPO following a
SDS-PAGE
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Since PPO activity is not adversely affected by SDS [24],
separation of PPO from the plant extract using SDS-PAGE is
possible. Fig. 1A depicts the present technique step by step for
visualizing the PPO activity from the whole mushroom extract.
After electrophoresis, conventional procedures including gel
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Fig. 1. Instant identification of PPO activity following a 10% SDS-PAGE: (A)
Following electrophoresis (step I), one side of the glass plates (7cm×8cm)
was removed (step II). The resolving gel was immediately placed onto the top
of a catechol-paper (step III). (B) Top view of visualized PPO activity (5?L
of mushroom extract) with different concentrations of immobilized catechol
(0.1–10%).
instantly observed in less than 1min, when laying the gel onto
the catechol-paper (Fig. 1B). The optimal concentration of cat-
echol immobilized was about 1%. Notably, 1% catechol-paper
gave a sharp chromogeneity, but prolonged blotting (>30min)
should be avoided to prevent “over exposure” of the catechol-
paper. This over exposure is a result of the in situ diffusion of
PPOinthepolyacrylamidegel.Insomecases,decreasingimmo-
bilized catechol concentration in the paper to 0.5% may reduce
this effect.
Fig. 2 shows the molecular form of PPO in the mushroom
extract superimposed to that of purified PPO standard. To eval-
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Fig. 2. Identification of PPO activity between the isolated PPO standard of
mushroomanditsextractonSDS-PAGEblot.Onesinglebandcorrespondingto
PPO standard (60units) or to that in mushroom extract (10?L) was observed.
The molecular form of PPO in the extract (5?L) spiked with the purified PPO
(30units) was superimposed to that of purified PPO standard.
Please cite this article as: Tsai-Mu Cheng et al., Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting, Journal
of Chromatography B (2006), doi:10.1016/j.jchromb.2006.08.046

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PPO. It is capable of cross-reacting with the PPO of selective
plantspecies.Thedatafurtherindicatethatthebandcorrespond-
ing to the PPO enzyme activity on the PAGE-blot to be a PPO.
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Fig. 3. Comparison of the PPO activity between SDS-PAGE bolt and con-
ventional solution assay: (A) Serial dilutions of purified mushroom PPO
(4–250units) were run for SDS-PAGE. Following electrophoresis, the gel was
placed onto the catechol-paper allowing the development of o-quinone product.
(B) The intensity of developed bands was determined by an image densitome-
try. (C) Serial dilutions of purified mushroom PPO with which the activity was
determined by a conventional solution assay. The linear relationship between
the PPO dose and activity is observed in both methods.
uate the sensitivity and linearity of SDS-PAGE blot, various
amounts of purified mushroom PPO were applied for elec-
trophoresis. Fig. 3 reveals the chromogeneity developed on the
paper to be linear to the amount of PPO loaded (16–250units),
which also correlates to PPO activity determined in a solution
assay. The lower limit of detected PPO activity on PAGE-blot
was approximately 31units.
TotestthefeasibilityofSDS-PAGEblotagainstotherspecies,
we randomly chose a total of 12 different fruits and vegetables
for the identification of PPO. Some typical examples are shown
in Fig. 4A, in which tomato possessed two molecular forms of
PPO at about 58 and 59kDa. These values are consistent to that
recentlyreported[25].Interestingly,themolecularformsofPPO
identified in oriental melon, tomato, and radish were identical
as identified by a Western blot analysis (Fig. 4B). The antibody
used for this immunoblot was prepared against a mushroom
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Table 1
PPOactivityindifferentfruitsandvegetablesasevaluatedbyconventionalPPO
and PAGE-blot assay
Species Conventional assaya
(percentage of maximal activity)
PAGE-blotb
(density, INT/mm2)
Peach
Mushroom
Lettuce
Oriental melon
Cucumber
Radish
Cauliflower
Tomato
Orange
Papaya
Pineapple
Guava
100.0
71.3
13.5
5.9
3.8
2.7
2.1
0.2
0
0
0
0
300
2000
3100
2000
2200
3100
100
3600
2100
2000
0
0
aPPO activity was determined using a conventional solution assay.
bPPO activity (arbitrary density) was determined by PAGE-blot as described
in Fig. 4. Quantum One Imaging Software was employed to evaluate the image-
intensity over the specific PAGE–blot bands.
