Kinetic characterization of the oxidation of chlorogenic acid by polyphenol oxidase and peroxidase. Characteristics of the o-quinone

Article abstract of DOI:10.1021/jf062081+

Chlorogenic acid is the major diphenol of many fruits, where it is oxidized enzymatically by polyphenol oxidase (PPO) or peroxidase (POD) to its o-quinone. In spectrophotometric studies of chlorogenic acid oxidation with a periodate ratio of [CGA]₀/[lO₄^-]₀ < 1 and [CGA]₀/[IO₄^-]₀ > 1, the o-quinone was characterized as follows: λ_max at 400 nm and ε = 2000 and 2200 M^-1 cm^-1 at pH 4.5 and 7.0, respectively. In studies of o-quinone generated by the oxidation of chlorogenic acid using a periodate at ratio of [CGA]₀/[lO₄^-]₀ > 1, a reaction with the remaining substrate was detected, showing rate constants of k = 2.73 ± 0.17 M^-1 s^-1 and k = 0.05 ± 0.01 M^-1 s^-1 at the above pH values. A Chronometric spectrophotometric method is proposed to kinetically characterize the action of the PPO or POD on the basis of measuring the time it takes for a given amount of ascorbic acid to be consumed in the reaction with the o-quinone. The kinetic constants of mushroom PPO and horseradish POD are determined.

Full text of DOI:10.1021/jf062081+

920 J. Agric. Food Chem. 2007, 55, 920−928
Kinetic Characterization of the Oxidation of Chlorogenic Acid
by Polyphenol Oxidase and Peroxidase. Characteristics
of the o-Quinone
J. MUN˜ OZ,† F. GARCIA-MOLINA,† R. VARON,§ J. N. RODRIGUEZ-LOPEZ,†
†
P. A. GARC´ıA-RUIZ,# F. GARC´ıA-CAÄ NOVAS,*,† AND J. TUDELA
GENZ: Grupo de Investigacio´n de Enzimolog´ıa, Departamento de Bioqu´ımica y Biolog´ıa
Molecular-A, Facultad de Biolog´ıa, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain;
Departamento de Qu´ımica-F´ısica, Escuela Polite´cnica Superior, Universidad de Castilla la Mancha,
Avenida Espan˜a s/n, Campus Universitario, E-02071 Albacete, Spain; and Departamento de Quimica
Orga´nica, Facultad de Quimica, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain
Chlorogenic acid is the major diphenol of many fruits, where it is oxidized enzymatically by polyphenol
oxidase (PPO) or peroxidase (POD) to its o-quinone. In spectrophotometric studies of chlorogenic
acid oxidation with a periodate ratio of [CGA]0/[IO4-]0 < 1 and [CGA]0/[IO4-]0 > 1, the o-quinone was
characterized as follows: λmax at 400 nm and ꢀ ) 2000 and 2200 M-1 cm-1 at pH 4.5 and 7.0,
respectively. In studies of o-quinone generated by the oxidation of chlorogenic acid using a periodate
at ratio of [CGA]0/[IO4-]0 > 1, a reaction with the remaining substrate was detected, showing rate
constants of k ) 2.73 ( 0.17 M-1 s-1 and k′ ) 0.05 ( 0.01 M-1 s-1 at the above pH values. A
chronometric spectrophotometric method is proposed to kinetically characterize the action of the PPO
or POD on the basis of measuring the time it takes for a given amount of ascorbic acid to be consumed
in the reaction with the o-quinone. The kinetic constants of mushroom PPO and horseradish POD
are determined.
KEYWORDS: Chlorogenic acid; polyphenol oxidase; peroxidase; ascorbic acid; o-quinone; molar
absorptivity
INTRODUCTION
oxidize CGA at different velocities of catalysis and with
different affinities (5).
Chlorogenic acid (CGA) is the major phenolic compound
found in many fruits, including apples, pears, cherries, and
apricots (1). The product resulting from the enzymatic oxidation
of CGA, the o-quinone (CGA-Q), has been reported to oxidize
other polyphenols, such as flavans, by a coupled oxidation
reaction (1). The enzymatic oxidation of polyphenols is
important because polyphenols are the precursors of several
biosynthetic pathways, including those of tannins, lignins, and
melanins (2).
The product formed by the enzymatic oxidation of CGA by
PPO and POD is CGA-Q, directly in the case of PPO (6) and
through the disproportion reaction of the semiquinone in the
case of POD (7).
