Modification of quercetin with l-cysteine by horseradish peroxidase

Article abstract of DOI:10.1080/10242422.2016.1247829

Horseradish peroxidase is a well-known member of the peroxidase family that catalyzes oxidation of flavonoids and phenolic substrates to free phenoxyl or semiquinone radicals. Aim of this study was to investigate in vitro oxidation of quercetin by horseradish peroxidase in the presence of l-cysteine as nucleophilic agent, and its influence on previously formed semiquinone- and quinone-type metabolites. The obtained results showed that in the reaction without l-cysteine several products were present, such as quercetin quinone methide, phloroglucinol carboxylic acid, protocatechuic acid, as well as quercetin heterodimer and derivates of quercetin heterodimer. On the other hand, in the presence of l-cysteine only three products were obtained, quercetin quinone methide and two new isomeric mono-cysteine derivatives of quercetin with mass exp. m/z 420.04 ± 0.1 [quercetin + cysteine–H]^– (theor. m/z 420.0389 [quercetin + cysteine–H]^–).

Full text of DOI:10.1080/10242422.2016.1247829

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Modification of quercetin with l-cysteine by
horseradish peroxidase
Sasa Savic, Silvio Keckes & Zivomir Petronijevic
To cite this article: Sasa Savic, Silvio Keckes & Zivomir Petronijevic (2016): Modification of
quercetin with l-cysteine by horseradish peroxidase, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2016.1247829
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Date: 22 November 2016, At: 11:51

Biocatalysis and Biotransformation, 2016; Early Online: 1–10
RESEARCH ARTICLE
Modification of quercetin with L-cysteine by horseradish peroxidase
SASA SAVIC1, SILVIO KECKES2 & ZIVOMIR PETRONIJEVIC1
2
1Faculty of Technology, University of Nis, Leskovac, Serbia and Analysis, Belgrade, Serbia
Abstract
Horseradish peroxidase is a well-known member of the peroxidase family that catalyzes oxidation of flavonoids and phenolic
substrates to free phenoxyl or semiquinone radicals. Aim of this study was to investigate in vitro oxidation of quercetin by
horseradish peroxidase in the presence of L-cysteine as nucleophilic agent, and its influence on previously formed
semiquinone- and quinone-type metabolites. The obtained results showed that in the reaction without L-cysteine several
products were present, such as quercetin quinone methide, phloroglucinol carboxylic acid, protocatechuic acid, as well as
quercetin heterodimer and derivates of quercetin heterodimer. On the other hand, in the presence of L-cysteine only three
products were obtained, quercetin quinone methide and two new isomeric mono-cysteine derivatives of quercetin with mass
exp. m/z 420.04 ꢀ 0.1 [quercetin + cysteine–H]– (theor. m/z 420.0389 [quercetin + cysteine–H]–).
Keywords: Quercetin modification, horseradish peroxidase, L-cysteine adducts, UHPLC-DAD-HESI-MS/MS
Introduction
as acute cytotoxicity, immunotoxicity and carcino-
genesis (Awad et al. 2001).
Quercetin (3,30,40,5,7-pentahydroxyflavone) is one of
the most abundant plant-derived polyphenols and
one of the most studied flavonoids (Awad et al. 2000;
Michels et al. 2004; Andrade-Filho et al. 2009). It
contains 3’,4’-dihydroxy structure of the B ring
(catechol B ring) and possesses a high-antioxidant
ability expressed through scavenging of free radicals.
However, although quercetin possesses a high-anti-
oxidant and free radical scavenging ability, sometimes
it can also act as pro-oxidant (Awad et al. 2000;
Nijveldt et al. 2001; Andrade-Filho et al. 2009). This
is possible because flavonoids have a catechol moiety
which provides potential for their efficient autoxida-
tion and/or enzymatic one- as well as two-electron
oxidation leading to the formation of semiquinone-
and quinone-type metabolites (Awad et al. 2001;
Andersen and Markham 2006). Resulting free phe-
noxyl, semiquinone and quinone radicals are Michael
acceptors which have electrophilic properties that can
bind cellular macromolecules (Bolton et al. 1998). In
addition, they are toxicological intermediates which
can create a variety of hazardous effects in vivo, such
Due to the abundance of some oxidases (poly-
phenol oxidase and peroxidase), process of flavonoid
oxidation in plant tissues is commonly present
(Pourcel et al. 2006; Kabeya et al. 2007). Incurred
products of oxidation can spontaneously react with
phenols, amino acids or proteins, yielding a complex
mixture of brown products. This process (known as
browning) is generally considered as harmful pro-
cess, because it can cause undesirable organoleptic
characteristics of the product, which may negatively
affect its nutritional value (Pourcel et al. 2006). This
information indicates that it is necessary to stop this
unwanted reactions, but the question is how, and is it
possible?
The knowledge about electrophilic properties of
previously formed semiquinone- and quinone-type
metabolites opens the possibility for studying the toxic
metabolites in the presence of a nucleophile which has
capability to scavenge the semiquinone radical and
reduce the adverse effects of created radicals. In
previous studies, flavonoid oxidation by peroxidases,
in the presence of thiol compound as nucleophile, has
Correspondence: Sasa Savic, Faculty of Technology, University of Nis, Leskovac, Serbia. Tel: +381 16247203. Fax: +381 16242859. E-mail:
sasa.savic@tf.ni.ac.rs
(Received 11 April 2016; accepted 4 August 2016)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2016 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.1080/10242422.2016.1247829

