Novel screening assay for antioxidant protection against peroxyl radical-induced loss of protein function

Article abstract of DOI:10.1002/jps.20881

Oxidative damage to proteins, implicated amongst other in the etiology and progression of Parkinson's disease (PD) and Alzheimer's disease (AD), results in the loss of specific biological protein function. A simple, sensitive, and cost-effective fluorimetric test to assess the antioxidant capacity of new chemical entities to protect proteins from loss of activity caused by reactive oxygen species (ROS) was developed using alkaline phosphatase (ALP) as model protein. Protein oxidation was induced by 2,2′-azobis(2- methylpropionamidine) dihydrochloride (AAPH) and the decrease in catalytic activity of ALP to hydrolyze 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) was monitored as a marker of protein degradation. According to their capacity to protect ALP from peroxyl radical-induced activity loss, ten reference antioxidants were divided into three classes, namely efficient (pIC₅₀ > 5 for quercetin, chlorogenic acid, caffeic acid, mangiferin, and resveratrol), intermediate (4 < pIC₅₀ ≤ 5 for melatonin, trolox, and ascorbic acid), and poor antioxidants (pIC₅₀ < 4 for glutathione and D-mannitol). Multifunctional drugs, having the ability to interact with several disease-related targets are of interest in PD. Therefore, the capacity of three catechol-O-methyltransferase (COMT) inhibitors, entacapone, nitecapone, and tolcapone to protect ALP from oxidative damage was also investigated and found to be very similar to the most potent reference antioxidants.

Full text of DOI:10.1002/jps.20881

DRUG DISCOVERY INTERFACE
Novel Screening Assay for Antioxidant Protection against
Peroxyl Radical-Induced Loss of Protein Function
FRANCESCA BERTOLINI, LAURA NOVAROLI, PIERRE-ALAIN CARRUPT, MARIANNE REIST
LCT-Pharmacochemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne,
30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
Received 12 September 2006; revised 13 December 2006; accepted 22 December 2006
Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20881
ABSTRACT: Oxidative damage to proteins, implicated amongst other in the etiology
and progression of Parkinson’s disease (PD) and Alzheimer’s disease (AD), results in the
loss of specific biological protein function. A simple, sensitive, and cost-effective
fluorimetric test to assess the antioxidant capacity of new chemical entities to protect
proteins from loss of activity caused by reactive oxygen species (ROS) was developed
using alkaline phosphatase (ALP) as model protein. Protein oxidation was induced
by 2,20-azobis(2-methylpropionamidine) dihydrochloride (AAPH) and the decrease in
catalytic activity of ALP to hydrolyze 4-methylumbelliferyl phosphate (4-MUP) to
fluorescent 4-methylumbelliferone (4-MU) was monitored as a marker of protein degra-
dation. According to their capacity to protect ALP from peroxyl radical-induced activity
loss, ten reference antioxidants were divided into three classes, namely efficient
(pIC50 > 5 for quercetin, chlorogenic acid, caffeic acid, mangiferin, and resveratrol),
intermediate (4 < pIC50 ꢀ 5 for melatonin, trolox, and ascorbic acid), and poor antiox-
idants (pIC50 < 4 for glutathione and D-mannitol). Multifunctional drugs, having the
ability to interact with several disease-related targets are of interest in PD. Therefore,
the capacity of three catechol-O-methyltransferase (COMT) inhibitors, entacapone,
nitecapone, and tolcapone to protect ALP from oxidative damage was also investigated
and found to be very similar to the most potent reference antioxidants. ß 2007 Wiley-Liss,
Inc. and the American Pharmacists Association J Pharm Sci 96:2931–2944, 2007
Keywords: in vitro models; fluorescence spectroscopy; proteins; alkaline phospha-
tase; peroxyl radicals; oxidation; antioxidants; COMT inhibitors; kinetics; multi-
functional hits
INTRODUCTION
Reactive oxygen species (ROS) including super-
oxide, hydroxyl radicals, hydrogen peroxide,
nitric oxide, and peroxyl radicals are normal by-
Abbreviations: PD, Parkinson’s disease; AD, Alzheimer’s
disease; ALP, alkaline phosphatase; AAPH, 2,20-azobis(2-
methylpropionamidine) dihydrochloride; ROS, reactive oxygen
species; COMT, catechol-O-methyltransferase; 4-MUP, 4-
methylumbelliferyl phosphate; 4-MU, 4-methylumbelliferone;
DMSO, dimethyl sulfoxide; BBB, blood brain barrier; CNS,
central nervous system; BChE, butyrylcholinesterase; HSA,
human serum albumine; BSA, bovine serum albumine.
Correspondence to: Marianne Reist (Telephone: þ41 22 379
33 65; Fax: 41 22 379 33 60;
products of cellular physiology, and their intended
functions range from host defense (e.g., the
oxidative superoxide burst) to neuronal signal
transduction (e.g., NO as neuronal messenger).1
However, they also have the ability, either
directly or indirectly, to damage biomolecules,
including lipids, DNA, and proteins. ROS may,
for example, alter the function of proteins by
E-mail: marianne.reistoechslin@pharm.unige.ch)
Journal of Pharmaceutical Sciences, Vol. 96, 2931–2944 (2007)
ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
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BERTOLINI ET AL.
changing their tertiary structure, or destroy
structural proteins. Although there are several
defense systems that balance the ratio between
generation and detoxification of ROS, endogenous
and exogenous triggers may cause the over-
production of ROS or the impairment of these
antioxidant defense systems, leading to oxidative
stress.1,2
to assess antioxidant properties of compounds are
needed. Numerous in vitro methods to quantify
antioxidant protection of proteins have been
developed.21 The majority of these assays are
based on the investigation of structural damages,
such as the detection of carbonyl groups in the
structure of the protein,22 protein fragmentation
and aggregation,23,24 as well as amino acid
side chain modifications.25 Only a few in vitro
methods were designed to investigate the effects
of potential antioxidants on loss of activity of a
given protein (e.g., enzyme) induced by ROS.