The evolution of CGA-Q has been studied by several authors
(6-9). For example, it has been shown that CGA-Q generates
H2O2 in the medium and CGA (7, 8), which suggests that POD
may be involved in the process of enzymatic browning, the
enzyme working at the expense of the H2O2 generated by PPO
in its action on CGA (7). The production of H2O2 in the
melanogenesis pathway associated with L-dopa and dopamine
oxidation has also been revealed (10).
However, the great instability of the CGA-Q has hindered
its characterization. Very low absorptivity coefficients on the
order of 970 M-1 cm-1 have been proposed (8), as have several
pathways for CGA-Q evolution from the addition of water to
generate a trihydroxylated compound, which would be oxidized
by another molecule of CGA-Q (6, 9), to the reaction of CGA-Q
with molecules of substrate to generate polymers (9).
Spectrophotometric measurements of PPO or POD enzymatic
activity on CGA may give erroneous results, precisely because
of the instability of the CGA-Q, for which reason several authors
The browning damage caused to the tissues of fruits and
vegetables during postharvest handling and processing is one
of the main causes of quality loss (3). The main enzymes
involved in these processes are polyphenol oxidase (EC
1.14.18.1; PPO) and peroxidase (EC 1.11.1.7; POD) (4).
Because CGA is the major component of many fruits, its
enzymatic oxidation has attracted much attention. For example,
it has been demonstrated that tobacco leaf and mushroom PPOs
* Corresponding author (fax +34 968 363963; e-mail canovasf@um.es).
† Departamento de Bioqu´ımica y Biolog´ıa Molecular-A, Universidad de
Murcia.
§ Departamento de Qu´ımica-F´ısica, Universidad de Castilla la Mancha.
# Departamento de Quimica Orga´nica, Universidad ded Murcia.
10.1021/jf062081+ CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/17/2007

Oxidation of Chlorogenic Acid by PPO and POD
J. Agric. Food Chem., Vol. 55, No. 3, 2007 921
Figure 3. Representation of absorbance versus concentration of CGA-
Figure 1. Representation of the rate of oxidation of CGA by PPO and
POD. The velocity was monitored by measuring the variation in absorbance
at 400 nm. Experimental conditions: phosphate buffer, 30 mM, pH 7.0;
Q. Values of absorbance λ ) 400 nm were obtained by oxidation with
-
periodate at ratios of [CGA]0/[IO4
]0 < 1. Experimental conditions: acetate
buffer, 30 mM, pH 4.5; phosphate buffer, 30 mM, pH 7.0. The concentration
acetate buffer, 30 mM, pH 4.5, at 25 °C; [POD] ) 2 nM; [H2O2]0 ) 0.25
mM. (Inset) To measure the rate of oxidation of CGA by PPO at the
-
of [IO4
]
was 1.5 mM, and CGA concentration was the same as
0
CGA-Q.
same pH values, the experimental conditions were the same except [PPO]0
)
35 nM.
Scheme 1
or polarographic (monitoring oxygen consumption using a
Wilson oxygraph equipped with a Clark electrode) (15). The
results described above can be explained by the instability of
the CGA-Q.
Because CGA is the major compound in many fruits, several
methods have been designed to monitor PPO activity, sometimes
termed chlorogenic acid oxidase, measuring the disappearance
of chlorogenic acid at 325 nm (1). A method based on
differential spectrophotometry has been designed for PPO,
whereby the reaction is followed in the ultraviolet wavelength
range during enzymatic polyphenol oxidation (16). Although
measurements obtained in this way closely agree with the
measurements of oxygen uptake done with the manometric
method, the method can be applied only at low concentrations
of CGA (16).
The study of CGA and its products is important because of
the abundance of this compound: for example, 3.4-14 mg/
100 mg of fresh weight in potatoes, 12-31 mg/100 mL in apple
juice, 89 mg /100 mg in dry tea shorts, and 250 mg per cup of
coffee (17, 18). Furthermore, it has been attributed anticarci-
nogenic (19, 20), antimutagenic (21), and antiinflammatory (22)
properties. Recently, too, the antinitrosating properties of CGA,
which serves as an efficient nitrite scavenger and a suppresser
of nitrosamine formation in the gastric compartment (23-25),
have also been emphasized. The formation of CGA adducts with
glutathiones has also been characterized (26).
-
Figure 2. Oxidation of CGA by periodate at ratios of [CGA]0/[IO4
] < 1.
