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Preparative Biochemistry and


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Some Biochemical Properties


of Polyphenol Oxidase from
Celery
Hlya Yagar

Department of Chemistry , Trakya University ,


22030, Edirne, Turkey
Published online: 18 Aug 2006.

To cite this article: Hlya Yagar (2004) Some Biochemical Properties of Polyphenol
Oxidase from Celery, Preparative Biochemistry and Biotechnology, 34:4, 387-397,
DOI: 10.1081/PB-200031054
To link to this article: http://dx.doi.org/10.1081/PB-200031054

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PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY


Vol. 34, No. 4, pp. 387397, 2004

Some Biochemical Properties of Polyphenol


Oxidase from Celery
Hulya Yagar*
Department of Chemistry, Trakya University, Edirne, Turkey

ABSTRACT
Polyphenol oxidase (PPO, EC 1.14.18.1) was extracted from celery roots
(Apium graveolens L.) with 0.1 M phosphate buffer, pH 7.0. The PPO was
partially purified by (NH4)2SO4 and dialysis. Substrate specificity experiments were carried out with catechol, pyrogallol, L -DOPA, p-cresol,
resorcinol, and tyrosine. The Km for pyrogallol, catechol, and L -DOPA
were 4.5, 8.3, and 6.2 mM, respectively, at 258C. Data for Vmax/Km
values, which represent catalytic efficiency, show that pyrogallol has
the highest value. The optimum pH and temperature were determined
with catechol, pyrogallol, and L -DOPA. Optimum pH was 7.0 for catechol and L -DOPA, and 7.5 for pyrogallol. Optimum temperatures for
maximum PPO activity were 258C for pyrogallol, 408C for catechol,
and 458C for L -DOPA. Heat inactivation studies showed a decrease in
enzymatic activity at temperatures above 608C. The order of inhibitor

*Correspondence: Hulya Yagar, Department of Chemistry, Trakya University, 22030,


Edirne, Turkey; E-mail: hulyagar@yahoo.com.
387
DOI: 10.1081/PB-200031054
Copyright # 2004 by Marcel Dekker, Inc.

1082-6068 (Print); 1532-2297 (Online)


www.dekker.com

Yagar

388
effectiveness was: L -cysteine . ascorbic acid . glycine . resorcinol .
NaCl.
Key Words: Polyphenol oxidase; Celery; Apium graveolens; Isolation;
Characterisation.

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INTRODUCTION
Celery (Apium graveolens L.) is a popular herb and vegetable in Europe
and the Mediterranean region; particularly the roots are often used as a vegetable in Turkey. One of the main problems associated with preserving the
vegetable is the enzymatic browning, which is catalysed by polyphenol
oxidase (PPO). Polyphenoloxidases (EC.1.14.18.1), also referred to as catecholoxidases, are widely distributed in the plant kingdom[1,2] and are known
as the main class of enzymes involved in the browning of damaged fruits
and vegetables.[3] Because the enzymatic browning results in an unpleasant
appearance and concomitant development of off-flavours, it is an economic
problem for producers and consumers.
Polyphenoloxidase is a copper-containing enzyme which catalyses two
reactions. Cresolase reactions catalyse the oxidation of monohydric phenols,
such as tyrosine and o-cresol, to form a hydroxyl group at the ortho position.
Catecholase involves the removal of two hydrogen atoms from an o-diphenol
such as catechol, chlorogenic acid, or 3,4-dihydroxyphenylalanine to form the
corresponding o-quinone. It has been related to enzymatic browning in several
plant tissues, including banana,[4] apple,[5] artichoke,[6] kiwi fruit,[7] edible
burdock,[8] head lettuce,[9] pears,[10,11] palmito,[12] cocoa bean,[13] oil
bean,[14] apricot,[15] quince,[16] Starking apple,[17] cabbage,[18] and tea
leaf.[19]. Little has been reported about celery PPO. We observed that celery
roots have the highest rate of enzymatic browning.
Our objective in this study was to isolate PPO from celery roots and
to study the characteristics of the enzyme at various pHs and temperatures.
Substrate and inhibitor effects were also studied. The enzymatic activity in
the celery products was carried out in the presence of air/oxygen.

