Kinetic studies on the manganese(II) complex catalyzed oxidation of epinephrine

Article abstract of DOI:10.1016/j.molcata.2013.02.003

The manganese complex [Mn₂(HL)₂](BPh ₄)₂ was found to be a selective catalyst for the oxidation of epinephrine (a catecholamine derivative) to adrenochrome at room temperature in sodium carbonate-bicarbonate buffer. The epinephrine auto-oxidation rate significantly increases upon the addition of manganese complex. The kinetics of reaction was studied by spectrophotometric method, monitoring the increase in concentration of adrenochrome product. According to the proposed mechanism, O₂ binding to the manganese complex is followed by the formation of a ternary catalyst-dioxygen-substrate complex as active intermediate, which decomposes in a rate-determining step, generating the adrenochrome.

Full text of DOI:10.1016/j.molcata.2013.02.003

Journal of Molecular Catalysis A: Chemical 372 (2013) 66–71
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Journal of Molecular Catalysis A: Chemical
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Kinetic studies on the manganese(II) complex catalyzed oxidation of epinephrine
I.Cs. Szigyártóa,∗, L. Szabób, L.I. Simándia
a Research Centre for Natural Sciences, Hungarian Academy of Sciences, Institute of Molecular Pharmacology, Department of Biological Nanochemistry,
1025 Budapest, Pusztaszeri Street 59-67, Hungary
b Research Centre for Natural Sciences, Hungarian Academy of Sciences, Institute of Materials and Environmental Chemistry,
Department of Functional and Structural Materials, 1025 Budapest, Pusztaszeri Street 59-67, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
The manganese complex [Mn2(HL)2](BPh4)2 was found to be a selective catalyst for the oxida-
tion of epinephrine (a catecholamine derivative) to adrenochrome at room temperature in sodium
carbonate–bicarbonate buffer. The epinephrine auto-oxidation rate significantly increases upon the
addition of manganese complex. The kinetics of reaction was studied by spectrophotometric method,
monitoring the increase in concentration of adrenochrome product. According to the proposed
Received 27 September 2012
Received in revised form 21 January 2013
Accepted 5 February 2013
Available online xxx
mechanism, O2 binding to the manganese complex is followed by the formation of
catalyst–dioxygen–substrate complex as active intermediate, which decomposes in a rate-determining
step, generating the adrenochrome.
a ternary
Keywords:
Epinephrine
Manganese(II) complex
Catechol oxidase
Kinetic studies
Functional model
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
physiological function, it is necessary to develop small molecu-
lar weight metal complexes for catechol oxidase as models, and
The catalytic activation of dioxygen by transition metal com-
plexes has been attracting interest because of its relevance to
biological oxidation and elucidation of the reaction mechanism via
structural or functional modelling of oxidoreductase enzymes. One
of the most important substrate groups are catechol and phenol
derivatives. Catechol oxidase (EC 1.10.3.1) catalyzes the oxidation
of catechol to o-quinone and it is involved in the formation of
melanin pigments and in the enzymatic browning of fruits [1–3].
Extensive research was done to elucidate the reaction mechanism
of copper, iron, cobalt and manganese complexes as functional
models systems [4–9].
Related to catecholase activity is the ability of tyrosinase (EC
1.14.18.1) to promote the oxidation of epinephrine (also known
as adrenaline, H4A) to adrenochrome (AC). Adrenaline is produced
in the medulla of the adrenal glands and plays an important role
as neurotransmitter in the central nervous system. The reduction
of catecholamine level caused by the exposure of cells to reac-
tive oxygen species (ROS) contributes to Alzheimer’s, Parkinson’s
and inflammation diseases. The catecholase oxidation reaction is
applied in medical diagnosis for the determination of the hor-
monally active catecholamines [10]. In order to understand their
different methods for determining catecholamines.