0
-
Experimental conditions: [CGA]0
)
0.15 mM; [IO4
]
0
)
2.25 mM; acetate
buffer, 30 mM, pH 4.5. (Right inset) Oxidation in phosphate buffer, 30
2.25 mM. The measurements
were made every 60 s. (Left inset) To measure the evolution of the CGA-Q
-
mM, pH 7.0, [CGA]0
)
0.11 mM; [IO4
]
0
)
-
generated by oxidation with periodate at ratios of [CGA]0/[IO4
] < 1, the
0
experimental conditions were as follows: phosphate buffer, 30 mM, pH
-
7.0; [IO4
] ) 7.5 mM; and [CGA]0 was varied [(a) 0.3 mM; (b) 0.45 mM;
0
(c) 0.6 mM; (d) 0.75 mM; (e) 0.9 mM].
In this work, we study some of the properties of CGA-Q and
some kinetic characteristics of its evolution; we also propose a
chronometric spectrophotometric measurement method that
kinetically characterizes the enzymes responsible for its oxida-
tion, including PPO and POD.
have proposed kinetic mechanisms of inhibition by excess of
substrate (1, 2, 11-14). In the same way, studies involving the
inhibition of mushroom PPO using CGA as substrate have
concluded that the type and degree of inhibition depend not
only on the inhibitor but also, and especially, on the method
used to measure the enzymatic activity, which may be spec-
trophotometric (following the increase in absorbance at 420 nm)
MATERIALS AND METHODS
Materials. Chlorogenic acid, mushroom tyrosinase (3320 units/mg),
and horseradish peroxidase (HRP isoenzyme C type IV, 280 units/mg)

922 J. Agric. Food Chem., Vol. 55, No. 3, 2007
Mun˜oz et al.
were obtained from Sigma Chemical Co. H2O2 was obtained from
Scharlau (Madrid, Spain). The substrate was prepared in dilute
phosphoric acid.
Other Methods. Protein was determined according to Bradford’s
method using bovine serum albumin as standard (27).
Determination of Enzymatic Activity. Enzymatic activity was
determined spectrophotometrically using the method described under
Results and Discussion. The only condition is that there must be an
absorbance increase (due to product formation) that can be measured
once the ascorbic acid is consumed. Preliminary experiments were
carried out in the absence of ascorbic acid with the PPO/O2/CGA and
POD/H2O2/CGA systems, recording spectrophotometric scans to iden-
tify in which zone of the spectrum, particularly the visible zone, the
greatest changes in absorbance occurred. The wavelength chosen at
both acid and neutral pH was 400 nm. Peroxidase activity was followed
in the same way.
Kinetic Data Analysis. Initial rate values (V0) were calculated from
triplicate measurements at each reducing substrate concentration. The
reciprocals of the variances of V0 were used as weighting factors in
the nonlinear regression fitting of V0 versus [CGA]0 data to the
Michaelis-Menten equation. The fitting was carried out using Mar-
quardt’s algorithm (28) implemented in the Sigma Plot 9.0 program
for Windows (29).
NMR Assays. 13C NMR spectra of chlorogenic acid were recorded
at pH 4.5 and 7.0 on a Varian Unity spectrometer (300 MHz) using
2H2O as solvent. Chemical shift values (δ) were determined relative to
those of tetramethylsilane (δ ) 0). The maximum accepted error for
each peak was (0.1 ppm.
RESULTS AND DISCUSSION
Enzymatic Oxidation of Chlorogenic Acid. CGA is the
main acid found in fruits, including apples and pears (1).
Peroxidase catalyzes the oxidation of the o-diphenols to
semiquinones, which disproportionate to generate the corre-
sponding o-quinone, regenerating substrate at the same time
(30). The enzymatic oxidation of CGA by POD/H2O2 and PPO/
O2 transforms the substrate into one product with a maximum
at 400 nm. CGA is oxidized by PPO to its corresponding
o-quinone (CGA-Q), which shows a maximum at 400 nm (8).
When the enzymatic reaction is followed at this wavelength,
during the oxidation of CGA both with POD/H2O2 and with
PPO/O2, the results shown in Figure 1 and its inset are obtained.
The explanation of the results shown in Figure 1 has been given
as an inhibition by excess of substrate (1, 2, 11-14). However,
the characteristics of o-quinones described in other works (31)
led us to look at the instability of the CGA-Q and its possible
control to enable the kinetic characterization of this important
substrate. For this, we carried out the following series of
experiments.
Oxidation of CGA by Sodium Periodate. It is known that
sodium periodate oxidizes different o-diphenols in a way similar
to that followed by PPO (32). Because our aim was to
characterize POD and PPO, which can show pH maxima
running from acidic (pH 4.5), in the case of POD, to close to
neutrality (pH 7.0) for mushroom PPO (33), oxidations were
carried out using sodium periodate at both pH values to
characterize CGA-Q.