EXPERIMENTAL
Isolation of Polyphenoloxidase
Celery roots (Apium graveolens L.) were obtained from the local market
of Edirne, Turkey and were stored at 48C. For the preparation of the crude

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Biochemical Properties of Polyphenol Oxidase from Celery

389

extract, 100 g of celery was cut quickly into thin slices and homogenised with
100 mL of 0.1 M phosphate buffer, pH 7.0, containing 10 mM ascorbic acid
and 0.1% polyvinylpyrrolidone, in a Waring Blender for 3 min. The homogenate was filtered through glass wool and the filtrate was centrifuged at
20,000  g for 30 min at 48C by using a Sanio MS 60 ultracentrifuge. The
supernatant was brought to 20 80% (NH4)2SO4 saturation with solid
(NH4)2SO4, pH 7.0. The precipitated PPO was separated by centrifugation
at 20,000  g for 30 min. The precipitate was dissolved in a small volume
of 0.1 M phosphate buffer, pH 7.0, and dialysed at 48C in the same buffer
for 6 hr with three changes of buffer during dialysis.

Protein Determination
Protein concentration was determined by the method of Lowry, using
bovine serum albumin as an internal standard.[20]

Enzyme Assay
PPO activity was determined by measuring the increase in absorbance at
420 nm (475 nm for L -DOPA) in a spectrophotometer (Shimadzu UV-160 A)
in the presence of air/oxygen. The reaction mixture contained 0.2 mL of
enzyme solution (5.306 mg protein/mL) and 2.8 mL of 0.02 M substrate
solution in 0.05 M phosphate buffer, pH 7.0, at 258C. The blank sample
contained only 3.0 mL of substrate solution. Reaction velocity was computed
from linear slopes of absorbance time curve.[21] One unit of PPO activity
was defined as the amount of enzyme that caused an increase in absorbance
of 0.001 per min.

Substrate Specificity
PPO activity was determined by using six potential substrates (catechol,
pyrogallol, L -DOPA, p-cresol, resorcinol, and tyrosine). All substrate solutions were prepared at 0.02 M in 0.05 M phosphate buffer, pH 7.0. Activity
assays were performed in duplicate measurements, employing the standard
reaction mixture but with different substrate.
Kms and Vmaxs were calculated with the Lineweaver and Burk plot, with
substrate concentration ranging from 0.002 to 0.01 M in the standard reaction
mixture. Substrates examined included catechol, pyrogallol, and L -DOPA.

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Optimum pH and Temperature of Celery PPO


The PPO activity was determined using three different substrates
(catechol, pyrogallol, and L -DOPA) in the pH range of 4 7 0.1 M citrate/
0.2 M phosphate and 0.1 M phosphate, and in a pH range of 7 9 in Tris
HCl buffer. PPO activity was assayed using the standard reaction mixture,
but changing the buffer.
To determine optimum temperature of PPO, the enzyme activity was
measured in the temperature range of 15808C by using various substrates. Substrate solution was heated to the test temperature and the enzyme was added.

Temperature Stability of Celery PPO


The temperature stability of celery PPO was determined by placing
2.0 mL of the enzyme solution in a test tube, in a water bath, at the appropriate
temperatures, which ranged 25 808C. Enzyme solution, 0.2 mL, was withdrawn at appropriate time intervals and rapidly cooled in an ice bath. Then,
the PPO activity was assayed as described above, at pH 7.0 and 258C;
0.02 M catechol solution was used as the substrate solution.