2
S. Savic et al.
been examined (Awad et al. 2000, 2001, 2002).
Obtained results indicate that glutathione has a
capacity to scavenge the obtained semiquinone rad-
ical, regenerate the flavonoid and generate oxidized
glutathione and reactive oxygen species responsible
for the toxicity (Galati et al. 1999; Metodiewa et al.
1999; Awad et al. 2000, 2002).
Enzymatic reaction was performed in a manner
that phosphate buffer and water were always the first
added in a tube. Then, the solution of quercetin was
added, but not much earlier before the start of the
reaction, due to sensitivity to light. Thereafter, the
solution of L-cysteine was added, followed by the
solution of HRP, and finally the reaction began by
adding the solution of H2O2 with efficient mixing on
the vortex. The assay medium (5 mL) contained
80 mM quercetin, 0.4 mM L-cysteine (when it was
present), 0.1 mM hydrogen peroxide (H2O2),
0.8 nM HRP, and 50 mM sodium phosphate buffer
(pH 7.0). The blank reference contained all reagents
except the hydrogen peroxide, which was replaced by
distilled water.
The aim of this study was to investigate in vitro
oxidation of quercetin by horseradish peroxidase
(HRP, EC 1.11.1.7.) in the presence of L-cysteine, as
well as the influence of L-cysteine on previously
formed semiquinone- and quinone-type metabolites
in oxidation reaction of quercetin. We selected
L-cysteine because it is semi-essential a-amino acid
that is present in plant tissues and human body. It
has a good potential as nucleophilic agent due to a
thiol group present in the cysteine structure (O’Hair
et al. 1998). We believe that results from this study
could be beneficial because a new compound formed
between L-cysteine and quercetin may possess a
better solubility in aqueous solutions and, at the
same time, can keep all positive properties of
quercetin. In addition, to the best of our knowledge,
enzymatic modification of quercetin with L-cysteine
using HRP has not been reported to date.
UHPLC-DAD-HESI-MS/MS analysis
UHPLC-DAD-HESI-MS/MS analysis was per-
formed by using a Thermo Scientific liquid chroma-
tography system (UHPLC) composed of
a
quaternary pump with a degasser, a thermostated
column compartment, an autosampler, and a diode
array detector connected to LCQ Fleet Ion Trap
Mass Spectrometer (Thermo Fisher Scientific, San
Jose, CA) equipped with heated electrospray ionisa-
tion (HESI). Xcalibur (version 2.2 SP1.48) and
LCQ Fleet (version 2.7.0.1103 SP1) software were
used for instrument control, data acquisition, and
data analysis. Separations were performed on a
Hypersil gold C18 column (50 ꢂ 2.1 mm, 1.9 mm)
obtained from Thermo Fisher Scientific.
Materials and methods
Chemicals and preparation of sample solutions
Horseradish peroxidase (298 U/mg; using pyrogal-
lol), quercetin, dimethyl sulfoxide (DMSO) and
L-cysteine were obtained from Sigma–Aldrich
Chemie GmbH (Steinheim, Germany). Acetonitrile
and water were purchased from Fisher Scientific
(Loughborough, UK) (LC-MS and HPLC grade,
respectively). Formic acid was from Carlo Erba
Reagents S.r.l. (Cornaredo, Italy).
UHPLC-DAD-HESI-MS/MS analysis was per-
ˇ
ˇ
formed according to Keckes et al. (2013) with slight
modifications. The mobile phase consisted of (A)
water +0.2% formic acid and (B) acetonitrile.
A linear gradient program at a flow rate of
0.250 mL/min was used 0–2 min from 10% to 20%
(B), 2–4.5 min from 20% to 90% (B), 4.5–4.8 min
for 90% (B), 4.8–4.9 min from 90% to 10% and 4.9–
12.0 min for 10% (B). The injection volume was
10 mL, and the column temperature was maintained
at 25 ꢃC. The separated compounds were detected at
a wavelength of 230, 270, 320, and 360 nm, and each
online spectrum was recorded within the range of
200–800 nm. The mass spectrometer was operated
in a negative mode. HESI-source parameters were as
follows: source voltage 4.5 kV, capillary voltage –48 V,
tube lens voltage –100.40 V, capillary temperature
300 ꢃC, sheath and auxiliary gas flow (N2) 32 and 8
(arbitrary units), respectively. MS spectra were
acquired by full-range acquisition covering 150–700
m/z. For fragmentation study, a data-dependent scan
was performed by deploying the collision-induced
dissociation (CID). The normalized collision energy
A stock solution of quercetin (10 mM) and the
L-cysteine (20 mM) were prepared in DMSO and
distilled water, respectively. A 2 mM stock solution of
HRP was prepared by dissolving the 0.34 mg of the
solid HRP in 10 mL of cold 50 mM phosphate buffer
pH 6.0. The enzyme concentration was calculated
using a e403 ¼102.0 mMꢁ1cmꢁ1
.
Spectrophotometric assays
The UV–VIS assays were carried out using VARIAN
Cary-100 Spectrophotometer controlled with
VARIAN Cary-100 UV-Winlab software, in quartz
cuvettes dimensions 1 ꢂ 1 ꢂ 4.5 cm. The total
amount of the reaction mixture was 5 mL. The
reaction mixture was scanned at wavelengths of
200–800 nm. All measurements were performed at
20 ꢃC. The blank contained 100 mM phosphate
buffer pH 7.0.