Oxidation of CGA with Periodate at a Ratio of [CGA]0/
[IO4-]0 < 1. When oxidation was carried out at pH 4.5 with
[CGA]0/[IO4-]0 < 1, the spectra of CGA-Q showed a maximum
at λ ) 400 nm. Successive scans indicated that in these
conditions CGA-Q is stable during the measurement time.
However, at pH 7.0, the CGA-Q evolves to generate two
isosbestic points, indicating stoichiometric evolution (Figure
2, right inset). This behavior might be the result of attack by a
hydroxyl or by another o-quinone (6), and the adduct would be
freshly oxidized by periodate (Figure 3).
Figure 4. Spectrophotometric scans of CGA-Q generated with
-
-
periodate at different [CGA]0/[IO4
]
ratios: (A) [CGA]0/[IO4 ]
0
0
ratio 2, [CGA]0 0.5 mM, pH 4.5, acetate buffer, 30 mM
)
)
[(inset) pH 7.0, phosphate buffer, 30 mM]; (B) [CGA]0/
-
[IO4
]
ratio
)
6, [CGA]0
)
1.5, pH 4.5 [(inset) pH 7.0]; (C)
mM, pH 4.5 [(inset)
0
-
[CGA]0/ [IO4
pH 7.0].
]
ratio 8, [CGA]0 ) 2
)
0

Oxidation of Chlorogenic Acid by PPO and POD
J. Agric. Food Chem., Vol. 55, No. 3, 2007 923
Figure 7. Enzymatic oxidation of CGA: (a) oxidation of CGA with PPO
Figure 5. Kinetic of evolution of CGA-Q generated by oxidation with
0.2
[H2O2]0
oxidation of CGA with POD 20 nM, at pH 7.0 and [H2O2]0
curves a d, [CGA]0 3 mM. (Left inset) Spectrophotometric scans of
µ
M at pH 4.5;(b) oxidation of CGA with POD 20 nM at pH 4.5 and
0.5 mM; (c) oxidation of CGA with PPO 0.2 M at pH 7.0; (d)
0.5 mM. In
-
periodate at ratios of [CGA]0/[IO4
]
)
0 > 1. Experimental conditions: acetate
0.12 mM. Concentrations of [CGA]0:
)
µ
-
buffer, 30 mM, pH 4.5, [IO4
]
0
)
(a) 1 mM; (b) 2 mM; (c) 3 mM; (d) 4 mM; (e) 5 mM; (f) 6 mM; (g) 7 mM;
(h) 8 mM. (Inset) Representation of apparent constant with respect to
CGA concentration.
−
)
evolution of CGA-Q generated enzymatically at pH 4.5. (Right inset)
Spectrophotometric scans of evolution of CGA-Q generated enzymatically
at pH 7.0.
Figure 6. Kinetic of evolution of CGA-Q generated by oxidation with
-
periodate at ratios of [CGA]0/[IO4
]
> 1. Experimental conditions:
] ) 0.2 mM. Concentrations of
0
0
-
phosphate buffer, 30 mM, pH 7.0, [IO4
Figure 8. Measurement of PPO and POD activity at pH 4.5; representation
CGA: (a) 0.5 mM; (b) 1 mM; (c) 1.10 mM; (d) 1.5 mM; (e) 2 mM; (f) 2.25
mM; (g) 3 mM. (Inset) Representation of apparent constant with respect
to CGA concentration.
of V0 versus [CGA]0: (a) PPO 25 nM; (b) POD 2 nM at [H2O2]0
) 0.1
mM. (Inset) Experimental recordings of isosbestic point λ ) 480 nm, of
CGA-Q generation at pH 4.5. Substrate concentration: (a) 0.25 mM; (b)
0.5 mM; (c) 0.75 mM; (d) 1.5 mM; (e) 2.5 mM; (f) 2.75 mM; (g) 3 mM.
Experiments were carried out at different concentrations of
CGA at pH 7.0, and the evolution of CGA-Q with time was
recorded at λ ) 400 nm. The kinetic of this reaction was
complex (Figure 2, left Inset).
The above experimental approach with periodate at ratios of
[CGA]0/[IO4-]0 < 1 allowed us to calculate the molar absorp-
tivity coefficient of the CGA-Q at the different pH values studied
(Figure 3), the values at 400 nm being 2000 and 2200 M-1
cm-1 at pH 4.5 and 7.0, respectively. When CGA was oxidized
at a ratio of [CGA]0/[IO4-]0 > 1, similar values were obtained.