Various studies have assessed the acidity constants of the cate-
cholamine derivatives by different methods such as potentiometric
[11] or spectrophotometric titrations and point-by-point analysis
[12]. Several metal–adrenaline complexes and its chemical behav-
ior have been studied by many research groups. It was found that
the coordination of catecholamines (adrenaline and noradrenaline)
with copper-, nickel-, manganese-, and zinc(II) ions is toward
the phenolic groups in aqueous solution [13]. Gergely et al. [14]
have determined the stability constants for the above mentioned
catecholamine–metal complexes. At a metal ion:ligand ratio of 1:1
and at higher pH, the participation of the side-chain donor atoms in
complex formation increase and a polynuclear complex may also
be formed. In the presence of excess ligand, mainly the phenolic
hydroxyl groups take part in the coordination [15].
Methods, such as chromatography [16], fluorescence [17],
chemiluminescence [18], and capillary electrophoresis [19]
are used to distinguish catecholamine derivatives. Chen and
co-workers [20] have developed a rapid and sensitive new chemi-
luminescence method for the determination of catecholamines.
Reactive oxygen species generated in potassium periodate –
luminol system by gallic acid, acetaldehyde and manganese(II)
were scavenged by adrenaline and dopamine. Another chemi-
luminescence method was proposed for the determination of
adrenaline by Kohji et al. [21]. The light emission of the oxidation
was increased after the addition of a manganese(II) catalyst.
∗
Corresponding author. Tel.: +36 1 4381100.
E-mail addresses: szigyarto.imola.csilla@ttk.mta.hu (I.Cs. Szigyártó),
simandi@chemres.hu (L.I. Simándi).
1381-1169/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcata.2013.02.003

I.Cs. Szigyártó et al. / Journal of Molecular Catalysis A: Chemical 372 (2013) 66–71
67
H
N
N
N
H3C
H3C
CH3
OH
OH
O
HO
HO
O2-
O2
Mn2+
B(C6H5)4
2
H2N+CH3
CH3
N+
CH3
N
N
-
OH
- 4 e
O
O
2
+
- 4 H
Scheme 1. Structure of the [Mn2(HL)2](BPh4)2 complex.
Scheme 2. Auto-oxidation reaction of adrenaline.
The determination of adrenaline and other catecholamines
have been also investigated by numerous electroanalytical tech-
niques [22–24]. The stability constants of lanthanum(III) [25] and
yttrium(III) [26]–adrenaline complexes have been determined by
potentiometric and spectrophotometric studies. In all the inves-
tigated La(III)–catecholamine systems, metals have been found
to interact with the phenolic group, but one of the pheno-
late protons does not dissociate, as in the Y(III) complexes. The
yttrium–catecholamine complexes show similar behavior as the
calcium analogues, making them useful in vitro studies. Oxo-
vanadyl(IV) can catalyze the oxidation of adrenaline [27] by
forming a vanadyl-tris(adrenaline) species.
Under physiological conditions the auto-oxidation of adrenaline
is very slow, however, its rate can be increased with pH or by
catalysts [28,29]. Oxidation of adrenaline by different metal salts
in phosphate buffer at pH 7.0 was studied by Chaix et al. [30].
They have observed that the formation of adrenochrome as product
depends upon the formation of an adrenaline–metal complex. The
maximal velocity of oxygen consumption in case of copper(II)-ion
was observed at a ratio of Cu(II)/adrenaline of the order of ½, while
manganese showed maximum at a concentration equal to that of
adrenaline. Copper possessed the greatest catalytic activity in the
oxidation of catecholamines among nickel and manganese; and
iron possessed the least [31]. Futhermore manganese(III) was found
more efficient catalyst than manganese(II) or manganese(IV) [32].
Iron(III) gives much more stable complexes with adrenaline, than
with dopamine, especially the [Fe(H2L)]3+ species in acidic medium
[33]. Intermediates of adrenaline oxidation catalyzed by ceric-, iron
ions, permanganate, ferricyanide and ceruloplasmin were detected
by electron paramagnetic resonance [34] and fluorescence spectro-
metric analysis [35].
5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from
Sigma–Aldrich. Analytical reagent-grade chemicals were used for
buffer preparation.
