Differential activation of a latent polyphenol oxidase mediated by sodium dodecyl sulfate

Article abstract of DOI:10.1021/jf050505e

A kinetic study of the activity of soluble and membrane-bound latent polyphenol oxidase (PPO) extracted from beet root (Beta vulgaris) was carried out. For the first time, two types of behavior (hyperbolic and sigmoid) are reported in the same enzyme for

Full text of DOI:10.1021/jf050505e

J. Agric. Food Chem. 2005, 53, 6825−6830 6825
Differential Activation of a Latent Polyphenol Oxidase Mediated
by Sodium Dodecyl Sulfate
FERNANDO GANDIÄA-HERRERO, MERCEDES JIMEÄ NEZ-ATIEÄ NZAR, JUANA CABANES,
FRANCISCO GARCIÄA-CARMONA, AND JOSEFA ESCRIBANO*
Departamento de Bioqu´ımica y Biolog´ıa Molecular A, Unidad Docente de Biolog´ıa, Facultad de
Veterinaria, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain
A kinetic study of the activity of soluble and membrane-bound latent polyphenol oxidase (PPO)
extracted from beet root (Beta vulgaris) was carried out. For the first time, two types of behavior
(hyperbolic and sigmoid) are reported in the same enzyme for PPO activation by the surfactant sodium
dodecyl sulfate (SDS), depending on substrate nature. A kinetic model based on cooperative systems
is developed to describe the activation effect of SDS, enabling the determination of the number of
surfactant molecules binding to the enzyme in the activation process. The results indicate that the
active site of the enzyme is not affected by SDS and that a stepwise conformational change favors
the access of hydrophobic substrates compared to hydrophilic ones. Differential activation of PPO
mediated by SDS may be of relevance in the control of PPO activity since the enzyme is able to
express activity toward a specific substrate while remaining latent to others.
KEYWORDS: Polyphenol oxidase; tyrosinase; latency; activation; sodium dodecyl sulfate; Beta vulgaris;
betalains
INTRODUCTION
The implication of PPO in the secondary metabolism of
betalains has been proposed in plants belonging to the order
Caryophyllales (11-13), like beet (Beta Vulgaris). Interest in
betalains has grown since their antiradical activity was char-
acterized (14-16) and they are widely used as additives in the
food industry because of their natural colorant properties and
absence of toxicity, even at high concentrations (17, 18).
Polyphenol oxidase, PPO (monophenol, o-diphenol:oxygen
oxidoreductase; EC 1.14.18.1) is a copper-containing enzyme
that catalyzes two different reactions using molecular oxygen:
the hydroxylation of monophenols to o-diphenols (monophe-
nolase activity) and the oxidation of the o-diphenols to o-
quinones (diphenolase activity) (1, 2). This enzyme has been
found in microorganisms, animals, and plants, and thus PPO is
responsible not only for browning in plants but also for
melanization in animals. In the plant kingdom PPO has been
detected in most fruits and vegetables and is predominantly
located in the chloroplast thylakoid membranes (3-6). However,
it is not an intrinsic membrane protein and it can be released
from the thylakoids by sonication, mild detergent treatment, or
proteases. The enzyme has also been detected in soluble
fractions in homogenates from different vegetables (7).
PPO is a very important enzyme in the food industry since
during the processing of fruits and vegetables any wounding
may cause cell disruption and lead to quinone formation. The
appearance of food and beverages may be affected but also the
taste and nutritional value, often decreasing the quality of the
final product (8, 9). Because of the considerable economic and
nutritional loss induced by enzymatic browning in the com-
mercial production of fruits and vegetables, numerous studies
have been devoted to the biochemical and catalytic properties
of PPO (7, 10).
One unusual and intriguing characteristic of this enzyme is
its ability to exist in an inactive or latent state (19, 20). PPO
can be released from latency, or activated, by a variety of
treatments or agents, including acid and base shock (21, 22),
anionic detergents, such as sodium dodecyl sulfate (SDS) (5,
19, 23), proteases (3, 24), and fatty acids (25).