Oxidation of CGA with Periodate at a Ratio of [CGA]0/
[IO4-]0 > 1. Although the above experimental conditions of
oxidation periodate at ratios of [CGA]0/[IO4-]0 < 1 are suitable
for calculating the molar absorptivity coefficient, neither PPO
nor POD works in these conditions. For any kinetic character-
ization [CGA]0 . [E]0 must be accomplished, and, because to
maintain the steady state the substrate concentration should not
vary much, then [CGA-Q] , [CGA]0. These are conditions
similar to those obtained by oxidation at ratios of [CGA]0/
[IO4-]0 > 1.
Several oxidations of CGA with periodate were carried out
with different [CGA]0/[IO4-]0 ratios of 2-8. Figure 4A (ratio
2), Figure 4B (ratio 6), and Figure 4C (ratio 8) show the scans
obtained for oxidation by periodate. Note the isosbestic point

924 J. Agric. Food Chem., Vol. 55, No. 3, 2007
Mun˜oz et al.
reference
Table 1. Kinetic Constants That Characterize the Action of PPO on CGA
substratea
CGA
pH
method
k
cat (s
)
K
m (mM)
k6b (M 1 s
)
KmO
(
µ
M)
δ3
δ5
2
-
1
-
-1
4.5
4.5
7.0
7.0
7.0
7.0
7.0
isosbestic point
chronometric
chronometric
2 consumption
2 consumption
2 consumption
2 consumption
135
121
176
842
641
878
439
±
±
±
±
±
±
±
12
7
1.30
1.38
0.87
0.12
1.41
0.10
0.72
±
±
±
±
±
±
±
0.10
0.11
0.08
0.02
0.20
0.01
0.01
(1.04
(8.77
(2.03
(7.02
(4.55
(8.78
(6.10
±
±
±
±
±
±
±
0.8)
1.0)
0.5)
1.5)
0.8)
1.2)
1.1)
×
×
×
×
×
×
×
105
104
105
106
105
106
105
5.8
5.3
7.6
35.8
28.0
40.5
21.5
±
±
±
±
±
±
±
0.12
0.10
0.08
3.20
3.10
4.00
3.10
145.9
148.8
this paper
this paper
this paper
34
34
34
10
25
20
50
25
146.4
146.4
146.2
146.6
146.9
149.3
144.1
144.1
146.6
145.7
MeCAT
TBC
CAT
O
O
O
O
dopamine
34
a CGA, chlorogenic acid; MeCAT, 1-methylcatechol; TBC, 1-tert-butylcatechol; CAT, pyrocatechol. b Binding constant of CGA to Eox (Scheme 3).
Table 2. Kinetic Constants That Characterize the Action of POD on CGA
KSm (mM)
KmH
(
µ
M)
k
5 (M 1 s
)
k
a (s
)
k2b (s
)
δ3
δ4
reference
this paper
2O2
-
1
-
-
1
-
1
-1
substrate
CGA
pH
4.5
method
k
cat (s
)
+
+6
isosbestic point
chronometric
880
910
±
±
40
22
0.30
0.52
±
±
0.05
0.05
25
26
20
128
125
129
27
±
1
(3.00
(3.80
±
±
1.32)
1.52)
×
104
1.57
1.67
×
×
103
2000
2000
145.9
148.8
4.5
±
1
×
104
103
this paper
chronometric
7.0
7.0
7.0
7.0
7.0
674
2155
2146
2200
447
±
±
±
±
±
13
0.48
1.30
2.60
2.30
16.80
±
±
±
±
±
0.05
0.10
0.20
0.10
1.30
±
±
±
±
±
1
8
7
8
4
(2.60
(1.66
(8.25
(9.56
(2.66
±
±
±
±
±
1.02)
0.20)
1.02)
1.06)
0.45)
×
×
×
×
×
104
106
105
105
104
1.02
>2.00
>2.00
>2.00
5.36
×
×
×
×
×
103
104
104
104
102
2000
2000
2000
2000
2000
146.4
146.4
146.2
146.6
146.9
149.3
144.1
144.1
146.6
145.7
this paper
MeCAT
TBC
CAT
chronometric
chronometric
chronometric
chronometric
105
100
150
42
31
31
31
31
dopamine
a Transformation constant of CGA in CGA-SQ for POD-II (Scheme 4). b Data taken from ref 31.
is maintained at 475 nm and pH 4.5, with clear stoichiometry
in the evolution of CGA-Q. When oxidation was carried out at
pH 7.0, there was an isosbestic point at low concentrations of
CGA, but this was broken when concentrations were raised
(Figure 4A-C, inset). In theory, these experiments show that
CGA-Q does not evolve with a clear stoichiometry at pH 7.0.
Kinetic Evolution of the o-Quinone. Oxidation of CGA with
periodate at a ratio of [CGA]0/[IO4-]0 > 1 at acid pH (4.5)
generates CGA-Q, which evolves with a clear stoichiometry
(Figure 4). The kinetic of this evolution is shown in Figure 5.
