Pitfalls in the ABTS Peroxidase Activity Test: Interference of Photochemical Processes

Article abstract of DOI:10.1021/acs.inorgchem.8b02525

ABTS (2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid) oxidation to form its radical cation in the presence of H₂O₂ is frequently used as a test for determining the peroxidase activity of enzyme mimics. Detailed studies using s

Full text of DOI:10.1021/acs.inorgchem.8b02525

Communication
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Pitfalls in the ABTS Peroxidase Activity Test: Interference of
Photochemical Processes
†
†
†
,‡
́
́
́
́
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Andrea Liberato, M. Jesus Fernandez-Trujillo, Angeles Manez, Marcelino Maneiro,*
Laura Rodríguez-Silva,‡ and Manuel G. Basallote*,†
†
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Departamento de Ciencia de los Materiales e Ingeniería Metalurgica y Química Inorganica, Facultad de Ciencias, Universidad de
́
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Cadiz, Avda. Republica Saharahui s/n, Puerto Real, 11510 Cadiz, Spain
‡
́
Departamento de Química Inorganica, Facultade de Ciencias, Campus de Lugo, Universidade de Santiago de Compostela, Avda.
Alfonso X s/n, Lugo 27002, Spain
S
* Supporting Information
artificial mimics (1−3, Chart 1) that differ in their neutral or
ABSTRACT: ABTS (2,2′-azinobis(3-ethylbenzothiazo-
line)-6-sulfonic acid) oxidation to form its radical cation
in the presence of H2O2 is frequently used as a test for
determining the peroxidase activity of enzyme mimics.
Detailed studies using salen-type Mn(III) complexes show
that photochemical processes involving H2O2, ABTS, and
the complex itself can lead to erroneous results. The
ionic nature and in the spacers between the phenyl rings.
Chart 1. Structure of Complexes 1−4
•
capability of the complexes to act as OH scavengers can
be also relevant when the mechanism of their biological
activity is considered.
ontrol of H2O2 levels in biological systems by antioxidant
C
enzymes as catalases, peroxidases, and glutathione
peroxidases1 is critical to avoid oxidative stress, which causes
numerous diseases and aging.2 The diammonium salt of 2,2′-
azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) is a
water-soluble trap for radical species commonly used for
peroxidase assays3 with both natural peroxidases4 and enzyme
mimetics.5 Peroxidase mimics may constitute an exogenous
source of peroxidases for living organisms to neutralize
overproduction of H2O2,6 but they also attract interest in
industrial processes under environmentally friendly catalytic
methods.7 Hundreds of compounds have been evaluated by
the ABTS test since the 70s,8 including some reported by us.9
ABTS is colorless and reacts readily with H2O2 in the presence
of a catalyst to yield a green radical cation, ABTS•+, with
several absorption bands that can be used for quantitative
determinations.10 Although the stability of ABTS and ABTS•+
may vary depending on the reaction conditions,11 reproducible
results are usually obtained with this method, which allows
establishing and comparing the peroxidase-like activity of
artificial mimics.
These compounds had been previously reported as peroxidase-
like catalysts on the basis of the results for the standard ABTS
peroxidase test.9 For comparative purposes, the well-known
EUK-134 complex (4), which acts as scavenger for hydrogen
peroxide, and has been tested against different oxidative
pathologies and commercialized as a potent antioxidant,6a,12,13c
was also included in the study.
During the course of the work we found that spectral
scanning experiments with solutions containing ABTS, H2O2,
and one of the complexes at concentrations typically used in
the ABTS test yield systematically different results depending
on the instrument used (see Table 1 and the SI). The
appearance of the bands typical of ABTS•+ is observed with
any of the instruments, but the experiments showed that those
bands initially increase and then decrease in a slower process.