Examples are the assays proposed by Salvi et al.26
and by Halliwell et al.27 The former is based on a
fluorimetric test using the peroxyl radical-induced
decrease of the esterase activity of butyrylcholi-
nesterase (BChE)-contaminated human serum
albumin (HSA) as a marker of oxidative damage.26
The latter was developed to assess the peroxyni-
trite scavenging capacity of test compounds and is
based on the spectrophotometric determination
of the activity of a1-antiproteinase to inhibit
elastase.27
In this work, a convenient, simple, sensitive,
and cost-effective 96-well plate fluorimetric
method to assess the antioxidant capacity of
new chemical entities to protect proteins from
loss of activity caused by peroxyl radicals was
developed. Alkaline phosphatase (ALP), from calf
intestinal mucosa28 was used as model protein
and the antioxidant activity of a compound
was measured by its ability to preserve catalytic
effectiveness of ALP despite oxidative stress.
Protein oxidation by peroxyl radicals was
induced by 2,20-azobis(2-methylpropionamidine)
dihydrochloride (AAPH),29 and the decrease in the
catalytic activity of ALP to hydrolyze 4-methy-
lumbelliferyl phosphate (4-MUP) to fluorescent 4-
methylumbelliferone (4-MU) was monitored as a
marker of protein degradation.
Oxidative stress contributes to the pathophy-
siology of different human disorders, including
cancer, atherosclerosis, pulmonary dysfunction,
and several aging-related neurodegenerative
diseases such as Alzheimer’s disease (AD) and
Parkinson’s disease (PD).3 Indeed, free radical-
induced damage to biomolecules has been exten-
sively described as a hallmark of AD and PD,
unsaturated fatty acids and proteins present in
synaptic membranes being particularly suscepti-
ble to ROS.3 The role of protein oxidation in
the etiology and progression of aging-related
neurodegenerative disorders is indicated by the
increased level of protein carbonyls measured in
brain samples of AD3–5 and PD patients.6 Brain
regions show specific changes in this regard, and
carbonyl levels correlate well with tangles.7
To develop more efficient drugs for the treat-
ment of complex diseases in which free radicals
and protein oxidation might be a significant, but
not exclusive pathogenic factor, antioxidants
that also have other pharmacological effects are
desired.8 Indeed, because of the multifaceted
etiology of AD and PD, new therapeutic strategies
are based on the development of multifunctional
drug candidates that are able to interact with
several disease-related targets. Advantages of
multifunctional drugs seem to be a more pre-
dictable pharmacokinetic profile, a lower risk of
drug–drug interactions, and major compliance by
the patients due to a simplification of the
therapeutic regiment with less side effects.9,10
Multifunctional drugs with antioxidant proper-
ties might thus well be the answer to complex
diseases implicating free radical-induced damage
to biotargets. Multifunctional compounds with
antioxidant properties are already being devel-
oped and their activity against various diseases,
such as cancer,11,12 neurodegenerative,13–18 or
cardiovascular diseases,9,10,19,20 has been estab-
lished.
The capacity to protect ALP from peroxyl
radical induced damage of ten reference com-
pounds (1 to 10) and three catechol-O-methyl-
transferase (COMT) inhibitors, bearing
a
nitrocatecholic structure,30 tolcapone (11), enta-
capone (12), and nitecapone (13) was investigated
(Fig. 1).
Recently, entacapone and tolcapone were com-
mercialized for their COMT inhibitor properties
as adjunct to the standard levodopa/decarboxy-
lase inhibitor therapy for PD.30 Nitecapone had
been tested on healthy volunteers but not been
commercialized so far.30 Catechols and nitroca-
techols are recognized as groups possessing
To obtain multifunctional antioxidants, two
strategies can be followed: (i) screen drug collec-
tions and/or compound databases, (ii) couple an
antioxidant group with other pharmacophores.9
For both approaches, convenient screening assays
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ANTIOXIDANT PROTECTION AGAINST LOSS OF PROTEIN FUNCTION
2933
Figure 1. Structure of reference antioxidants and COMT inhibitors examined.
antioxidant properties. Indeed, the ability of
entacapone to chelate ferric ion and other transi-
tion metals has already been reported,31 as well as
the capacity of entacapone and nitecapone to
scavenge nitric oxide radicals.32 Moreover, differ-
ent authors have described the remarkable ability
of nitecapone to act as a ROS scavenger and to
inhibit lipid peroxidation.33,34 However, the capa-
city of COMT inhibitors to protect proteins from
oxidation was not reported so far. In this study,
the antioxidant capacity of these compounds
to protect ALP from peroxyl radical-induced
activity loss was found to be equal to the one of
the most potent reference compounds. The ability
of COMT inhibitors to protect proteins from
oxidative damage is of interest in the therapy
of PD.
MATERIALS AND METHODS
Materials
ALP from calf intestinal mucosa (EC. 3.1.3.1),
[9001-78-9], 4-MUP disodium salt, AAPH (CAS #:
2997-92-4), 4-MU, glycine, magnesium chloride
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BERTOLINI ET AL.
hexahydrate, sodium hydroxide, sodium chloride,
melatonin (6), ascorbic acid (8), and dimethyl
sulfoxide (DMSO) of microbiological quality
were purchased from Fluka (Buchs, Switzerland).