In these experiments, different initial concentrations of CGA
were oxidized with the same concentration of sodium periodate
and the evolution of the CGA-Q was monitored at λ ) 400
nm. The apparent constant of the evolution of CGA-Q increased
with increasing concentrations of CGA, suggesting that the
quinone reacted with the substrate according to the mechanism
shown in Scheme 1, where P may be a dimer.
versus [CGA]0, giving a second-order rate constant (k′) of 0.05
( 0.01 M-1 s-1. Note that at longer times the absorbance at
400 nm increases, perhaps because the adduct is oxidized by
the CGA-Q, to give a new quinone, in agreement with ref 9.
These results would be in agreement with a rupture of the
isosbestic point in Figure 4A-C (inset), because several
reactions would overlap in time.
Enzymatic Oxidation of Chlorogenic Acid. When the CGA
is oxidized at pH 4.5 with high enzyme concentrations, the
recordings of Figure 7 are obtained. With PPO, Figure 7, trace
a, the absorbance at 400 nm increases until all of the oxygen is
used up, after which the CGA-Q disappears as a result of the
reaction with the substrate, giving a kinetic similar to that shown
in Figure 5. How the CGA-Q evolves is shown in Figure 7
(left inset); the isosbestic point is similar to that obtained for
the oxidations with sodium periodate at a ratio of [CGA]0/
[IO4-]0 > 1 (Figure 4A-C). Similar results were obtained with
POD [Figure 7 (trace b and left inset)].
The rate of disappearance of CGA-Q is
If oxidation is carried out at pH 7.0, we obtain trace c with
-d[CGA-Q]
PPO and trace d with POD, again similar to that obtained with
) kapp[CGA-Q]
(1)
-
dt
oxidation using sodium periodate at a ratio of [CGA]0/[IO4
]
0
> 1. The absence of an isosbestic point (Figure 7, right inset)
confirms the nonstoichiometric evolution of the CGA-Q during
the oxidation with both enzymes at this pH.
In light of the above results, the kinetic characterization of
CGA with enzymes such as mushroom PPO at its optimum pH
(7.0) is difficult, and the results have been interpreted as an
inhibition through excess of substrate (1, 2, 11-14). Something
similar occurs with POD/H2O2 (Figure 1).
and so
with
appt
[CGA-Q]t ) [CGA-Q]0 e -k
(2)
(3)
kapp ) k[CGA]0
In this work we propose two methods for kinetically
characterizing CGA as a substrate for PPO and POD, which
have different pH optima. Both methods are based on the
monitoring of the evolution of the quinone, but two cases can
be distinguished.
(a) Kinetic Characterization at Acid pH. In this case, the
isosbestic point obtained for the evolution of CGA-Q is selected
as measurement wavelength (Figure 8). The wavelength of 480
nm is obtained in Figure 7 (right inset), and once the molar
absorptivity coefficient has been obtained (ꢀi ) 360 M-1 cm-1),
Analysis of the experimental recordings (Figure 5) according
to eq 2 provides kapp, and its dependence on [CGA]0 is shown
in the inset of Figure 5, giving a second-order rate constant (k)
of 2.73 ( 0.17 M-1 s-1
.
When CGA is oxidized in conditions by periodate at a ratio
of [CGA]0/[IO4-]0 > 1 but at pH 7.0, the recordings shown in
Figure 6 are obtained, and the disappearance of CGA-Q can
be followed as in Figure 5. Analysis of the data using eq 2
gives us the value of k′app, which increases with increasing
concentrations of CGA. The inset of Figure 6 depicts k′app

Oxidation of Chlorogenic Acid by PPO and POD
J. Agric. Food Chem., Vol. 55, No. 3, 2007 925
the recordings of Figure 8 (inset) and their analysis give the
values of kcat and Km, which are shown in Table 1 for PPO and
in Table 2 for POD.
The following expression is obtained for the rate of radical
formation:
2kcat[H2O2]0[CGA]0[E]0
(b) Kinetic Characterization at Acid or Neutral pH. This
spectrophotometric method does not involve measuring CGA-Q
because of its instability. However, it may be prevented by using
ascorbic acid (AH2), according to Scheme 2 for PPO.
However, it is not possible to follow the disappearance of
AH2 because the spectra of CGA and AH2 overlap at the
different pH values assayed (results not shown), and so we
propose the following chronometric method.