Modification of quercetin with L-cysteine by horseradish peroxidase
3
of the CID cell was set at 25 eV. All compounds were
identified according to the corresponding spectral
characteristics: mass spectra, accurate mass, and
characteristic fragmentation.
371 nm, as well as the formation of the already
existing absorption band at 257 nm, were observed.
Based on this, it can be concluded that quercetin is a
substrate for HRP, and as Makris and Rossiter
(2002) reported, the obtained products are probably
semiquinone- and quinone-type metabolites.
In order to identify products formed during the
reaction of quercetin oxidation by HRP, UHPLC-
DAD-HESI-MS/MS method was used. UV-chro-
matogram of quercetin incubation in the presence of
HRP is shown in Figure 2. The results obtained by
the reaction of quercetin oxidation by HRP revealed
that several products were present. The identified
compounds from the reaction of quercetin oxidation
by HRP, labeled as peaks 1–7 following the elu-
tion order in the UV chromatogram, are shown
in Table 1.
Results and discussion
Oxidation of quercetin by HRP
Based on previous studies, it is known that absorp-
tion spectrum of quercetin expresses two major
peaks, one belonged to the band I as a result of
absorption of catechol B-ring in UV-A range,
between 360 and 370 nm (lmax ¼ 365 nm,
e ¼ 28.400 Mꢁ1 cmꢁ1), and the other one belonged
to the band II as a result of absorption of benzoyl
A–C system in UV-C range, around 260 nm (lmax
¼ 256 nm, e ¼ 28.300 Mꢁ1 cmꢁ1) (Fenoll et al. 2003;
Dehghan et al. 2011).
The first two peaks labeled as 1 and 1’ at retention
time 5.55 and 5.79 min and mass at m/z 329.14 and
329.17, respectively, were identified as derivates of
quercetin quinone methide. MS fragmentation of
these two peaks with mass m/z 329.14 and 329.17
[quercetin quinone methide +2CH3–H]– yielded two
ion fragments at m/z 299 [quercetin quinone
methide–H]– and m/z 151 [quercetin quinone
methide–148–H]– that is characteristic ion fragment
Figure 1 shows the change in absorption spectrum
of the reaction mixture during the reaction of
quercetin oxidation by HRP. By analyzing the UV–
VIS absorption spectrum of this reaction mixture,
the presence of two absorption bands at 371 and
255 nm (Figure 1) can be noted. By comparing the
shape and position of these bands with previously
published results, it can be concluded that they
originate from quercetin (Fenoll et al. 2003;
Dehghan et al. 2011). During the time, the decrease
of absorption bands that originate from quercetin, at
ˇ ˇ
for quercetin (Keckes et al. 2013). Peak 2 with mass
at
m/z
210.07
[phloroglucinol
carboxylic
acid + acetonitrile–H]– after MS fragmentation
yielded two ion fragments at m/z 169 [phloroglucinol
Figure 1. The UV–VIS absorption spectrum of the reaction medium during the reaction of quercetin oxidation by HRP.