Caffeic acid (2), chlorogenic acid (3), trolox
(6-hydroxy-2,5,7,8-tetraethylchroman-2-carboxylic
acid) (7) were obtained from Aldrich (Buchs,
Switzerland). Quercetin dihydrate (1), mangiferin
(4), resveratrol (5), mannitol (9), and glutathione
(10) were from Sigma–Aldrich (Buchs, Switzer-
land). Tolcapone (11), entacapone (12), and
nitecapone (13) were kindly donated by Prof.
Alberto Gasco (Dipartimento della Scienza e
tecnologia del Farmaco, University of Turin,
Italy). Methanol of HPLC grade was from Prolabo
(Fontenay-sous-Bois, France), and ethanol was
obtained from Schweizerhalle Chemie AG (Basel,
Switzerland). All chemicals were of analytical
grade and all solutions were prepared in demi-
neralized and purified water obtained with
the Elix 3 Millipore water purifying system. All
incubations and measurements were performed
in black polypropylene 96-well plates, obtained
from Millian (Geneva, Switzerland). During
incubations, the plates were sealed with elasto-
mer 96-well cap mats from Nunc (Roskilde,
Denmark).
KM value), the plate was shaken for 50 s and the
fluorescence (lEx 360 ꢁ 20 nm, lEm 460 ꢁ 20 nm)
was monitored at room temperature for 15 min
using a Bio-Tek FLX 800 microplate fluorescence
reader (top reading) and KC4 v3.3 software (Bio-
Tec Instruments, Inc., Winooski, VT). Product
formation was quantified by comparing the
fluorescence emission of the samples to that of
known amounts of 4-MU (standard solutions of
0.05–1 mM).
To obtain the kinetic parameters KM and vmax
,
hydrolysis rates for duplicates of six concentra-
tions of 4-MUP (final concentrations in the
reaction mixture ranging from 0.8 to 50 mM,
prepared in buffer) were determined. Data
analysis was performed with Prism V4.0 (Graph-
Pad Software, Inc., San Diego, CA). The kinetic
parameters KM and vmax were obtained by curve
fitting according to the classical Michaelis–Men-
ten equation.
Optimization of Oxidation Conditions: AAPH
Concentration and Oxidation Time
ALP was oxidized at 408C by the peroxyl radical
generator AAPH. To determine optimal oxidation
conditions, the influence of AAPH concentration
and oxidation time at 408C on the activity loss of
ALP was investigated. 146 mL of buffer, 20 mL of
ALP stock solution (20 mU/mL in buffer), as well
as 4 mL of cosolvent or buffer were preincubated
for 15 min at 40 ꢁ 0.18C using a Heidolph
Titramax 1000 incubator (Geneva, Switzerland).
For test samples, oxidation was initiated by
addition of 10 mL of AAPH solution prepared in
buffer (final concentrations in the reaction mix-
ture of 0.3–5 mM) and followed by incubation at
40 ꢁ 0.18C with continuous shaking at 150 rpm,
for 30–180 min. During incubations 96-well plates
were sealed with elastomer 96-well cap mats to
avoid sample evaporation. After oxidation, the cap
mats were removed and samples were cooled down
to room temperature (5 min). For the controls,
that is, to obtain the ALP activity in the absence of
Determination of ALP Hydrolytic Activity in the
Absence and Presence of Cosolvent
The reaction followed to assess the catalytic
activity of ALP was the enzymatic dephosphor-
ylation of 4-MUP to fluorescent 4-MU. Enzymatic
hydrolysis rates of 4-MUP were determined by a
continuous spectrofluorimetric assay (Fig. 2). 96-
Well plates were filled with 156 mL of buffer (0.1 M
glycine/NaOH-buffer pH 8.3, containing 1 mM of
MgCl2 and 1 mM of ZnCl2), 20 mL of ALP stock
solution (20 mU/mL in buffer) as well as 4 mL of
cosolvents or buffer for the control without
cosolvent. After addition of 20 mL of substrate
(4-MUP) solution, prepared in buffer at a final
concentration of 5 mM (concentration close to the
Figure 2. Dephosphorylation of 4-methylumbelliferyl phosphate (4-MUP) to 4-methyl-
umbelliferone (4-MU) catalyzed by alkaline phosphatase (ALP).
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ANTIOXIDANT PROTECTION AGAINST LOSS OF PROTEIN FUNCTION
2935
oxidation, 10 mL of AAPH solution (final concen-
trations in the reaction mixture of 0.3–5 mM)
were added just before the addition of MUP.
The hydrolytic activity of oxidized (samples)
and nonoxidized (controls) ALP was measured
by spectrofluorimetry as described above. The
remaining hydrolytic activity of oxidized ALP was
calculated as the percentage of ALP activity
obtained in the absence of oxidation but in the
presence of AAPH during the activity determina-
tion, which was assumed to be 100% (see
discussion below).
using Prism V4.0 (GraphPad Software, Inc., San
Diego, CA). All pIC50 values were determined in
triplicate from at least ten test compound
concentrations.
As a control, the ALP inhibitory activity of test
compounds in the absence of AAPH was assessed.
The percentage of inhibition of ALP was deter-
mined after incubation of the enzyme (final
concentration of 2 mU/mL) with test compounds
(final concentrations of 6.25–125 mM) for 90 min at
40 ꢁ 0.18C.