The use of SDS is of particular interest since few enzymes
are known to be activated by this detergent, while many are
inactivated. Thus PPO is active at high SDS concentrations (19),
which would denature many other enzymes. The activation
process by SDS detergent was found to involve a reorganization
of protein tertiary structure (19, 26, 27). A limited conforma-
tional change due to binding of small amounts of SDS may
induce or initiate the activation of the latent enzyme, and it was
reported that the low optimum pH obtained for the latent enzyme
was abolished in the presence of SDS. The possible relationship
between these two activating factors (SDS and pH) has been
studied in broad bean, grape, and lettuce PPOs (5, 28-30).
Furthermore, the reversibility of the SDS-mediated activation
of latent PPO from peach has been demonstrated by use of
cyclodextrins as an agent able to trap SDS molecules (31). With
respect to the physiologically relevant counterpart of the
* Corresponding author: phone +34 968 364762; fax +34 968 364147;
e-mail pepa@um.es.
10.1021/jf050505e CCC: $30.25
© 2005 American Chemical Society
Published on Web 07/28/2005

6826 J. Agric. Food Chem., Vol. 53, No. 17, 2005
Gand´ıa-Herrero et al.
detergent, it has been suggested that lipids might fulfill this role
(32), and SDS activation of tyrosinase has been demonstrated
in vivo with Terfezia claVeryi sections (33). However, a kinetic
study of the PPO activation by SDS depending on substrate
nature has not been carried out.
Thus, the aim of this paper was to study the variations in the
kinetic behavior of the latent beet root PPO through the
activation process mediated by SDS, by use of different types
of substrates. For the first time, we report a differential activation
of PPO by SDS depending on the substrate nature. This kinetic
behavior is modeled and interpreted in terms of a conformational
change that modifies the access of the substrates to the active
site of the enzyme but does not affect the structure of the active
site itself.
and diphenolic substrates. The selected compounds were the
L-tyrosine/L-DOPA and tyramine/dopamine pairs along with the
hydrophobic diphenols catechol, 4MC, and 4tBC. The in-depth
characterization of the enzymatic activation was carried out on
the latent PPO obtained from soluble and membrane fractions
of red beet root. The effect of the anionic detergent SDS has
been studied in relation to the surfactant concentration, pH, and
the nature of the substrate used to follow the enzymatic activity.
pH Study and Degree of Activation of PPO by SDS. pH
is a determining factor in the expression of enzymatic activity;
it alters the ionization states of amino acid side chains or the
ionization of the substrate. PPO activity was measured, at
different pH values, in the absence or presence of 0.69 mM
SDS. At this detergent concentration the enzyme was fully
active, as later demonstrated, and the concentration is below
the cmc (critical micelle concentration) for the detergent at the
experimental conditions (19, 38).
MATERIALS AND METHODS
Plant Material. Beta Vulgaris roots (granadina variety) were grown
in an ecological plantation with no addition of pesticides. The roots
were harvested, sliced, frozen in liquid nitrogen, and stored at -80 °C
until use.
A pH variation in the absence and presence of SDS was
carried out on the soluble PPO for all the selected substrates
(Figure 1). In the absence of SDS, almost no activity was
detected at acid pH values for most of the substrates (Figure
1) with the exception of the more hydrophobic ones, 4MC
(Figure 1E) and 4tBC (Figure 1F). All the substrates showed
an optimum pH over pH 5.5 under these conditions.
Chemicals. PPO substrates and SDS were obtained from Sigma
(Barcelona, Spain). All other chemicals were of analytical grade.
Subcellular Fractionation. Soluble and membrane fractions were
obtained from red beet root slices, as previously described (34). Briefly,
a beet root homogenate was centrifuged at 1000g for 10 min. The pellet,
containing the wall fraction, was discarded, and the supernatant was
centrifuged at 120000g for 40 min. The supernatant was considered as
the soluble fraction, and the pellet, the membrane fraction. The soluble
fraction was brought up to 35-85% (NH4)2SO4. The salt content was
removed by dialysis against 10 mM sodium phosphate buffer, pH 7.0.
