Spectrochimica Acta Part A 79 (2011) 15

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

The protective effect of salicylic acid on lysozyme against riboavin-mediated

photooxidation
Kun Li a,1,2 , Hongbao Wang b,1,2 , Lingli Cheng a,2 , Hui Zhu a,2 , Mei Wang c,2 , Shi-Long Wang a,
a
b
c

School of Life Science and Technology, Department of Chemistry, Tongji University, Shanghai 200092, PR China
Department of Cardiology, Tongji Hospital, Tongji University, Shanghai 200065, PR China
Department of Chemistry, Tongji Hospital, Tongji University, Shanghai 200065, PR China

a r t i c l e

i n f o

Article history:
Received 1 July 2010
Accepted 6 December 2010
Keywords:
Salicylic acid
Riboavin
Oxidative pressure
Laser ash photolysis
Triplet state
Photosensitizer

a b s t r a c t
As a metabolite of aspirin in vivo, salicylic acid was proved to protect lysozyme from riboavin-mediated
photooxidation in this study. The antioxidative properties of salicylic acid were further studied by using
time-resolved laser ash photolysis of 355 nm. It can quench the triplet state of riboavin via electron
transfer from salicylic acid to the triplet state of riboavin with a reaction constant of 2.25 109 M1 s1 .
Mechanism of antioxidant activities of salicylic acid on lysozyme oxidation was discussed. Salicylic acid
can serve as a potential antioxidant to quench the triplet state of riboavin and reduce oxidative pressure.

1. Introduction
Riboavin, present in free form or as avin mononucleotide
(FMN) and avin adenine dinucleotide (FAD) in cells, is an
important and efcient endogenous cellular photosensitizer. The
photosensitization process is classied into two major types of
reaction. Type I involves the direct interaction of the triplet state
of photosensitizer with a substrate, and Type II involves reaction
between sensitizer and oxygen to form reactive oxygen species
(ROS). The photochemical action of a sensitizer in Type II generally refers to electron transfer and energy transfer, thereby yielding
the hydroperoxyl/superoxide ion radicals (HO2 /O2 ) and singlet
molecular oxygen (1 O2 ), respectively [13]. For riboavin, values
of 0.49 and 0.01 have been reported for the quantum yields of
formation of 1 O2 and O2 , respectively [4]. RF-sensitized photomodication of biomolecule is a complex system, showing a
mixed Type IType II photooxidative mechanism. The triplet state
of riboavin (3 RF*) can directly oxidize amino acids and proteins
[59]. ROS are involved in oxidative damage to biomolecules such
as proteins which are recognized as major biological targets of
oxidative damage [10,11]. Damages to protein were proved to be

Corresponding author. Tel.: +86 21 65982595; fax: +86 21 65982286.

E-mail addresses: liangliang2419@126.com (K. Li), chenglingli 6@hotmail.com
(L. Cheng), zhuhui979.student@sina.com (H. Zhu), wm@mail.tongji.edu.cn
(M. Wang), wsl@tongji.edu.cn (S.-L. Wang).
1
These authors contributed equally to this work.
2
Tel.: +86 21 65982595; fax: +86 21 65982286.
1386-1425/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.12.008