2.2. Methods
All experiments were done at 25 ◦C in 0.05 M sodium
carbonate–bicarbonate buffer at pH 10.2. The spectrophotometric
studies were performed using a Hewlett-Packard 8453 diode array
UV–vis spectrophotometer thermostated at 25 ◦C with Grant LTD
6G circulating water bath. Kinetic measurements were carried out
by spectrophotometric monitoring the increase in the concentra-
tion of the strongly absorbing adrenochrome product (ꢀ = 480 nm,
ε = 4020 M−1 cm−1) [41] in a quartz cuvette with a 1 cm optical
path.
In a typical experiment 0.1 ml of 5 × 10−4 M solution of man-
ganese complex and 0.15 ml of 9 × 10−3 M solution of adrenaline
substrate were mixed in carbonate buffer, for a final volume of
3 ml. Directly after addition of adrenaline, the UV–vis spectra were
recorded.
EPR spectra of reacting solutions were recorded on a Bruker
ELEXYS E500 CW-EPR spectrometer. Spectra were accumulated
over the initial 30 min of the reaction time. Hydroxyl radical pro-
duced was identified by means of spin-trapping method with EPR
detection. Simulations of the spectra were carried out using the
program developed by Rockenbauer et al. [42].
3. Results and discussion
3.1. Oxidation of adrenaline
Recently, new type of catalysts (mesoporous silica nanoparticles
[36], metal porphyrin-immobilized anion-exchange resin [37]) and
different transition metal complexes [38] have been developed as
functional catecholase enzyme models for oxidation of adrenaline.
According to our recent findings, the dioximato-manganese(II)
dimer [Mn2(HL)2](BPh4)2 (1) {where H2L is the quinqueden-
The auto-oxidation of adrenaline to adrenochrome is known
to take place with increasing pH, as well as the conversion is a
multistep process [43–45]. Misra et al. [28] have observed that at
higher pH the mechanism involves a superoxide-mediated free rad-
ical chain reaction. Scheme 2 illustrates the oxidation process of
adrenaline to adrenochrome.
tate dioxime ligand HON C(CH3)C(CH3)
N
CH2CH2)2NH} acts
The oxidation product adrenochrome exhibits a strong absorp-
tion band at 480 nm. The course of the auto-oxidation reaction was
followed by UV–vis spectroscopy over the first 6 min. Fig. 1 shows
the time evolution of the absorbance spectra of the oxidation of
adrenaline to adrenochrome in carbonate–bicarbonate buffer.
The spectra reveal that, in the beginning of the reaction, the
solution presents a band with maximum absorbance at 294 nm
characteristic for adrenaline solution. The reactive species in this
process is the superoxide (O2−.), which occurs at high pH. It binds
adrenaline affording the adrenaline semiquinone (245 nm), which
then oxidizes further to the o-quinone (340 nm). The cycliza-
tion reaction of adrenaline-quinone is fast and the leucochrome
oxidizes quickly to adrenochrome (480 nm). An additional band
appears at 375 nm after 240 s corresponding to the adrenochrome
tautomer.
as a catecholase mimic in methanol, converting the 3,5-di-tert-
butylcatechol to the corresponding o-quinone [39]. In MeOH
solution the dimer 1 dissociates to monomeric form [Mn(HL)]+
(2). We have now found that complex 1 (see Scheme 1) is also
a functional model for catechol oxidase, converting adrenaline to
adrenochrome. In the present paper the results of the kinetic study
on this functional model system are presented.
2. Experimental
2.1. Materials
The ligand 3,3’-(3-aza-pentanediyldiimino)-bis-butan-2-one
dioxime (H2L) was prepared according to ref. [40]. The octahedral
complex [Mn2(HL)2](BPh4)2 (1) was synthesized and characterized
by a procedure developed by us [39].
Adrenaline and superoxide dismutase were purchased from
Sigma–Aldrich (Hungary). A stock solution of adrenaline (9 mM)
was prepared in 0.01 M hydrochloric acid and stored at 4 ◦C.