As AAPH is still present during the measure-
ment of the hydrolytic activity of ALP it could
influence the activity of ALP either directly by
enzyme inhibition, or indirectly by inactivation
due to the amount of free radicals generated
during the 15 min of activity determination at
room temperature. Hence, the influence of AAPH
on the hydrolytic activity of ALP during the
15 min of 4-MUP dephosphorylation was assessed
at room temperature and at 48C by measuring the
production of 4-MU by nonoxidized ALP in the
presence and absence of a final AAPH concentra-
tion of 5 mM. The experiment at 48C was
performed to keep the production of free radicals
low and to assess principally ALP inhibition by
AAPH.
RESULTS AND DISCUSSION
ALP Hydrolytic Activity in the Absence and
Presence of Cosolvents
Choice of Reaction Conditions to Assess the
Catalytic Activity of ALP
ALP from calf intestinal mucosa (EC. 3.1.3.1) was
chosen as model protein to assess antioxidant
protection against ROS-induced loss of protein
function because it is readily available at low
cost. In addition, the reaction it catalyzes, that is,
dephosphorylation of orthophosphoric monoe-
sters,35 is well known and can easily be followed
by spectrophotometric and fluorimetric detection.
Indeed, although the physiologic function of ALP
is still uncertain, its catalytic activity is widely
used for the diagnosis of liver and bone dis-
eases.35,36 In the present work, the reaction
followed to assess the catalytic activity of ALP
was the enzymatic hydrolysis of 4-MUP to
fluorescent 4-MU (Fig. 2).
The catalytic activity of ALP was assessed at pH
8.3 and room temperature. The optimum of
the catalytic activity of ALP being at basic pH
values, a pH of 8.3 is an acceptable compromise
between a good ALP activity and a pH not too
far from physiological conditions. Preliminary
experiments had shown that hydrolysis rates of
4-MUP to 4-MU by ALP were the same at room
temperature and at 378C, indicating that for this
reaction, temperature changes between 258C and
378C did not influence reaction rates. ALP activity
determination at room temperature rather than
at 378C was chosen to minimize the temperature-
dependent production of radicals generated by
the amount of AAPH still present during the
measurement of the hydrolytic activity of ALP
(see below).
Determination of Antioxidant
Activity of Compounds
The antioxidant activity of a compound was
determined by its ability to preserve the catalytic
effectiveness of ALP despite the presence of
peroxyl radicals generated by AAPH. Volumes
and concentrations of buffer, ALP and AAPH
solutions were the same as described above. Only
the 4 mL of cosolvent or buffer were replaced by
4 mL of DMSO or buffer solutions of the test
compounds (final concentrations in the reaction
mixture of 0.1–100 mM). After preincubation of 15
min at 40 ꢁ 0.18C, protein oxidation by AAPH
(final concentration 5 mM) followed for 90 min at
40 ꢁ 0.18C. Enzymatic hydrolysis rates were
measured as described above and the protective
activity of the test compounds for each concentra-
tion was calculated as the percentage of ALP
activity obtained in the absence of oxidation
(considered to be 100%). The antioxidant capacity
of test compounds was expressed as pIC50 (ꢂlog
IC50) and was calculated by curve fitting according
to the classical sigmoidal dose–response equation
The criterion for the choice of the ALP
concentration was to obtain reliably detectable
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2936
BERTOLINI ET AL.
amounts of product formed, with the lowest
possible ALP amount. At a final ALP concentra-
tion of 2 mU/mL and substrate (4-MUP) concen-
trations of 0.8–50 mM, 0.6–37.5 mM of 4-MU was
produced in 15 min. These product concentrations
correspond to fluorescence values which fall
within the appropriate dynamic range of relative
fluorescence units when the highest possible
sensitivity for fluorescence measurements was
selected on the microplate fluorescence reader.
Finally, the kinetic parameters KM and vmax for
the dephosphorylation of 4-MUP to 4-MU at pH
8.3 and room temperature were determined by
means of the Michealis–Menten rectangular
hyperbola. A KM value of 4.33 ꢁ 0.26 mM and a
vmax of 0.3 ꢁ 0.05 mM ꢃ minꢂ1ꢃmg proteinꢂ1 were
obtained. A substrate concentration close to the
three cosolvents. Due to its good solubilizing
capacity, DMSO at a final concentration of 2% was
selected as cosolvent for all assays. As a control,
the influence of this final DMSO concentration on
the kinetic parameters of 4-MUP dephosphoryla-
tion by ALP was determined. The KM and vmax
values in the presence or absence of 2% of DMSO
were found to be the same within experimental
errors (Tab. 1), confirming that a 2% final DMSO
concentration did not influence the catalytic
activity of ALP. Moreover, it was also verified,
that the addition of DMSO at a final concentration
of 2% did not modify the pH of the reaction
medium.
Peroxyl Radical-Induced Loss of ALP Function
K
M value, that is, 5 mM, was chosen to assess the
Choice and Optimization of Oxidation Conditions
catalytic activity of ALP for all further experi-
ments.
Appropriate oxidation conditions are of crucial
importance for the present screening test. For the
determination of the antioxidant capacity of a
compound to protect ALP from free radical-
induced activity loss, a remaining ALP activity
after oxidation of about 50% was considered
optimal, because it is important to give substan-
tial but not complete enzyme inactivation for the
pIC50 determination.27 Indeed, it is advantageous
to have reaction conditions that allow detecting
antioxidant but also prooxidant activity of a test
compound.