Both the magnitude of the absorbance changes and the time
scale drastically depend on the instrument used, as illustrated
by the kinetic traces in Figure 1 at wavelengths corresponding
to maxima in the spectrum of ABTS•+. The time required to
achieve the maximum concentration of the radical is only 35 s
Manganese complexes with salen, and related ligands
containing different spacers between the aromatic rings, are
ROS scavengers whose catalytic and pharmacological proper-
ties have been studied for over 20 years.6a,12 It is well-
established that they protect cells from oxidative damage in
animal models and lead to benefits in Alzheimer’s and
Parkinson’s diseases, stroke, motor neuron disease, multiple
sclerosis, and excitotoxic neural injury.13 To gain insight into
the kinetic and mechanistic details of the peroxidase-like
activity of salen-type Mn(III) complexes, we selected three
Received: September 5, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.8b02525
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Communication
Table 1. ABTS•+ Yield Obtained under Different
Illumination Conditions for Solutions with Different
Compositions
than in the experiments with the conventional spectropho-
tometer. The observation of a maximum for the bands of
ABTS•+ is somewhat surprising because it is considered a
persistent radical, stable even in solutions containing dissolved
oxygen.14 The negligible effect of oxygen was confirmed in
experiments using oxygen-depleted solutions, which yielded
results similar to those in the presence of air, both in the
amount of ABTS•+ formed and the time course of its
absorption bands. Thus, the disappearance of ABTS•+ must
be caused by its disproportionation3a and/or its interaction
with other species, probably including H2O2 and/or the metal
complex. Actually, it has been reported that disproportionation
and overoxidation of ABTS•+ leads to the red azodication
ABTS2+ that decomposes yielding a complex mixture of
products.15 Those processes have been reported to require
several hours for completion, but the SF kinetic traces indicate
that they can occur in a much faster time scale, probably
because they are also photochemically activated.16 In any case,
the present results indicate that the peroxidase activity
estimated with this test depends on both the instrument
used and the time at which absorbance readings are taken,
which constitutes a serious limitation when the activity of
different compounds has to be compared.
a
ABTS•+ yield
b
c
d
e
compounds
UV−vis
SF
455 nm filter
f
1 + ABTS + H2O2
2 + ABTS + H2O2
3 + ABTS + H2O2
4 + ABTS + H2O2
ABTS
0.06
0.08
0.04
0.09
0
0.68, 0.66
0.62
0.64
0.21
0.56
0.35
0
0.68
0.68
0.10
ABTS + H2O2
ABTS + 1
ABTS + 2
ABTS + 3
ABTS + 4
0.05
0
0
0
0
0.33
0.58
0.35
0.26
0
0.02
0.06
0.07
0.14
0.38
a
Quotient between the maximum concentration of ABTS•+ and the
b
initial concentration of ABTS. Solvent, acetonitrile with 2.5% of
water; initial concentrations, [ABTS]0 = (1.6−4.8) × 10−5 M,
c
[complex]0 = 2.5 × 10−5 M, [H2O2]0 = 0.05 M. Conventional UV−
d
e
vis spectrophotometer. Stopped-flow instrument. Stopped-flow
f
instrument using a 455 nm cut-on long pass filter. Oxygen-depleted
solutions.
For additional information, the behavior of the three
reagents involved in the test (ABTS, H2O2, and the artificial
mimic) was tested separately and in pairs. Solutions of the
complexes 1−4 do not show detectable spectral changes when
subjected to the SF illumination conditions. However, the
•
photochemical generation of OH and other radicals from
H2O2 (eq 1) is well-known,17 and the photochemical
generation of ABTS•+ from ABTS (eq 2) in aqueous solution
was reported more recently, although with a lower quantum
yield.14 Despite these reports, the possibility of interferences
from photochemical processes in the ABTS peroxidase test is
usually ignored. In our case, ABTS solutions only show the
formation and subsequent decay of small amounts of ABTS•+
when the SF instrument is used (Table 1). Solutions
containing ABTS and H2O2 yield higher concentrations of
ABTS•+, which can be explained by considering that the higher
quantum yield of H2O2 leads to rapid formation of •OH
radicals that react with ABTS (eq 3). In any case, the amount
of ABTS•+ formed in the experiments with ABTS, either alone
or with H2O2, is smaller than for solutions containing ABTS,
H2O2, and any of the complexes, thus suggesting participation
of the Mn species in the process. This suggestion was
confirmed in experiments using mixtures of ABTS and one of
the complexes, which showed yields intermediate between
those observed for ABTS alone and for solutions containing all
three reagents (Table 1).
Figure 1. Kinetic traces at 400 (red) and 750 (blue) nm obtained for
the ABTS peroxidase test using compound 1 with a conventional
spectrophotometer (bottom, note the different scales of the main
figure and the inset) and with a stopped-flow instrument (top). The
blue dashed line corresponds to an SF experiment using a 455 nm cut-
on long pass filter. Solvent, acetonitrile with 2.5% of water;
temperature, 25.0 °C; initial concentrations, [ABTS]0 = 3.1 × 10−5
M, [complex]0 = 2.5 × 10−5 M, [H2O2]0 = 0.05 M.