The catalytic activity of the dioximato-manganese complex was
examined in carbonate–bicarbonate buffer at 25 ◦C. The catalytic
solution was prepared by mixing 5.00 × 10−4 M methanol solution
of 1 with 0.05 M carbonate buffer (methanol gave 3.3% of the final
volume). When 1.67 × 10−5 M solution of complex was added to a

68
I.Cs. Szigyártó et al. / Journal of Molecular Catalysis A: Chemical 372 (2013) 66–71
Fig. 3. EPR spectra EPR spectra of the DMPO-OH adduct, (a) experimen-
tal spectrum, and (b) simulated spectrum using the parameters g = 2.0055,
aH = 14.25 G, aN = 14.84 G in carbonate buffer, T = 25 ◦C, [Mn]o = 2.50 × 10−5 M,
[H4A]o = 9.00 × 10−4 M.
Fig. 1. Auto-oxidation of adrenaline. Time dependent spectra of adrenaline auto-
oxidation at pH 10.2, [H4A]o = 3.00 × 10−4 M, T = 25 ◦C. Spectra 1–5 (from bottom
up) were taken at 30 s and spectra 5–9 at 60 s intervals.
(DMPO–OH) adduct, shown in Fig. 3. Improvement in the signal
to noise ratio by the accumulation of 200 successive spectra pro-
vided a good quality spectrum. The ESR parameters obtained from
the simulation of the spectrum was compared with the literature
values [46].
solution in which the auto-oxidation of adrenaline was in progress,
it brings about a remarkable increase (ca. 5-fold) in the oxidation
rate. This acceleration is illustrated by the absorbance measured at
480 nm displayed in Fig. 2 as a function of time. An introduction
time (25 s) was required to generate active oxygen radicals, as can
be seen at the beginning of Section 1.
The same experiment done without adrenaline has shown that
the slope of absorbance at 480 nm vs. time is essentially zero (data
not shown). These results clearly demonstrate the catalytic effect
of the manganese complex.
The monitoring of the EPR spectra during the reaction provides
information upon the nature of manganese species present and on
the reaction intermediates. After methanol solution of manganese
complex was added to the carbonate buffer (total volume 3 ml) at
room temperature, the typical six-line pattern of monomeric Mn(II)
disappeared. Using DMPO as spin-trapping agent, the catalytic
solution exhibited a typical ESR signal of hydroxyl radical–DMPO
3.2. Kinetic measurements
The kinetics of the adrenaline oxidation were determined by the
method of initial rates through monitoring the increase in the char-
acteristic adrenochrome absorption band at 480 nm as a function of
time. The initial rates of adrenochrome formation were determined
as a function of the catalyst and substrate concentration. The data
were taken at an interval of 2 s and the conversion was measured
up to 10 min in each case. Observed initial rates were expressed
as M s−1 by taking the extinction coefficient of adrenochrome into
account and calculated from the initial slope of the curve describing
the evolution of product versus time. The results are shown in Tables
S1–S2 (see Supplementary information), where each value shown
is the average of three individual runs (reproducible to within 3%).
To determine the dependence of the rates on the catalyst con-
centration, solutions of the substrate were treated with increasing
amounts of 2. A second order dependence was found for the initial
rate vs. the catalyst concentrations (Fig. 4).
The dependence of initial rate of the adrenochrome formation
on the initial concentration of substrate at fixed catalyst concen-
tration ([Mn]o = 2.50 × 10−5 M) is illustrated in Fig. 5.
Under this experimental condition, saturation type curve
was observed for the initial rate vs. adrenaline concentration. A
kinetic treatment on the basis of the Michaelis–Menten model,
originally developed for enzyme kinetics, was applied. The kinetic
parameters were calculated using the Lineweaver–Burk plot (or
double reciprocal plot, Fig. 6).
The calculated Michaelis constant (KM
) and Vmax are
1.04 × 10−3 M and 6.84 × 10−7 Ms−1
,
kcat = 2.74 × 10−2 s−1 and
(kcat/KM) = 26.35 M−1s−1
. Our result is comparable to those
reported for other model systems [30–32,43], but lower than in
the case of silica nanoparticles [36].
Based on results from the kinetic measurements shown in
Figs. 3 and 4 and the Lineweaver–Burk plot (Fig. 6) we suggest a
general reaction mechanism shown in Scheme 3.