3.5. Conventional PPO activity assay versus PAGE-blot
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In the next experiment, we measured the PPO activity using
a conventional solution assay in those randomly chosen fruits
and vegetables. Table 1 shows that most of their PPO activity,
including the tomato, was low or not detectable, except that of
mushroom and peach. It should be noticed that the low PPO
activity does not necessarily correspond to the actual PPO level
insomespecies.Forexample,arecentstudyshowsthatanabun-
dant amount of PPO is present in tomato [24]. Interestingly,
more than half of the species that possessed the “low” PPO
activity in the solution assay had revealed significant levels of
enzyme activity on our SDS-PAGE blot. The overall results of
the enzyme activity identified in PAGE-blot and solution assay
are given in Table 1. We therefore speculated that there was an
endogenous PPO inhibitor in the presence of the extract. The
inhibitor (if any) might be dissociated from the PPO while run-
ning PAGE.
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3.6. Presence of endogenous inhibitor of PPO in plant
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In the next experiment, we addressed the existence of natural
PPO inhibitors in some tested fruits and vegetables. First, using
tomatoandradish,wedemonstratedthattheirextractswereable
to inhibit the PPO activity of mushroom in a dose-dependent
manner when assessed on catechol-paper (Fig. 3, inserts). Sec-
ond, using alcohol to remove the proteins from the tomato and
radish extracts, we found the inhibitor to be present in the
alcohol-soluble fraction with a dose-dependent inhibitory activ-
ity by a PPO solution assay (Fig. 5). Gandia-Herrero et al. have
shown that an aldehyde derivative of cucumber, 2,6-nonadienal,
can inhibit mushroom PPO with a Kiof 3.4mM [26]. In a pre-
liminary study, we passed the alcohol-soluble fraction through
an Amicon membrane of 3000-kDa cut-off (Millipore, Cork,
Ireland). The pass-through was then heated at 95◦C for 10min,
of which the inhibitory activity was totally retained (data not
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Please cite this article as: Tsai-Mu Cheng et al., Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting, Journal
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Fig. 5. Inhibitory effect of alcohol soluble-fraction from tomato and radish
extract on mushroom PPO activity. Each fraction was incubated with 10?L
of purified mushroom PPO at 24◦C for 10min prior to the solution assay (see
Section2).Insert:5?Lofthereactionmixturewerespottedonacatechol-paper.
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Fig. 4. Typical examples of PPO identified by PAGE-blot and Western blot: (A) PPO activity of plant extracts showing different molecular forms was identified by
a SDS-PAGE blot. (B) PPO proteins were identified by a Western blot using a mouse polyclonal antibody prepared against mushroom PPO. This antibody could
cross-react with the PPO of oriental melon, tomato, and radish, but not with all the tested species.
shown).Itsuggeststhattheinhibitorisheatstablewithamolec-
ular weight less than 3000kDa. Whether the chemical nature
of the inhibitors found in our study is also an aldehyde analog
deserves further study. Nonetheless, this PAGE-blot technique
in combining with the PPO solution assay could also be used
for an initial search for a possible natural inhibitor from plants
(Table 1).
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4. Conclusion
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Instead of a Western blot, the ready-for-use catechol-paper
can be used for an initial identification of PPO after a gel elec-
trophoresis without any additional liquid reagents for instant
visualization. It is also convenient to monitor the PPO fraction
while isolating PPO over a column chromatography. For exam-
ple, Fig. 6 shows a one-step isolation procedure for mushroom
PPO on HPLC Sepharose G-200, in which the fractions con-
taining PPO are exhibited on the catechol-paper. If desired, the
catechol can be co-immobilized with 20mM proline to produce
a dark purple prolyl-quinone adduct for increased sensitivity
(data not shown). The latter procedure has been used in PPO
solution assay [27].
We demonstrated the use of this electrophoretic-blot tech-
nique for instant identification of PPO in fruits and vegetables.
Because PPO is denatured at high temperature, pre-heating the
sample for routine SDS-PAGE should be avoided.
Furthermore, in view of recent studies indicating that tyrosi-
naseisresponsibleforhyperpigmentationinhumans,tyrosinase
inhibitors have become increasingly important in medical [28]
and cosmetic products [29]. A widely used method in the food
and beverage industries to control browning is to add reducing
agents such as sulfites, which chemically reduce the o-quinones
to less reactive colorless diphenols. These compounds, how-
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Please cite this article as: Tsai-Mu Cheng et al., Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting, Journal
of Chromatography B (2006), doi:10.1016/j.jchromb.2006.08.046