The method uses the antioxidant power of AH2, which reacts
rapidly with the CGA-Q generated by the enzyme (31) (Scheme
2). The steady-state rate is V0, which is rapidly reached when
[O2]0 and [CGA]0 . [E]0 in the case of PPO or when [H2O2]0
and [CGA]0 . [E]0 in the case of POD, fulfilling the following
mass balance:
V0 )
(12)
k
k
cat[H2O2]0 + cat[CGA]0 + [H2O2]0[CGA]
k5
k1
This equation can be rearranged as follows:
app,CGA
max
V
[CGA]0
V0 )
(13)
(14)
Kampp,CGA + [CGA]0
where
2kcat[H2O2]0[E]0
app,CGA
max
V
)
k
k
cat + [H2O2]0
k1
V0t - [AH2] ) CGA-Q
(4)
k
cat[H2O2]0
k5
The total quantity of material entering the system is V0t, where
t is the time during which the enzyme acts. In the presence of
ascorbic acid, the CGA-Q is reduced to CGA, until no AH2
remains. The straight line absorbance versus t cuts axis X at
CGA-Q ) 0, and so eq 4 is transformed into
Kampp,CGA
)
(15)
cat + [H2O2]0
k1
and
V0τ - [AH2] ) 0
Reorganising eq 5 gives
(5)
k2k6
kcat
)
(16)
(17)
(18)
k2 + k6
[AH2]0
V0 )
(6)
kcat
τ
KCmGA
)
k5
Taking into account the kinetic mechanisms of PPO and POD
described below, some conditions can be established so that
this chronometric method provides suitable values for parameter
V0, thus permitting these enzymes to be characterized.
The diphenolase activity of PPO is described in Scheme 3
(33), where Em, Ed, and Eoxy represent the oxygenated and
deoxygenated forms and another with a peroxide group,
respectively. V0 is given by (33)
kcat
KHm O
)
2
2
k1
Characterization of PPO. The experimental conditions in
the presence of AH2 imply that the concentration of reducing
substrate (CGA) remains constant during the lag phase, although
[O2] will vary with the stoichiometry 1O2:2CGA:2AH2. Thus,
the permitted concentration of AH2 in the experiment will be
e0.1[O2], so that O2 consumption will be <10% and V0 will
not vary, and the steady-state will be reached.
V
max [CGA]0[O2]0
V0 )
KCmGA kOs + Km [CGA]0 + KmCGA[O2]0 + [CGA]0[O2]0
O2
2
(7)
Kinetic Experiments To Accurately Estimate the Initial
Rate. To apply the method, it is necessary to take into account
the following points: (a) limitation of the consumption (as
mentioned above, the concentration of O2 determines the suitable
concentration of AH2; thus, [AH2]0 e [O2]0 should be used);
(b) length of the lag period [the minimum length of the lag
period must be greater than the “dead time” of the measurement,
τd (12 s); we opted for τ g 3 τd, which was achieved by varying
[E]0 (see eq 6); however, τ should not be so long that changes
in the blank samples (due to the chemical reaction of O2 with
AH2) become significant]; (c) quantity of AH2 used in the assays
[this quantity is limited by O2 consumption (see above)].
Validation of Initial Rate Determination. Effect of Enzyme
Concentration. At constant concentrations of both substrates and
AH2, the lag period diminishes as the enzyme concentration
increases, resulting in an increase of V0 (see eq 6) (results not
shown).
where
2k3k7
Vmax ) 2kcat[E]0 )
[E]0
(8)
(9)
k3 + k7
k-8
KsO
)
)
2
k8
kcat
k8
KmO
(10)
(11)
2
kcat
KCmGA
)
k6
POD activity on the reducing substrates (CGA) in the
presence of H2O2 is described by Scheme 4 (30), where POD
stands for the free enzyme, POD-I is compound I, POD-II is
compound II, and CGA-SQ is the semiquinone radical corre-
sponding to the CGA.
Effect of Substrate Concentration. When the concentrations
of the other reagents are kept constant and the concentration of
substrate (CGA) is increased, the lag period becomes shorter
because V0 increases, according to eq 6 (Figure 9).

926 J. Agric. Food Chem., Vol. 55, No. 3, 2007
Mun˜oz et al.