The water-soluble diazo compound AAPH was
chosen as free-radical generator because it under-
goes spontaneous thermal decomposition to gen-
erate peroxyl radicals at constant rate,29 which
allows to easily vary and control the amount of
free radicals formed during oxidation. Peroxyl
radicals are thought to play an important role in
the pathogenesis of numerous diseases as they
are formed in a variety of physiological and
pathological processes such as lipoperoxida-
tion.37,38 Moreover, this radical generator was
preferred to an oxidation system involving metal
ions as it allows us to test selectively the free
radical-scavenging properties of an antioxidant
compound without implicating its metal-ion-
chelating capacities. Indeed, the catalytic activity
of ALP requires the presence of magnesium and
zinc28 and the reaction buffer therefore contains
these ions at 1 mM concentration.
Influence of Organic Cosolvents on
the Hydrolytic Activity of ALP
A number of antioxidants tested in this study were
not sufficiently water-soluble to be examined
without cosolvent. To assess the influence of
cosolvents on the enzymatic activity of ALP,
methanol, ethanol, and DMSO have been added
to the reaction mixture at four different concen-
trations (0.25%, 0.5%, 1%, and 2%). The results
obtained have been compared with the hydrolytic
activity of ALP in the absence of cosolvent.
Figure 3 shows that at all concentrations tested
no significant decrease (p > 0.05) in the hydrolytic
activity of the enzyme was observed for any of the
AAPH decomposes unimolecularly to form two
carbon-centered radicals and N2 gas. In the
presence of O2 some of these carbon-centered
radicals recombine to give stable products, but
Figure 3. Nonsignificant influence of cosolvents at
different final concentrations (0% &, 0.25%
, 0.50%
, 1%
, 2% &) on the hydrolytic activity of ALP
(p > 0.05).
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ANTIOXIDANT PROTECTION AGAINST LOSS OF PROTEIN FUNCTION
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Table 1. Influence of Incubation Time and DMSO on the Kinetic Parameters (KM and
v
max) of 4-MUP Dephosphorylation by ALP
Incubation time
(min) (408C)
DMSO (%)
AAPH (mM)
KM (mM)a
vmax (mMꢃminꢂ1ꢃmgꢂ1 a
)
0
90
90
0
0
2
0
0
0
4.33 ꢁ 0.26
4.15 ꢁ 0.21
4.12 ꢁ 0.74
0.31 ꢁ 0.05
0.32 ꢁ 0.05
0.31 ꢁ 0.01
aMeans ꢁ standard errors (n ¼ 2) [mMꢃminꢂ1ꢃmg proteinꢂ1].
many of them diffuse apart and react rapidly
with oxygen molecules to give peroxyl radicals,29
which are usually considered to be the initiating
species responsible for the oxidation.39 The rate of
carbon-centered radical formation is determined
primarily by temperature and AAPH concen-
tration and only to a minor extent by solvent
and pH.29
In this study the optimal temperature was
selected to be 408C. This choice relies on the
following considerations. Around the physiologi-
cal temperature, a 58C increase doubles the rate of
radical production,40 thus a higher temperature
shortens the oxidation time. On the other hand,
with temperatures above 408C the accuracy of
the results was considerably lower due to volume
variations caused by evaporation during the
incubation (results not shown). In addition,
incubation of ALP at temperatures up to 408C
did not alter its catalytic activity. Indeed, after
incubation of the enzyme for 90 min at 408C, the
kinetic parameters KM and vmax were the same
within experimental errors than without incuba-
tion (Tab. 1).
formation of free radicals continued, although to a
minor extent as the temperature is about 158C
lower during activity determination than during
oxidation. The presence of AAPH could thus
influence the activity of ALP either directly by
enzyme inhibition, or indirectly by inactivation
due to the amount of free radicals generated
during the 15 min of activity determination at
room temperature. To assess the influence of
AAPH on the hydrolytic activity of ALP during the
15 min of 4-MUP dephosphorylation, the produc-
tion of 4-MU by nonoxidized ALP in the absence
and presence of a final AAPH concentration of
5 mM was determined at room temperature and at
48C. The experiment at 48C was performed to keep
the production of free radicals low and to assess
principally ALP inhibition by AAPH. The hydro-
lytic activity of ALP was 19 ꢁ 5% (room tempera-
ture) and 22 ꢁ 3% (48C) lower in the presence of
5 mM AAPH compared to its activity in the
absence of AAPH. Hence, AAPH at the concentra-
tion used to induce ALP oxidation had also an
activity as enzyme inhibitor. On the other hand,
the amount of radicals generated by 5 mM AAPH
during 15 min at room temperature seems not
To vary the amount of free radicals formed and
thus to determine suitable oxidation conditions,
ALP oxidation was performed at 408C with
different AAPH concentrations (0.3–5 mM) for
various incubation times (30–180 min). As
expected, Figure 4 shows that with the increase
in the oxidant concentration there is a decrease in
the hydrolytic activity of ALP which depends also
on oxidation time. With AAPH concentrations up
to 1.25 mM no substantial ALP inactivation
could be obtained, even for incubation times up
to 180 min. A final AAPH concentration of 5 mM
and 90 min of oxidation time allowed obtaining an
optimal remaining ALP activity after oxidation of
about 50%. The oxidation conditions chosen for all
further experiments were thus ALP incubation
with 5 mM AAPH for 90 min at 408C.
Figure 4. Effect of increasing AAPH concentrations
at different oxidation times (30 min &, 60 min
,
AAPH was still present during the measure-
90 min , 120 min , and 180 min &) on the hydrolytic
ment of the hydrolytic activity of ALP and the
activity of ALP.
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BERTOLINI ET AL.
important enough to induce a detectable activity
loss of ALP as the influence of AAPH was the same
within experimental error at room temperature
than at 48C.