The extraction of PPO from the membrane fraction was carried out
through treatment with Triton X-114 (34). To avoid possible activation
of the PPO enzyme by endogenous proteases, PMSF (phenylmethane-
sulfonyl fluoride) and benzamidine hydrochloride were added before
and after the dialysis to give a final concentration of 1 mM.
Spectrophotometric Assays. Monophenolase and diphenolase ac-
tivities were determined spectrophotometrically at 25 °C by measuring
the appearance of reaction products in the medium. Catechol, 4-me-
thylcatechol (4MC), and 4-tert-butylcatechol (4tBC) oxidations were
followed by the appearance of the o-benzoquinone product (ꢀ ) 1450,
1350, and 1150 M-1 cm-1, respectively) at 390 nm (catechol) and 400
nm (4MC and 4tBC). Aminechrome formation for L-tyrosine and L-3,4-
dihydroxyphenylalanine (L-DOPA) (ꢀ ) 3600 M-1 cm-1) was moni-
tored at 475 nm, and for tyramine and dopamine (ꢀ ) 3300 M-1 cm-1),
at 480 nm (35). SDS studies were performed in 50 mM sodium
phosphate buffer, pH 6.5. Other conditions are detailed in the text and
figure captions. The percentage of enzyme activation was calculated
as % activation ) [(activity - latent activity)/(maximum activity -
latent activity)] × 100. Spectrophotometric measurements were per-
formed in a Kontron Uvikon 940 spectrophotometer (Kontron Instru-
ments, Zurich, Switzerland).
In the presence of SDS, the optimum pH value was the same
for the monophenol as for the corresponding diphenol, and this
value did not change in the absence of SDS. Thus, the optimum
pH was 6.0 for the L-tyrosine/L-DOPA pair (Figure 1B,C) and
6.5 for tyramine (Figure 1A) and dopamine. The activation
degree reached in the presence of SDS was 4-fold for L-tyrosine
and tyramine, whereas for L-DOPA and dopamine it was 2-fold.
For monophenols, the characteristic lag period (2) was also
affected by the pH and a decrease could be observed when pH
was increased, in both the absence and presence of SDS in the
reaction medium (results not shown). With respect to hydro-
phobic substrates, the enzyme exhibited an optimum pH of 5.5
toward 4MC and 4tBC (Figure 1E,F). For catechol, the
optimum pH observed was 7.0 (Figure 1D). A significant
difference in the degree of activation by SDS was not found
(around 2-fold) for these substrates. Thus the activation degree
was higher for the enzyme acting on monophenols than on
diphenols.
The pH profiles for all substrates studied in PPO from the
membrane fraction were similar to those obtained with the
soluble enzyme. However, the degree of activation changed
dramatically. Beet root PPO bound to membrane was fully
latent. In all cases the degree of activation was higher than 100-
fold, in contrast to the less than 4-fold activation found for the
soluble PPO. Figure 2 shows the pH profiles obtained for the
most hydrophobic substrate, 4tBC (Figure 2A), and for the most
hydrophilic L-tyrosine/L-DOPA pair (Figure 2B, inset). It is
noticeable that, in the absence of SDS, the membrane PPO
showed activity at acidic pH values (optimum pH ) 4.5).
However, this effect was not observed in the soluble PPO that
was not totally latent (Figure 1C,F). Other authors (5, 19, 21,
23) have proposed that PPO activity can be induced by acid
shocking, depending on the enzyme source. The presence of
SDS eliminated the low optimum pH.
Experiments were performed in triplicate and the mean and standard
deviations were plotted.
Kinetic Data Analysis. The values of Km and Vm on different
substrates were calculated from measurements of the steady-state rate,
Vss, which was defined as the slope of the linear portion of the product
accumulation curve. The lag period was estimated as the intercept on
the abscissa axis obtained by extrapolation of the linear portion. Kinetic
data analysis was carried out by linear and nonlinear regression fitting
(36), by use of SigmaPlot Scientific Graphing for Windows version
8.0 (2001; SPSS Inc., Chicago, IL).