4
S. Savic et al.
Figure 2. UV chromatogram reaction of quercetin oxidation by HRP.
Table 1. Identified products of quercetin oxidation by HRP in absence of L-cysteine.
m/z [M–H]–
Peak no.
tR, min
MS/MS
Compound
1, 10
2
5.55, 5.79
6.67
329.14, 329.17
210.07
299, 151
169, 126
Quercetin quinone methide +2CH3 groups
Phloroglucinol carboxylic acid + acetonitrile
from mobile phase
3
4
5
6
6.93
7.04
7.34
7.47
453.25
617.11
646.40
601.13
299.04, 299.11, 299.24
299, 153, 151, 109
601, 301
601, 301
301
Quercetin quinone methide + protocatechuic acid
Quercetin heterodimer + OH group
Quercetin heterodimer +3CH3 groups
Quercetin heterodimer
7, 70, 700
7.64, 7.73, 7.82
151
Quercetin quinone methide I, II and III
carboxylic acid–H]– that represent quasi-molecular
ion for phloroglucinol carboxylic acid and at m/z 126
[phloroglucinol carboxylic acid–43–H]– that is char-
acteristic ion fragment for this trihydroxybenzoic
acid. Peak 3 with mass m/z 453.25 [quercetin
quercetin heterodimer at m/z 601 [quercetin hetero-
dimer–H]– and ion fragment at m/z 301.2 [quercetin
heterodimer–300–H]–, representing quercetin, as
well as ion fragment at m/z 272 [quercetin hetero-
dimer–329–H]–. Most probably this ion fragment is
result of quercetin quinone cleavage, formed through
autoxidation and/or disproportionation, following by
CO removal, which gives benzofuran derivative
(Gu¨ls¸en et al. 2007). Due to the presence of two
ion fragments characteristic for quercetin, at m/z 151
[quercetin quinone methide–148–H]– and m/z 179
[quercetin quinone methide ꢁ120 ꢁ H]ꢁ, last three
peaks labeled as 7, 7’, and 7’’ at retention times 7.64,
7.53, and 7.82 min, with mass m/z 299.04, 299.11,
and 299.24 [quercetin quinone methide–H]–,
were identified as quercetin quinone methide I, II,
and III, respectively. Presence of identified com-
pounds in the reaction of quercetin oxidation
by HRP was in accordance with the previously
reported results (Schreiber 1974; Ma et al. 1997;
Makris and Rossiter 2002; Gu¨ls¸en et al. 2007).
quinone
methide + protocatechuic
acid–H]–
yielded ion fragments at m/z 299 [quercetin quinone
methide–H]–, m/z 151 [quercetin quinone methide–
148–H]– – characteristic ion for quercetin, m/z 153
[protocatechuic acid–H]– that represent quasi-
molecular ion of protocatechic acid, and ion frag-
ment at m/z 109 [protocatechuic acid–44–H]–
–
characteristic ion for protocatechuic acid as the result
of CO2 loss. Peaks 4, 5 and 6 were identified as
derivates of quercetin heterodimer. After MS frag-
mentation of peaks 4 and 5, quasi-molecular ion of
quercetin heterodimer at m/z 601 and its ion
fragment at m/z 301 [quercetin heterodimer–300–
H]– were observed, indicating that these two com-
pounds (peaks 4 and 5) are derivates of quercetin
heterodimer. Peak 6 gave quasi-molecular ion of