According to the proposed mechanism (1)–(6) in Scheme 3,
step (1) is a pre-equilibrium dimerization of the monomeric
Fig. 2. Manganese catalyzed oxidation of adrenaline. Rate of the auto-oxidation
(Section 1) and its increase upon the addition of Mn complex (Section 2). Rate mea-
sured spectrophotometrically by monitoring the increase of absorbance at 480 nm
(1 cm cell). Section 1: [H4A]o = 4.50 × 10−4 M, T = 25 ◦C. Section 2: same as 1 but with
[Mn]o = 1.67 × 10−5 M added.

I.Cs. Szigyártó et al. / Journal of Molecular Catalysis A: Chemical 372 (2013) 66–71
69
Fig. 4. Initial rate vs. catalyst concentration. Dependence of the initial rate on the
square of concentration of catalyst. [H4A]o = 3.00 × 10−4 M, T = 25 ◦C, in air.
Fig. 6. (Initial rate)−1 vs. (substrate)−1 concentration. Lineweaver–Burk plot (all
investigations were carried out three times with constant catalyst concentration).
MnII(HL)(MeOH), in which the dimeric MnII/MnII catalyst is gener-
ated. This is followed by the reversibly binding dioxygen molecule
to the dimer, affording the superoxomanganese(III) complex. The
latter is capable to bind to the substrate in step (3), by cleavaging the
nitrogen bond from the pseudo axial position (N5) in the complex.
The Mn-substrate-O2 active ternary complex (X) decomposes in
rate-determining step (4) to hydrogen peroxide and leucochrome,
which is rapidly oxidized to the product adrenochrome and man-
ganese dimer complex regenerates. Final fast step (6) describes how
The concentrations of [MnII], [MnII2(H2A)] species and substrate
consumption are negligible. Combination of the mass balance with
the expressions for equilibria in steps (2) and (3) leads to the kinetic
equation (Eq. (3)) for the mechanism (1)–(6)
k4K1K2K3[Mn]2[O2]o[H4A]o
0
Vin
=
(3)
1 + K3[H4A]o
where [Mn]o and [H4A]o are the initial concentrations of added
dioximato-manganese(II) and adrenaline, respectively.
In our experiments the dioxygen concentration was held con-
stant, but Nachtman and co-workers [47] have shown that the rate
of oxidation was increased in 95% dioxygen relative to air. The
mechanism found consistent with the observed kinetic behaviour
of dioximato-manganese(II) is in several aspects similar to the
mechanism of dioxygen activation by divalent transition-metal
complexes [48].
intermediate O2 is converted to OH−.
−
In this catalytic process, steps (2) and (3) can be regarded as
pre-equilibria and (4) as rate determining step. To derive a kinetic
equation corresponding to the proposed mechanism (Scheme 3),
one needs the mass balance for the manganese species and for H4A,
which can be expressed by Eqs. (1) and (2).
[Mn]o = [MnII] + [MnII2] + [MnIII2(O2)] + [X] + [MnII2(H2A)]
(1)
3.3. Superoxide dismutase activity
[H4A]o = [H4A] + [X] + [MnII2(H2A)] + AC
(2)
Superoxide dismutase (EC 1.15.1.1) (SOD), is an enzyme capable
to protect living organisms from oxidative damage by dismuta-
tion of superoxide radical into oxygen and hydrogen peroxide.
−
It is possible to test the involvement of O2 in the oxidation of
adrenaline. This popular assay of superoxide and its variations have
been attracting interest in the last few decades [49–51]. The super-
oxide ion is generated indirectly in the oxidation at alkaline pH, to
serve as substrates for SOD. Because SOD slows down the rate of
adrenochrome formation, it is considered as a potent inhibitor of
this oxidation. This assay does not work if dopamine is used instead
of adrenaline, since the quinone cyclization reaction is much slower
and the reduction of the quinone by superoxide predominates.
2 [Mn(HL)]+
[(HL)2Mn2]2+ + O2
[(HL)2Mn2III(O2)]2+
[(HL)2Mn2III(O2)(H4A)]2+
[(HL)2Mn2(H2A)]2+ + O2
[Mn2(HL)2]2+
[(HL)2Mn2III(O2)]2+
[(HL)2Mn2III(O2)(H4A)]2+
[(HL)2Mn2II(H2A)]2+ + H2O2
[(HL)2Mn2]2+ + AC + O2- + H+
O2 + OH- + OH.