Protein Determination. Protein concentration was determined
according to the Bradford Bio-Rad (Hercules, CA) protein assay with
serum albumin as standard (37).
Differences in the pH profile found for beet root PPO
depending on the presence of SDS for different substrates
suggest that the activating effect of the detergent may be related
to substrate nature. Therefore, the subsequent experiments were
carried out to elucidate the mechanism for SDS activation.
RESULTS AND DISCUSSION
The activation process of beet root PPO mediated by the
anionic detergent SDS was studied with several monophenolic

Latent Polyphenol Oxidase Mediated by SDS
J. Agric. Food Chem., Vol. 53, No. 17, 2005 6827
Figure 1. Effect of pH on the soluble PPO in 50 mM sodium acetate or sodium phosphate buffer (pH 3.5
−7.5) in the presence (
O) or absence (b) of
0.69 mM SDS. The reaction medium at 25
mM 4MC (E), 15.0 mM 4tBC (F), and 5
°
C contained 3.0 mM tyramine (A), 0.75 mM tyrosine (B), 3.8 mM -DOPA (C), 80.0 mM catechol (D), 10.0
L
µ
g/mL soluble PPO for diphenols and 50 µg/mL for monophenols.
Figure 2. Effect of pH on the membrane-bound PPO in 50 mM sodium
acetate or sodium phosphate buffer (pH 3.5 7.5) in the presence ( ) or
absence ( ) of 0.69 mM SDS. The reaction medium at 25 C contained
12.4 g/mL PPO extracted with TX-114 and 15.0 mM 4tBC (A), 3.8 mM
-DOPA (B), or 0.75 mM tyrosine (B, inset).
Figure 3. Percentage of soluble PPO activation at different SDS
concentrations in the presence of catechol ( ), 4MC ( ), or 4tBC (
(A) or -tyrosine ( ), -DOPA ( ), tyramine ( ), or dopamine ( ) (B).
The reaction medium at 25 C contained 50 mM sodium phosphate, pH
−
O
1
0
3
2)
b
°
L
O
L
b
9
µ
°
L
6.5, 5 mg/mL soluble PPO for diphenols and 50 mg/mL for monophenols,
Effect of SDS Concentration Depending on the Substrate
and 80.0 mM catechol, 10.0 mM 4MC, or 15.0 mM 4tBC (A) or 0.75 mM
Nature. The effect of SDS concentration on the enzymatic
activity at a saturating concentration for each substrate was
studied. The soluble PPO activity showed hyperbolic behavior
with the hydrophobic substrates catechol, 4MC, and 4tBC
tyrosine, 3.8 mM L-DOPA, 3.0 mM tyramine, or 5.0 mM dopamine (B).
(Figure 3A). However, PPO was activated in a sigmoidal
manner with increasing SDS concentrations toward the L-

6828 J. Agric. Food Chem., Vol. 53, No. 17, 2005
Gand´ıa-Herrero et al.
tyrosine/L-DOPA and tyramine/dopamine pairs (Figure 3B). The
lag period observed in the monophenolase activity was also
modified by the presence of SDS, reaching a minimum at SDS
concentrations over 0.5 mM, at which 100% activation was
observed (results not shown).
In the presence of hydrophobic substrates, the curvature of
the hyperbole was related to the degree of hydrophobicity (4tBC
> 4MC > catechol). Furthermore, the maximum activation for
the hydrophobic substrates was reached at low SDS concentra-
tions (near 0.1 mM) at which the degree of activation was
negligible for the hydrophilic substrates tyramine, dopamine,
L-tyrosine, and L-DOPA (Figure 3B). Therefore, as can be
observed in Figure 3, for the soluble PPO, the behavior of the
enzyme activation and the SDS concentration at which maxi-
mum activity was reached was different for each substrate.
The same effect could be observed for the PPO solubilized
from the membrane fraction. Figure 4 shows the activity of
PPO toward the physiological substrate, L-DOPA, and toward
the most hydrophobic, 4tBC, at different SDS concentrations.