Figure 9. Chronometric method for characterizing enzymatic activity of
Figure 10. Chronometric method used for characterizing enzymatic activity
PPO on CGA; effect of substrate concentration. Experimental conditions:
of POD on CGA. The effect of the substrate concentration is presented.
acetate buffer, 30 mM, pH 4.5; [E]0
) 13.5 nM; [AH2]0 ) 75 µM. Substrate
Experimental conditions: acetate buffer, 30 mM, pH 4.5; [AH2]0
M; [H2O2]0 0.5 mM; [E]0 2 nM. Substrate concentrations: (a) 0.12
mM; (b) 0.20 mM; (c) 0.25 mM; (d) 0.5 mM; (e) 0.75 mM; (f) 1 mM. (Left
inset) Results obtained in phosphate buffer, 30 mM, pH 7.0, [AH2]0 50
M, [H2O2]0 0.50 mM, [E]0 2 nM. Substrate concentrations: (a)
) 50
concentration: (a) 0.35 mM; (b) 0.55 mM; (c) 0.75 mM; (d) 1.10 mM; (e)
1.85 mM; (f) 2 mM; (g) 2.25 mM; (h) 3 mM; (i) 3.15 mM. (Left inset)
Spectrophotometric recordings of chronometric method at constant [CGA]0
µ
)
)
)
×
[E]0 value. Experimental conditions: acetate buffer, 30 mM, pH 4.5.
-
6
× [E]0 ) 6.5 ×
10 mM2 and the concentrations of
µ
)
)
The value of [CGA]0
0.05 mM; (b) 0.10 mM; (c) 0.20 mM; (d) 0.25 mM; (e) 0.30 mM; (f) 0.35
mM; (g) 0.40 mM. (Right inset) Representation of steady-state rate versus
[CGA]0: (a) pH 4.5; (b) pH 7.0.
[CGA]0 and [E]0 were (a) 0.35 mM, (b) 0.55 mM, (c) 0.75 mM, (d) 0.95
mM, (e) 1.10 mM, (f) 2.25 mM and [E]0 (a) 17.5 nM, (b) 11.5 nM, (c) 8.5
nM, (d) 7 nM, (e) 6 nM, and (f) 3 nM, respectively, and [AH2]0
) 75 µM.
(Right inset) Representation of steady-state rate versus [CGA]0; (a) pH
4.5; (b) pH 7.0.
Scheme 4
Scheme 2
Scheme 3
the lag period becomes shorter because V0 increased, according
to eq 19 (Figure 10 and its left inset) at pH 4.5 and 7.0,
respectively.
To avoid the effects of V0 due to the blanks of the assays,
we propose that in a kinetic characterization where the substrate
varies and therefore V0, too, the τ may be very different. To
make the values of τ as similar as possible so that the oxidation
of AH2 by O2 or H2O2 takes place at the same time, we suggest
varying the enzyme and substrate concentrations at the same
time in such a way that their product ([CGA]0 × [E]0) is
constant. In this way τ remains practically constant and the
values of V0 can be normalized in accordance with the
corresponding factor (see Figure 9, left inset).
Kinetic Characterization of POD. In these experiments, the
same restrictions as for PPO are applied, except that, because
this enzyme consumes H2O2, the concentration of AH2 must
fulfill [AH2]0 e 0.1 [H2O2]0.
Furthermore, in agreement with the stoichiometry of Scheme
4, the expression of V0 is
Kinetic Constants. When the methods described above are
applied to the kinetic characterization of the substrate CGA, a
hyperbolic dependence is obtained for V0 versus [CGA]0 (Figure
9, right inset, and Figure 10, right inset). Analysis of the data,
as described under Materials and Methods, provided the
information described in Table 1 for PPO and in Table 2 for
POD.
The kinetic constants determined for the action of PPO on
CGA are compared with the values obtained for other o-
diphenols in Table 1 (33). The CGA, with its higher δ4, has a
lower V0, which reflects electronic effects. With regard to the
values of kcat, the presence of a voluminous group in C-1 (in
the case of TBC) provokes a great increase in Km, as discussed
in other studies (33). However, in the case of CGA, although
the volume of substituent is greater in C-1, perhaps the length
of the acid chain does not hinder the attack of the OH of C-4
on the copper of the active center. Note that the Km increases
as the pH falls, as in the case of mushroom PPO, with other
substrates (34), whereas Km hardly varies.
2[AH2]0
V0 )
(19)
τ
When the concentrations of the other reagents are kept
constant and the concentration of substrate (CGA) is increased,

Oxidation of Chlorogenic Acid by PPO and POD
J. Agric. Food Chem., Vol. 55, No. 3, 2007 927
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Received for review July 24, 2006. Revised manuscript received
November 28, 2006. Accepted December 1, 2006. This work was
supported in part by grants from the MCYT (Spain) Project AGL 2002-
01255 ALI, from the Fundacio´n Se´neca, and from the Fundacio´n
Se´neca/Consejer´ıa de Educacio´n y Universidades (Murcia) Project
00672/PI/04. F.G.-M. has a fellowship from the MEC (Spain), Reference
AP2003-0891. J.M. has a fellowship from the MEC (Spain) Reference
AP2005-4721.
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