Modification of quercetin with L-cysteine by horseradish peroxidase
5
Figure 3. Proposed structures of identified compounds in reaction of quercetin oxidation by HRP.
Proposed structures of identified compounds in the
reaction of quercetin oxidation by HRP are displayed
in Figure 3.
absorption bands: first at 295 and second at 258 nm
´
(Wojtas-Wasilewska et al. 1983). Hence, the appear-
ance of two absorption maximums at 296 and
257 nm in UV–VIS absorption spectrum of investi-
gated reaction mixture can be explained by the
formation of protocatechuic acid as one of the
reaction products. In addition, phloroglucinol car-
boxylic acid which possesses two absorption
Taking into account the absorption spectra of
identified compounds, the changes in UV–VIS
absorption spectrum of reaction of quercetin oxida-
tion by HRP could be explained (Figure 1). Namely,
it is known that protocatechuic acid shows two

6
S. Savic et al.
Figure 4. The UV–VIS absorption spectrum of the reaction medium during the reaction of quercetin oxidation by HRP in the presence of
L-cysteine.
Figure 5. UV chromatogram of the reaction of quercetin oxidation by HRP in the presence of L-cysteine.
Oxidation of quercetin by HRP in the presence
of L-cysteine
maximums at 296 and 260 nm was also identified in
the reaction mixture. Thus, it can be concluded that
the presence of phloroglucinol carboxylic acid was
additionally responsible for the increase of absorp-
tion maximums at 296 and 257 nm in UV–VIS
absorption spectrum of reaction mixture.
In order to identify the difference between the
reaction of quercetin oxidation by HRP in the
absence and in the presence of L-cysteine, both
reactions were performed under identical conditions.