Table 2. Percentage of ALP Inhibition by Various
Concentrations of Antioxidants and COMT Inhibitors
%ALP inhibition at various
antioxidant concentrations (mM)
As AAPH inhibits the activity of ALP, it is
important to note that its 4-MUP dephosphoryla-
tion rate determined in the absence of oxidation
but in the presence of 5 mM AAPH during the
activity determination has to be taken as refer-
ence activity of nonoxidized ALP. Indeed, only
activity loss by peroxyl radicals should be taken
into account to assess the protection by test
compounds. Hence, the activity of ALP incubated
at 408C for 15 min (preincubation time) plus
90 min (time of oxidation) without AAPH but in
the presence of 5 mM AAPH during the 15 min of
activity determination was assumed to be 100% to
calculate the remaining hydrolytic activity of
oxidized ALP.
Compounds
6.25
12.5
25
50
125
1 Quercetin
2 Caffeic acid
3 Chlorogenic acid
4 Mangiferin
5 Resveratrol
6 Melatonin
7 Trolox
8 Ascorbic acid
9 Mannitol
10 Glutathione
11 Tolcapone
12 Entacapone
13 Nitecapone
10
0
0
0
0
0
0
0
0
0
0
0
0
22
0
5
12
5
0
0
0
0
0
35
9
13
24
16
0
0
0
0
8
44
15
31
36
32
0
0
0
0
17
41
35
38
80
33
56
60
41
0
4
0
0
28
57
55
53
10
12
8
15
18
12
In addition, the stability of the substrate
(4-MUP) in the presence of 5 mM AAPH at room
temperature was verified. No difference was found
between the hydrolysis rate of 4-MUP preincu-
bated with AAPH for 30 min and 4-MUP
incubated without AAPH for the same time and
temperature conditions. This indicates that dur-
ing the activity determination of ALP (15 min) no
4-MUP oxidation or degradation occurs. Hence,
the lower rate of hydrolysis observed after ALP
oxidation is entirely due to its loss of activity
induced by peroxyl radicals and not to 4-MUP
degradation.
antioxidant protection of ALP against peroxyl
radical-induced activity loss had to be avoided.
Therefore, the highest concentrations of test
compounds used to determine the sigmoidal
dose–response curve of antioxidant protection
inhibited less than 20% of the catalytic activity
of ALP. This means that the highest concentration
used to determine the pIC50 value of antioxidant
protection was 25 mM for chlorogenic acid (3),
resveratrol (5), and the three COMT inhibitors,
20 mM for mangiferin (4) and 10 mM for quercetin
(1), respectively.
Determination of the Preincubation Time
Antioxidant Protection Against Peroxyl
Radical-Induced ALP Activity Loss
of Test Compounds With ALP before Oxidation
The influence on the antioxidant protection of
preincubating the test compounds for 15, 30, or
60 min with ALP prior to the addition of AAPH
was assessed for compounds 3, 6, 7, 11, 12, and 13.
The same antioxidant protection of ALP against
peroxyl radical-induced damage was obtained
after a preincubation time of 15, 30, and 60 min
for all tested compounds (data not shown). Thus, a
short preincubation time of 15 min was performed
in all assays.
Inhibition of the Hydrolytic Activity of
ALP by Antioxidants
First, it was verified whether the tested com-
pounds inhibit the catalytic activity of ALP at the
concentrations used to determine their antiox-
idant protection of ALP. Table 2 shows the
percentage of ALP inhibition at antioxidant
concentrations of 6.25–125 mM. As expected, none
of the tested compounds was a good inhibitor of
ALP. Four of the tested antioxidants, melatonin
(6), trolox (7), ascorbic acid (8), and mannitol
(9) showed no inhibition of ALP at concentrations
up to 125 mM. The other compounds, (1, 2, 3, 4, 5,
10, 11, 12, and 13), inhibited the catalytic activity
of ALP, but mainly at concentrations of 25 mM or
higher (Tab. 2). The interference of ALP inhibit-
ion with the determination of pIC50 values of
Antioxidant Protection of ALP by
Reference Compounds
The antioxidant protection of ALP against free
radical-induced activity loss was determined for
ten reference compounds, quercetin (1), caffeic
acid (2), chlorogenic acid (3), mangiferin (4),
resveratrol (5), melatonin (6), trolox (7), ascorbic
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ANTIOXIDANT PROTECTION AGAINST LOSS OF PROTEIN FUNCTION
2939
acid (8), D-mannitol (9), and glutathione (10)
Table 3. Antioxidant Activities (pIC50 Values) of
Reference Antioxidants and COMT Inhibitors to Protect
ALP Against Peroxyl Radical-Induced Loss of Catalytic
Activity (n ¼ 3)
(Fig. 1). Except 5, 6, and 9 which are neutral, all
compounds were present in anionic form at the
investigated pH.
A representative dose–response curve for mel-
atonin is shown in Figure 5. Table 3 summarizes
the pIC50 (ꢂlog IC50) values of antioxidant protec-
tion of ALP obtained for the tested reference
compounds together with literature values26 of
antioxidant protection of BChE- contaminated
HSA. According to their capacity to protect ALP
from peroxyl radical-induced activity loss, they can
be divided into three classes, namely efficient
(pIC50 > 5), intermediate (4 < pIC50 ꢀ 5), and poor
antioxidants (pIC50 < 4).