K1
K2
(1)
(2)
(3)
(4)
(5)
(6)
+
H4A
K3
k4
fast
fast
-.
H2O2 + O2
Fig. 5. Initial rate vs. substrate concentration. Dependence of the initial rate of
adrenochrome formation on the substrate concentration. [Mn]o = 2.50 × 10−5 M,
T = 25 ◦C, in air.
Scheme 3. Proposed reaction mechanism of the adrenaline oxidation catalyzed by
2.

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I.Cs. Szigyártó et al. / Journal of Molecular Catalysis A: Chemical 372 (2013) 66–71
In the presence of manganese complex we have investigated
the above mentioned inhibition reaction. To determine the IC50, a
solution containing adrenaline (3 × 10−4 M) and manganese com-
plex (1.67 × 10−5 M) was treated with increasing amounts of SOD
enzyme (Fig. 7b). As expected, the 50% inhibition was achieved at
higher SOD concentration, due to the catalytic effect of manganese
on the oxidation of adrenaline.
The manganese catalytic effects on the oxidation of adrenaline
to adrenochrome inhibited by SOD were measured at a constant
SOD concentration (corresponding to 50% inhibition). Under this
condition the oxidation rate increases with addition of manganese
as shown in Fig. 8.
We have found that the auto-oxidation of adrenaline
was inhibited by superoxide-dismutase in 0.05 M sodium
carbonate–bicarbonate buffer (pH 10.2) and IC50 = 4.28 × 10−9 M.
In the presence of manganese complex the 50% inhibition of
adrenaline oxidation was observed at 28.5 × 10−9 M SOD. These
results are consistent with the suggested reaction mechanism
(Scheme 3) and it can be conclude that the manganese complex
has no SOD-like activity.
Fig. 7. IC50 vs. SOD concentration. Inhibition percentage as
a function of
4. Conclusion
superoxide dismutase concentration. Conditions: [H4A]0 = 3.00 × 10−4 M; curve a:
136.8 ng/ml superoxide dismutase; curve b: 912.0 ng/ml superoxide dismutase,
[Mn]o = 1.67 × 10−5 M, T = 25◦C, in air.
In this work, we have carried out detailed kinetic studies on
functional catechol oxidase model based on manganese com-
plex, using adrenaline as model substrate. The catalytic effect is
due to the formation of a reactive ternary complex intermediate
([(HL)2Mn2III(O2)(H4A)]2+), which decomposes to adrenochrome.
The kinetic measurements have revealed second-order depend-
ence on the catalyst concentration and saturation type behaviour
with respect to the substrate concentration, as well as the
dioximato-manganese complex does not catalyze the dismutation
of superoxide, so it cannot be considered as a functional model of
superoxide dismutase.
Increasing amounts of SOD was added to adrenaline solution
(3 × 10−4 M), and the effect on the rate of adrenochrome formation
was recorded at 480 nm. The auto-oxidation of adrenaline at pH
10.2 was inhibited by SOD as shown in Fig. 7a. The SOD-activity
was characterized by determining the IC50 value (the concentra-
tion where inhibition of adrenochrome formation is 50%). The IC50
can be obtained from the plot of (v0 − vSOD)/v0 × 100% vs. enzyme
concentration, where v0 is the slope of the absorbance vs. time in
the absence of enzyme, whereas vSOD is the slope in the presence of
enzyme. The percent inhibition (%I) shows hyperbolic dependences
as a function of SOD concentration, as long as the maximum of inhi-
bition does not exceed 70%. This is probably due to the presence of
interfering intermediates which activated the alternative oxidative
pathways [28].
Acknowledgements
This work was supported by the Hungarian National Research
Fund (OTKA K60241). We thank Dr. L. Korecz for performing ESR
measurements and the valuable discussion on the spectra. The
authors acknowledge Dr. S. Kristyán for the valuable discussion and
providing language help.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.molcata.
2013.02.003.
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