Because PPO from the membrane fraction was fully latent, at
low SDS concentrations (0.1 mM) the enzyme activity was
practically null against L-DOPA, whereas the enzyme showed
maximum activity with 4tBC. Therefore, similar behavior was
found for both the membrane and soluble fractions. These results
strongly suggest that the conformational change needed for the
enzyme to express its maximum activity toward a substrate is
dependent on the substrate nature, with the binding of more
SDS molecules (and higher SDS concentrations) for the more
hydrophilic substrates being necessary.
Figure 4. Percentage of membrane PPO activation at different SDS
concentrations in the presence of 4tBC (
medium at 25 C contained 50 mM sodium phosphate, pH 6.5, 12.4 mg/
mL PPO extracted with TX-114, and 15.0 mM 4tBC or 3.8 mM -DOPA.
b) and L-DOPA (O). The reaction
°
L
the Hill coefficient (nH) relates to the number of SDS molecules
binding to the original latent enzyme:
max[A]n
H
V
V )
KH + [A]nH
nH
According to the proposed scheme, the presence of enzyme
forms with partial activity before the maximum activation for
each substrate is reached implies a lowering of the Hill
coefficient and the possibility of obtaining nonnatural values.
Thus, nH calculated from activity versus [SDS] graphs indicates
the minimum number of SDS molecules needed for the total
activation of the enzyme.
For hydrophobic substrates (which show a hyperbolic be-
havior), nH was 1, indicating that the enzyme is active toward
these molecules since the first SDS monomer is bound to its
surface at very low SDS concentrations. For hydrophilic
substrates, the sigmoidal curve is described by nH values ranging
from 2.2 to 6.1. For soluble PPO, the values of nH were 2.2
(tyramine), 2.7 (dopamine), 3.9 (L-tyrosine), and 4.2 (L-DOPA),
indicating that the number of SDS molecules needed for
activation is higher for more hydrophilic substrates. In the case
of the membrane-bound PPO, more SDS molecules were
necessary to show the maximum activity toward the most
hydrophilic substrates. The Hill coefficients determined were
3.5 (L-tyrosine), 3.7 (dopamine), and 6.1 (L-DOPA).
Model for PPO Differential Activation Mediated by SDS.
A general kinetic model can be suggested to explain the
differential activation of PPO depending on substrate nature
described for beet root. In the absence of SDS the enzyme
remains in a latent state with null (membrane fraction) or limited
(soluble fraction) activity. The mechanism can be described by
the following scheme, which is analogous to the model of
Koshland et al. (39):
k0
E + S T ES
98 E + P
1
k1
E + A 7K 8 EA + S T EAS
98 EA + P
A
2
k2
EA + A 7K 8 EA2 + S T EA2S
98 EA2 + P
A
Differences in the number of SDS monomers needed for
activating the enzyme (nH) make the differential activation of
latent PPO possible. These results corroborate that the ability
of SDS to activate the enzyme involves a limited conformational
change due to the binding of small amounts of SDS (19). The
access of hydrophobic substrates to the active site is favored
since the first molecules of SDS are bound to the enzyme, while
hydrophilic substrates require a deeper change for full access
(activity).
Differential activation of PPO mediated by SDS, which would
play an equivalent role to the physiological one fulfilled by lipids
in vivo (32), enables activity toward a substrate while the same
enzyme is latent to others. This may be of physiological
relevance, mainly for the membrane-bound form of PPO. The
data collected in the present study and the kinetic model
proposed are in agreement with differential activation and
positive cooperativity induced by SDS.
3
k3
EA2 + A 7K 8 EA3 + S T EA3S
98 EA3 + P
A
‚
‚
‚
n
EAn-1 + A 7K 8 EAn + S T EAnS
8 EAn + P
kn
9
A
The positive kinetic cooperativity observed in the steady-
state rate is due to the fact that the enzyme (E) undergoes
conformational changes toward a more active form (EAn)
induced by the binding of discrete molecules of the activator
(A). The SDS molecules are as monomers, since the detergent
concentration is below the cmc under the assay conditions. For
the same enzyme, the active form and thus the number of SDS
molecules (n) needed to express the maximum activity varies
depending on the nature of the substrate (S). Thus, a stepwise
mechanism for activation allows the existence of PPO activity
toward a substrate while the enzyme remains latent to others.