All compounds bearing a catechol moiety, that
is, quercetin (1), chlorogenic acid (2), caffeic acid
(3), and mangiferin (4) were shown to be efficient
antioxidants with pIC50 values > 5. Indeed, the
presence of a catechol moiety confers higher
stability to the aroxyl radical via hydrogen
bonding or by expanded electron delocalization.41
Also resveratrol (5), a polyphenolic compound
found in red wine, was classified as an efficient
antioxidant with a pIC50 value of ALP protection
of 5.67 (Table 3), which is in good agreement
with its free radical-scavenging properties
already reported.42 Indeed, it was found to
prevent Fe2þ, Cu2þ, and AAPH-induced lipid
peroxidation in rat liver microsomes and low-
density lipoproteins with pIC50 values between
5 and 6.43,44
a
pIC50
ALP
method
HSA
method
26
Compounds
1 Quercetin
2 Caffeic acid
3 Chlorogenic acid
4 Mangiferin
5 Resveratrol
6 Melatonin
7 Trolox
8 Ascorbic acid
9 Mannitol
10 Glutathione
11 Tolcapone
12 Entacapone
13 Nitecapone
6.09 ꢁ 0.01
5.90 ꢁ 0.04
5.87 ꢁ 0.17
5.87 ꢁ 0.12
5.67 ꢁ 0.03
5.00 ꢁ 0.07
4.62 ꢁ 0.08
4.37 ꢁ 0.04
<4
5.12 ꢁ 0.02
5.41 ꢁ 0.10
5.42 ꢁ 0.07
5.05 ꢁ 0.10
n.d.
4.43 ꢁ 0.04
4.85 ꢁ 0.10
4.74 ꢁ 0.07
n.d.
<4
n.d.
n.d.
n.d.
n.d.
5.69 ꢁ 0.03
5.43 ꢁ 0.02
5.41 ꢁ 0.05
a
ꢁStandard errors calculated by GraphPad prism on the
nonlinear sigmoidal regression.
activity loss at concentrations up to 100 mM and
were therefore classified as poor antioxidants
in this screening assay. For D-mannitol and
glutathione a lack of protection at the tested
concentrations is not surprising. Indeed, D-man-
nitol is known to readily scavenge free hydroxyl
radicals in solution45 but not other oxidant species
as peroxyl radicals and peroxynitrite.46 To obtain
protection by glutathione, which is widely recog-
nized as efficient endogenous compound to protect
biological targets against oxidative stress, higher
concentrations than 100 mM might be required. It
was reported that concentrations of 250 mM or
higher were needed to prevent proteins from
peroxyl radical-induced modifications.47,48
Melatonin (6), trolox (7), and ascorbic acid
(8) bearing one or no phenolic group showed
intermediate antioxidant properties with pIC50
values between 4 and 5.
D-mannitol (9) and glutathione (10) showed no
protection of ALP against peroxyl radical-induced
A comparison of the pIC50 values of antioxidant
protection of ALP obtained with the novel screen-
ing assay with those of antioxidant protection
of BChE-contaminated HSA reported by Salvi
et al.26 shows that the same classification of
reference compounds (1, 2, 3, 4, 6, 7, and 8) was
obtained with the two assays (Tab. 3).
Antioxidant Protection of ALP by COMT Inhibitors:
an Example of Multifunctional Compounds
The antioxidant capacity of three COMT
inhibitors bearing a nitrocatecholic structure,
Figure 5. Antioxidant protection of ALP against
AAPH-induced protein oxidation by melatonin.
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2940
BERTOLINI ET AL.
nitecapone (11), entacapone (12), and tolcapone
(13) (Fig. 1) to protect proteins from peroxyl
radical-induced damage was studied using the
screening assay developed. The COMT inhibitors
showed antioxidant potencies (pIC50 values) to
protect ALP from peroxyl radical-induced activity
loss above 5 and can thus be classified as efficient
antioxidants in this assay, like the four reference
catechols and the polyphenolic resveratrol
(Tab. 3).
Moreover, the capacity of 11, 12, and 13 to
protect ALP from peroxyl radical-induced activity
loss when added after the initiation of the
oxidation reaction was examined and compared
to the one of the three reference compounds
(chlorogenic acid (3), melatonin (6), and trolox (7)).
The antioxidants at final concentrations of 15 mM
for the catechols (3, 11, 12, 13), and 125 mM for
melatonin (6) and trolox (7), respectively, were
added 15 min before (Fig. 6a) or 30 min (Fig. 6b)
and 60 min (Fig. 6c) after the addition of AAPH. As
can be seen, from the moment they are present, all
six antioxidants completely protected ALP from
radical-induced activity loss, whether they were
added before or after initiation of the oxidation.
Indeed, when the compounds were added 15 min
before AAPH, a complete protection of ALP from
radical-induced activity loss was obtained for
the concentrations investigated (Fig. 6a). When
they were added after the initiation of the
oxidation, further activity loss of ALP was stopped
(Fig. 6b and c).
According to the equation established by Niki
et al.,29 the concentration of free radicals gener-
ated by 5 mM AAPH at 408C during 90 min can
roughly be estimated at 45 mM.29 Hence, the
presence of a concentration of 15 mM of antiox-
idants with a catechol moiety (3, 11, 12, and 13)
was able to protect ALP against free radical
concentrations up to 45 mM. On the other
hand, the concentration of trolox and melatonin
had to be eight times higher to obtain the same
protection.
The good antioxidant capacity to protect pro-
teins from oxidative damage found for the three
COMT inhibitors is in good agreement with their
ROS scavenging capacities previously reported.
Indeed, entacapone and nitecapone have been
shown to scavenge nitric oxide radicals32 and
nitecapone was shown to have a remarkable
ability to act as a ROS scavenger and to inhibit
lipid peroxidation.33,34
Figure 6. Remaining ALP hydrolytic activity, when
the antioxidants tolcapone (11), entacapone (12), nite-
capone (13), chlorogenic acid (3) (final concentration of
15 mM), melatonin (6), and trolox (7) (final concentra-
tion of 125 mM) were added 15 min before the initiation
of oxidation (a) or 30 min (b) and 60 min (c) after the
initiation of oxidation.