Significant amounts of ABTS•+ are formed from binary
mixtures of ABTS and the Mn complexes even when the filter
is used, which can be interpreted by considering that the Mn
complex is photochemically activated (eq 4). Although the
high intensity of the ABTS•+ bands hinders an analysis of the
spectral changes corresponding to the Mn complex, it must be
pointed out that related Mn(III) complexes have been shown
to be photoactive, the greatest activity being observed in the
450−600 nm wavelength range.18 On the other hand, solutions
containing H2O2 and one of the complexes showed spectral
changes (Figure 2) that also indicate the occurrence of a
photochemical process that leads to disappearance of the
complex (Table 2). Again the amount of complex decomposed
and the time required for its disappearance are strongly
with the stopped-flow (SF) instrument, which uses a
continuous source of white light, but it increases up to 21
min with the conventional spectrophotometer, which uses a
pulsed Xe lamp with a scanning monochromator that reduces
the amount of light reaching the sample. In addition, the
intensity of the ABTS•+ bands is higher with the SF
instrument. These observations clearly suggest that the striking
effect of the instrument used is caused by some photochemical
process, which was confirmed by the slower formation of
ABTS•+ in SF experiments using a 455 nm cut-on long pass
filter (Figure 1). Nevertheless, the amount of ABTS•+ formed
in the latter experiments is close to that achieved without the
filter despite the intensity of UV light being presumably smaller
B
DOI: 10.1021/acs.inorgchem.8b02525
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Communication
•
H2O2 + hν → OH + other radical species
(1)
ABTS + hν → ABTS•+
(2)
(3)
•
ABTS + OH → ABTS•+ + OH−
MnL + ABTS + hν → (MnL)′ + ABTS•+
(4)
(5)
•
MnL + OH → (MnL)″
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b02525.
■
S
Figure 2. Spectral changes observed during 2000 s with a stopped-
flow instrument for the reaction of complex 1 (4.0 × 10−5 M) with
H2O2 (2.5 × 10−2 M). Solvent, acetonitrile with 2.5% of water;
temperature, 25.0 °C.
Experimental details and selected figures illustrating the
experiments used for obtaining the data in Tables 1 and
2 of the main text, including absorption spectra (PDF)
Table 2. Percentage of Complex Decomposed upon
Reaction with H2O2 Using Different Illumination
Conditions
AUTHOR INFORMATION
Corresponding Authors
*E-mail: marcelino.maneiro@usc.es.
*E-mail: manuel.basallote@uca.es.
■
a b
,
complex decomposed
c
d
d
compound
UV−vis
SF
455 nm filter
ORCID
1
2
3
4
76
82
33
56
94
88
90
35
0
0
3
7
Manuel G. Basallote: 0000-0002-1802-8699
Notes
The authors declare no competing financial interest.
a
Solvent, acetonitrile with 2.5% of water; initial concentrations,
b
ACKNOWLEDGMENTS
The authors acknowledge the financial support provided by the
Spanish Ministerio de Economia y Competitividad and
FEDER funds from the European Union (CTQ2015-71470-
REDT and CTQ2015-65707-C2-2-P).
[complex]0 = 4.0 × 10−5 M, [H2O2]0 = 0.025 M. Estimated from the
absorbance decrease at 500 nm assuming that the reaction products
do not show significant absorption at this wavelength. After 8000
min. After 2000 s.
■
c
́
d
dependent on the illumination conditions, which suggests that
the complexes react with the photochemically generated OH
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Inorganic Chemistry
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(16) Further evidence on the strong effect of the illumination
conditions was obtained in SF experiments at single wavelength using
a monochromator and a photomultiplier detector set at wavelengths
corresponding to the different ABTS•+ bands. The ABTS•+ yields at
single wavelength (Table S2) are almost 1 order of magnitude lower
than when white light is used. Although those yields are quite similar
to those with the conventional spectrophotometer, the absorbance
maximum is achieved at times much shorter (c.a. 30 s) thus revealing
the occurrence of photochemical processes even under these
illumination conditions. As none of the three reagents are expected
to be excited at wavelengths as high as 844 nm, those findings suggest
that light triggers decomposition of the ABTS•+ formed by the
peroxidase activity of the complex.
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(19) Another point that requires additional investigation is to
determine at which extent the present conclusions can be extrapolated
to other compounds. In this sense, preliminary experiments with
horseradish peroxidase indicate that formation of ABTS•+ is much
faster than with the present Mn complexes, and experiments with
both the conventional spectrophotometer and the SF instrument
show its disappearance with time scales that also depend on the
instrument used, also affecting estimation of the amount of ABTS•+
formed.
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DOI: 10.1021/acs.inorgchem.8b02525
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