Modification of quercetin with L-cysteine by horseradish peroxidase
7
Figure 6. TIC chromatogram of reaction of quercetin oxidation by HRP in the presence of L-cysteine (A); MS fragmentation of two new
monoisomeric cysteinyl-quercetin products (B).
Figure 4 shows the changes in UV–VIS absorption
spectrum of reaction of quercetin oxidation by HRP
in the presence of L-cysteine. By analyzing the
changes in UV–VIS absorption spectrum, it can be
concluded that the decrease of absorption bands (at
371 and 255 nm) which originate from quercetin was
occurred during the reaction. At the same time, the
formation of two new absorption maximums at 295
and 332 nm, between isobestic points (271 and
343 nm), was observed, which indicates on the loss
of conjugation between A and B rings.
of this reaction is shown in Figure 5. Based on the
obtained results, it can be concluded that only three
products from previous reaction (in the absence of
L-cysteine, Figure 2) at retention times 5.81 and
6.92 min, with mass at m/z 329.19 [quercetin quin-
one methide +2CH3–H]– and m/z 301.20
[quercetin ꢁ H]ꢁ, respectively, and one new peak at
retention time 1.10 min, were present in this reac-
tion. However, the results obtained from mass
spectrometer confirmed that, actually, there were
two new products (Figure 6A), with similar retention
times at 1.01 and 1.12 min and almost identical MS
fragmentation (Figure 6B). Based on their masses
and MS fragmentations, it can be concluded that two
new quercetin adducts with L-cysteine were formed
As in the previous section, for identification of
reaction products of quercetin oxidation by HRP in
the presence of L-cysteine, UHPLC-DAD-HESI-
MS/MS method was used. The UV-chromatogram

8
S. Savic et al.
Figure 7. Proposed mechanism of quercetin modification with L-cysteine by HRP (According to Awad et al., 2000).

Modification of quercetin with L-cysteine by horseradish peroxidase
9
(exp. m/z 420.04 ꢀ 0.1 [quercetin + cysteine–H]–,
Conclusions
theor. m/z 420.03895 [quercetin + cysteine–H]–),
where ion fragments at m/z 301.1 and m/z 299.2
originate from quercetin and quercetin quinone
methides, respectively.
In summary, the results obtained from this study
showed that in the reaction of quercetin modification
with L-cysteine by HRP there are two new isomeric
products (exp. m/z 420.04 ꢀ 0.1 [quercetin + cyst-
eine–H]ꢁ), confirming that in this reaction we have
the formation of two isomeric mono-cysteine deriva-
tives of quercetin. According to our knowledge, this
is the first report about products of enzymatic
modification of quercetin with L-cysteine by HRP
and influence of L-cysteine on previously formed
o-quinones. Additionally, in the presence of L-cyst-
eine the amount of previously formed o-quinones
and quercetin quinone methides was significantly
lower, which suggests that L-cysteine has the ability
to scavenge o-quinones and its derivates. Bearing in
mind the hazardous effects of o-quinones, these
results could be particularly of importance for future
investigations and applications.
In addition, UV–VIS spectra of these two new
products were also similar and revealed an absorb-
ance peak at 292.9 and 293.1 nm, respectively
(Figure 6A, increment). Presence of this absorption
maximums at 293 nm in UV–VIS spectra of both
products was the best explanation for the formation
of absorption maximum at 295 nm in UV–VIS
absorption spectrum of reaction mixture when oxi-
dation of quercetin by HRP was performed in the
presence of L-cysteine (Figure 4).
From the Figure 5 it can be concluded that the
amount of other products was smaller, which
indicates that L-cysteine, like glutathione (Awad
et al. 2000), has the ability to scavenge the
previously obtained oxidation products of quercetin.
As can be seen, the products of reaction between
cysteine and by-products that were present in the
reaction of quercetin oxidation by HRP (Table 1;
Figures 1–3) not detected by UHPLC-DAD-HESI-
MS/MS method. The explanation for this
result could be that cysteine likely reacted
with the quercetin quinone methides before they
had the opportunity to undergo such secondary
reactions.
Declaration of interest
The authors report no declarations of interest.
This work was supported by the Ministry of
Education and Science of the Republic of Serbia
under Project No. TR 34012.
Position of two new compounds in UV-chro-
matogram can be explained by the fact that binding
of L-cysteine with the quercetin increases their
hydrophilicity because L-cysteinyl residue possesses
two hydrophilic (carboxyl and amino) groups. It is
important to note that the reaction of quercetin
modification with L-cysteine in the absence of HRP
was also investigated, where new quercetin adducts
with L-cysteine were not noticed. Based on this, it
can be concluded that HRP is responsible for the
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