Effect of Inhibitors
PPO activity was measured by using five different inhibitors (ascorbic acid,
cysteine, resorcinol, glycine, and sodium chloride) at the different concentrations with catechol as the substrate. The reaction mixture contained 0.02 M
catechol, 0.1 mL of inhibitor solution at the concentrations that were indicated
(Figs. 5 and 6), and 0.2 mL of enzyme solution. Inhibitor and substrate
solutions were prepared in 0.05 M phosphate buffer, pH 7.0. The percentage
of relative inhibition for each compound was compared with that of the control
(100% activity). The rate of the reaction was computed from the linear portion of
the curve absorbancetime, excluding lag-phases when they occurred.

RESULTS AND DISCUSSION


Celery PPO was partially purified by ammonium sulphate fractionation,
and dialysis. The partial purification resulted in about a threefold increase
in enzyme activity. Thus, in this discussion, it must be pointed out that
results were derived from a study of partially purified enzyme preparations
extracted from ammonium sulphate fractions. So, the properties reported

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Biochemical Properties of Polyphenol Oxidase from Celery

391

herein may be derived from a combination of isoenzymes and interaction with


nonenzymatic proteins. However, the properties of a partially purified
preparation can be a relevant to the food industry as for those of the purified
enzyme.[22]
Table 1 show that the total activity, Km, Vmax, and Vmax/Km data for celery
PPO. The partially purified celery PPO was active toward the o-diphenols:
pyrogallol, catechol, and L -DOPA but was not active toward the phenols:
tyrosine, resorcinol, and p-cresol. No reaction was observed for up to 1 hr
after the enzyme was mixed with tyrosine, resorcinol, and p-cresol. This
indicates that celery PPO has catecholase activity. It has been reported
that pear,[10] cocoa bean,[13] and apricot[15] have similar activities.
As shown in Table 1, the substrate with highest activity was pyrogallol,
followed by catechol and L -DOPA. Apparent affinities toward phenolic substrates shown in Table 1 for celery PPO are 8.3, 6.2, and 4.5 for pyrogallol,
L -DOPA, and catechol, respectively. In terms of physiological efficiency
(Vmax/Km), pyrogallol appeared to be the best substrate.
The Km of the celery PPO for pyrogallol was 4.5 mM as calculated from
the Lineweaver Burk plots (Fig. 1), while that of pyrogallol oxidase from
mandarin orange, oil bean seeds, and edible burdock were 7.1, 3.4, and
1.8 mM, respectively.[8 14]
The pH optimum was between 7.0 and 7.5 for catechol, L -DOPA, and
pyrogallol (Fig. 2). At pH . 9.0, the activity decreased very rapidly, except
for L -DOPA. The PPO system, in fruits, has been shown to be most effective
at neutral pH values.[3] It was reported that the pH optimum of PPO from
some sources also occurs in the range of pH 6.0 8.0. A maximum activity
at pH 7.0 was found in dAnjou pears,[10] burdock,[8] cocoa bean,[13] oil bean
seeds,[14] and Amasya apple.[5] Laurenco reported that the pH optimum of
palmito PPO occurred at 6.6. The optimum pH depends on genetic properties
(variety), nature of phenolic substrates, and extraction methods.[22]
Table 1.
Substrate

Substrate specificity of celery PPO.

Activity
(units/mL)

Km
(mM)

Vmax
(units/mL)

Vmax/Km

290
560
185
NA
NA
NA

8.3
4.5
6.2

400.0
666.6
222.2

48.19
148.13
35.83

Catechol
Pyrogallol
L -DOPA
Tyrosine
Resorcinol
p-Cresole
Note: NA: no activity.

Yagar

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392

Figure 1. Lineweaver Burk plots for celery PPO.