Because of the complex pathophysiology of PD,
new therapeutic strategies privilege drugs able to
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ANTIOXIDANT PROTECTION AGAINST LOSS OF PROTEIN FUNCTION
2941
interact with several disease-related targets,
amongst other oxidative stress.49 Indeed, at
present, the generally accepted hypothesis is that
oxidative processes play an essential role in
neurodegenerative diseases and that generation
of ROS is in some way involved in causing protein
oxidation. Regarding PD, peroxyl radicals,
although surely not the initiating ROS present,
are likely to be produced subsequently, for
example, in lipid and fatty acid peroxidation
reactions initiated by hydroxyl radicals or after
abstraction of hydrogen atoms by hydroxyl
radicals followed by addition of O2 to the formed
carbon-centered radicals.37,38 As peroxyl radicals
have a relatively long half-life compared to
other ROS, which favors diffusion from its site
of generation and reaction in other cellular
compartments, they might well play a role in
disease progression of neurodegenerative diseases
such as PD. The good antioxidant properties to
protect proteins from loss of activity induced by
peroxyl radicals of entacapone and tolcapone
emerged in this work might thus provide an
enhanced symptomatic efficacy in the treatment
of PD, these compounds being able to pass the
blood brain barrier (BBB).50 On the other hand,
the antioxidant properties of nitecapone are
not relevant for central nervous system (CNS)
protection against oxidative damage since it does
not penetrate in the brain.30,51
more cost effective. Indeed, the activity of only one
enzyme (ALP) is implicated. In contrast, in the
assay of Halliwell et al. the activities of two
enzymes (a1-antiproteinase and elastase) are
involved, and in the assay proposed by Salvi
et al., the activity of a contamination of albumin is
followed as marker of protein oxidation and a
fluoresce indicator is needed to assess the activity
of this contamination. This renders these assays
more complex and more expensive than that
proposed here.
Moreover, the activity of ALP was assessed by a
96-well microplate fluorescence reader allowing
us to test several compounds in parallel with a
good precision, sensitivity, and specificity. Indeed,
the benefits of fluorimetric over spectrophoto-
metric detection are the higher sensitivity, pre-
cision and specificity, eliminating the need to
correct for nonspecific chromogens. The assay
proposed by Halliwell et al.27 is based on spectro-
photometric determination of the chromogen
p-nitroaniline by measurement of the rise in
absorbance at 410 nm. Unfortunately, the color of
natural polyphenols is frequently yellow and
interferes with the dosage of p-nitroaniline.
Regarding the fluorimertic assay using BChE-
contaminated albumin as target, by the time of its
publication precision and sensitivity were the
same as those of the ALP assay (Tab. 3). However,
in the course of the last few years the albumin
Sigma A-1887 used by Salvi et al.26 was provided
more and more pure and did no longer contain
enough BChE contamination to detect a repro-
ducible activity. Thus, the precision of the
antioxidant capacity of compounds determined
with recently bought albumin is no longer
satisfying due to the very low rates of hydrolysis.
Hence, the general advantages of the proposed
screening assay to evaluate the antioxidant
capacity of new chemical entities to protect
proteins against free radical-induced damage
are therefore the easy handling of large amounts
of samples simultaneously, the need of little
material, and the quality of the results obtained.
The developed functional assay might be a
valuable complementary method to techniques
investigating specific structural damages in the
search of new compounds able to protect proteins
from oxidative stress.
Convenience of the Developed Screening Assay to
Assess Functional Damage to Proteins
The large majority of existing screening assays to
assess antioxidant capacity of compounds to
protect proteins against ROS are based on the
investigation of structural damages.21 Only very
few in vitro methods follow the activity of a given
protein (e.g., enzyme) to determine the antiox-
idant activity of test compounds. For example,
in the assay proposed by Salvi et al. BChE-
contaminated HSA is used as target of AAPH-
induced oxidation and its esterase activity as the
marker of protein damage.26 Another functional
test, based on the spectrophotometric determina-
tion of the activity of a1-antiproteinase to inhibit
elastase, was developed by Halliwell et al.27 to
assess the peroxynitrite scavenging capacity of
test compounds.
CONCLUSIONS
Compared to the two functional tests cited
above, the screening assay developed using ALP
as model protein is simpler, easier to handle, and
The fluorimetric screening method developed to
monitor the peroxyl radical-induced loss of ALP
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2942
BERTOLINI ET AL.
activity and its antioxidant protection was shown
to be convenient, simple, and accurate. It rapidly
allowed classifying ten reference compounds
according to their antioxidant capacity to protect
proteins from oxidative damage. Hence, the
screening assay proposed might be of interest
for the identification of multifunctional hits
potentially useful for the treatment of human
diseases where free radical-induced loss of protein
function is involved, including neurodegenerative
diseases.
For example, the good antioxidant capacity of
the COMT inhibitors tolcapone and entacapone to
protect proteins from oxidative damage evidenced
in this study might be of interest for the treatment
of PD. Indeed, the development of multifunctional
compounds has become one of the new therapeutic
strategies for the treatment of aging-related
neurodegenerative diseases.
9. Boschi D, Tron GC, Lazzarato L, Chegaev K,
Cena C, Di Stilo A, Giorgis M, Bertinaria M, Frut-
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new class of products endowed with antioxidant
and vasodilator properties. J Med Chem 49:2886–
2897.
10. Gasco A, Fruttero R, Rolando B. 2005. Focus
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