The Hill equation fits the suggested mechanism, as demonstrated
in the model of Koshland et al. (39), and for SDS activation,
Effect of SDS on Enzyme Kinetics. The effect of SDS on
the kinetic parameters was determined in order to characterize
in detail the behavior of the enzyme toward each substrate. To

Latent Polyphenol Oxidase Mediated by SDS
J. Agric. Food Chem., Vol. 53, No. 17, 2005 6829
monomers may constitute the basis of the differential activation.
Thus, the existence of a regulatory peptide not related to the
affinity of the enzyme fits with the data obtained in the present
study. Its presence can be assumed in relation to the “shield
region” described in hemocyanins to block the access of
phenolic compounds, and also proposed in the structural model
of a latent catechol oxidase (41, 42).
The results of this work regarding the differences in the pH
profile of PPO according to the presence of SDS, the behavior
of the enzyme activation for different substrates, and the SDS
concentration at which maximum activity was reached support
the existence of differential activation of PPO mediated by the
surfactant SDS, depending on the substrate nature. The presence
of sigmoidal or hyperbolic behavior in the activation process
of the same latent enzyme is reported. For the first time, the
conformational change provoked by the detergent is described
by a stepwise kinetic model based on cooperativity, which
allows the determination of the number of SDS molecules
binding to PPO in the activating process. The kinetic scheme
proposed might shed light on the mechanisms that regulate the
enzyme activity in vivo, since it describes how PPO is able to
express activity toward a specific substrate while remaining
latent to others.
Table 1. Kinetic Parameters for Soluble and Membrane-Bound PPO
Activitya
-
1
substrate
SDSb
Km (mM)
Vm (mM/min)
Vm/Km (min
)
optimum pH
Soluble Enzyme
-
-
3
3
-3
L
-tyrosine
−
+
−
+
−
+
−
+
−
+
−
+
−
+
2.79
1.98
2.48
2.32
0.27
0.35
0.34
0.35
8.94
7.77
1.85
1.60
6.59
4.04
2.73
8.73
×
×
10
10
0.98
4.41
×
×
10
10
6.0
6.0
5.5
6.0
6.5
6.5
6.5
6.5
7.0
7.0
5.0
5.5
5.5
5.5
-
3
L-DOPA
0.101
0.200
0.041
0.086
4.59 × 10
-
-
3
3
-
3
tyramine
dopamine
catechol
4MC
1.24
4.32
×
×
10
10
0.012
0.242
0.559
0.031
0.071
0.102
0.221
0.031
0.095
0.082
0.193
0.275
0.550
0.189
0.353
0.206
0.385
4tBC
Membrane-Bound Enzyme
-
3
-
3
L
-tyrosine
-DOPA
+
+
+
+
1.08
2.21
0.33
3.95
1.85
×
10
1.72
×
10
6.0
6.0
6.5
6.0
L
0.077
0.082
0.161
0.035
0.247
0.041
dopamine
4tBC
a All assays were performed in reaction media containing different substrate
concentrations in 50 mM sodium phosphate buffer, pH 6.5 at 25 C. The Vm values
are normalized with the same protein concentration (12.4
g/mL). b When SDS
°
µ
LITERATURE CITED
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Received for review March 7, 2005. Revised manuscript received June
8, 2005. Accepted June 23, 2005. The present work was supported by
two grants from the Ministerio de Ciencia y Tecnolog´ıa (Spain) and
FEDER (Project AGL2003-05647) and the Fundacio´n Se´neca, Conse-
jer´ıa de Agricultura, Agua y Medio Ambiente (Spain) and FEDER
(Project AGR/11/FS/02). F.G.-H. holds a fellowship from the Fundacio´n
Se´neca (Spain).
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