As shown Fig. 2, the various optimum temperatures were determined for


each substrate. The optimum temperature for the maximum PPO activity was
established at pH 7.0 for catechol and L -DOPA, and at pH 7.5 for pyrogallol as
substrate.
Maximum PPO activities were at 258C for pyrogallol, 408C for catechol,
and 458C for L -DOPA. When catechol was the substrate, PPO showed
maximum activity in the temperature range of 20 408C and decreased
gradually with increasing temperatures. Above 608C, PPO showed almost
no activity. When pyrogallol was used as substrate, the results were similar
(Fig. 3). This parameter for cocoa bean,[13] Amasya apple,[5] and burdock
PPO[8] were 458C, 158C, and 608C, respectively.
Temperature-stability profiles for partially purified celery PPO, presented
as the residual percentage activity, are shown in Fig. 4. Celery PPO lost about
70% of the original activity at 508C and about 85% of its activity at 608C for
30 min. These enzymatic properties were sim7ilar to those from palmito,[12]
pear,[10] cocoa bean,[13] and burdock.[8] As expected, at temperatures
.608C, the rate of inactivation was greater with increasing temperature.
However, the enzyme showed relatively high stability. It can be seen that
celery PPO is relatively thermostable.

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Biochemical Properties of Polyphenol Oxidase from Celery

Figure 2.

393

Effect of pH on celery PPO activity.

The effects of five inhibitors, namely NaCl, glycine, resorcinol, ascorbic


acid, and L -cysteine, on celery PPO activity, were studied. The enzyme
activity was markedly inhibited by L -cysteine and ascorbic acid. Catecholase
activity was also inhibited by resorcinol and glycine, but their inhibitory
powers were less affected.
From the data in Fig. 5, L -cysteine was the most effective PPO inhibitor
at a level very similar to that of ascorbic acid. In addition, the higher the
L -cysteine concentration was, the greater the inhibition was observed. It
has been reported that cysteine may react directly with sulfhydryl groups
with the reduction of o-quinone or with the other amino acid residues of
enzyme.[3 13]
The activity of PPO decreased as the concentration of ascorbic acid
increased (Fig. 5). Gomez-Lopez reported that avocado PPO had a similar
inhibitory effect. The mechanism of inhibition by ascorbic acid involves
the reduction of quinones, generated by PPO, back to phenolic compounds,
which arrest brown pigment formation until nearly all ascorbic acid is
depleted.[23] Two other mechanisms of inhibition, involving direct interaction

Yagar

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394

Figure 3.

Effect of temperature on celery PPO activity.

with the enzyme, have been reported, i.e., chelation of copper at the active site
and reduction of Cu to Cu.
As seen from Fig. 6, the PPO activity decreased as the concentration of
glycine and resorcinol increased. The inhibitor effect of glycine on PPO
activity is stronger than with resorcinol and NaCl. The inhibitory effect
of glycine could be explained in atleast two ways: by reacting with the
o-quinones and by chelating the copper at the active site of PPO.[24] Resorcinol added to concentrations as high as 100 mM was able to inhibit celery PPO
about 50%. Resorcinol resembles the structure of the substrates for PPO; these
types of inhibitors are typically competitive.
The activity of celery PPO decreased 10% for celery PPO, when using
0.8 M sodium chloride. No additional tests were done at the other NaCl concentrations because of the low inhibition achieved at the high concentration.

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Biochemical Properties of Polyphenol Oxidase from Celery

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Figure 4. Thermal stability profile of celery PPO. The enzyme solution was incubated
for various time intervals (5 30 min) at the specified temperature (25 808C) and
rapidly cooled. The activity was measured at 258C, was taken as 100%, and activities
which were measured (35 808C) were compared with the activity measured at 258C.

Figure 5.

The inhibitory effects of ascorbic acid and cysteine on celery PPO.

Yagar

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396

Figure 6.

The inhibitory effects of glycine and resorcinol on celery PPO.

From the data, it may be concluded that the inhibitory effect of NaCl was not
satisfactory. It is believed that the action of NaCl is due to the formation of a
complex between the halide ion and copper in the enzyme. Several works have
been reported on the effect of NaCl on PPO, in which a high inhibitor concentration was necessary to achieve inhibition. It was shown that the inhibition of
eggplant by 0.99 M sodium chloride was 90%, and inhibition from avocado by
0.8 M sodium chloride was 37%.[25]

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Received April 24, 2004
Accepted May 31, 2004
Manuscript 7401