2010 Elsevier B.V. All rights reserved.

associated with Alzheimers disease, Parkinsons disease, cancer,

and aging [12,13].
Antioxidants have been received great attention because they
are known to lower the risk of cardiovascular and other diseases
[1416]. Phenolic compounds, a kind of widely studied antioxidants, were proved to exhibit activity for eliminating oxidizing
radicals and scavenging triplet states, and show efcient protection against protein oxidation [7,1720]. Salicylic acid (SA),
a kind of phenolic compounds, was widely used as an additive in cosmetic industry. In pharmaceutical industry, salicylic
acid was commonly employed for sterilization, anticorrosion, and
also as an intermediate for synthesis of aspirin. Some evidences
suggest that the regular, long-term use of aspirin is associated
with reduced risks for developing cancers of the esophagus,
stomach, breast, ovary and lung [21,22]. In addition, aspirin has
been demonstrated to have chemopreventative effects towards
cataracts, inammation-induced tissue damage and circulatory,
Parkinsons and Alzheimers diseases [2327], and inhibit oxidative DNA strand breaks induced by ROS [28]. The pathogenesis
of these ailments has been linked to oxidative damage to tissue,
which indicated that aspirin might have antioxidant properties
[29]. Actually, the antioxidative properties of aspirin have been
studied and it can scavenge OH generated in free solution at almost
diffusion-controlled rates [23,29,30]. However, until now comparatively little direct evidences have been made to demonstrate the
role of SA as an antioxidant in preventing protein from oxidation.
In the body a substantial amount of aspirin is hydrolyzed to SA by
esterase [31]. Therefore, it is necessary to study the photophysical
and photochemical properties of SA, which may be helpful to give a

K. Li et al. / Spectrochimica Acta Part A 79 (2011) 15

new insight to the pharmacology of aspirin. Meanwhile, it can also

explain the reason of its application in cosmetic industry. The photoionization and photoexcitation mechanisms of salicylic acid had
been studied in our previous work [32]. The aim of this work was to
elucidate the antioxidative properties of SA in riboavin-mediated
photooxidation of protein. In this study, steady-state irradiation
and SDSPAGE was utilized to display the role of salicylic acid in
protecting lysozyme against photooxidation. Laser ash photolysis
was used to investigate the reaction between the triplet state of
riboavin and salicylic acid. The mechanism of antioxidant activities of salicylic acid was also explored.
2. Experimental
2.1. Chemical reagents
Riboavin and lysozyme were obtained from Sigma and tertbutyl alcohol (t-BuOH) was purchased from Fluka. All of them
were used as received. Salicylic acid and phosphate (analytic grade
reagent) were commercially available and used without further
purication. Unless otherwise indicated, all solutions were freshly
prepared with triply distilled water, buffered with 2 mM phosphate (pH 7.2). The solutions were saturated with high-purity N2
(99.99%), N2 O, or O2 (99.5%) for different purposes by bubbling
for at least for 20 min prior to each experiment.
2.2. Steady state irradiation and sodium dodecyl
sulfatepolyacrylamide gel electrophoresis (SDSPAGE)
A 500 W Xe lamp with a lter providing the light between
330 nm500 nm was used as the light source. Solutions (total volume of 200 L) for irradiation were placed in quartz cuvettes. Each
sample was irradiated for 40 min at room temperature under the
air and buffered with 0.02 M phosphate (pH = 7.2). Irradiated protein samples were subjected to SDSPAGE which was performed
by using a vertical electrophoresis unit (Tanon VE-180). The concentrations of separation gel and stacking gel were 15% and 4.5%,
respectively. Stained gels were subsequently scanned with gel
image system (Tanon 2500R), and the bands were qualied with
Quantity one software (Tanon GIS) to get intensities.
2.3. Laser ash photolysis experiments
Laser ash photolysis (LFP) experiments were carried out using
Nd:YAG laser of 355 nm light pulses with a duration of 5 ns and the
maximum energy of 70 mJ/pulse used as the pump light source.
A xenon lamp was employed as detecting light source. The laser
and analyzing light beam passed perpendicularly through a quartz
cell with an optical path length of 10 mm. The transmitted light
entered a monochromator equipped with an R955 photomultiplier.
The output signal from the Agilent 54830B digital oscillograph was
transferred to a personal computer for data treatment. The LFP
setup has been previously described [3335].
3. Results and discussion
3.1. Steady state irradiation and SDSPAGE
The RF-sensitized lyso photooxidation has been studied by
SDSPAGE [7,20]. When lyso was irradiated by visible light in the
presence of RF, appearance of dimers (26.2 kDa) and higher aggregates were observed. In this study, four different samples were
designed to establish the damage of lyso induced by RF. As shown
in Fig. 1, lyso and SA show nearly no absorption in the region
330500 nm. Therefore, under the irradiation of the light the main

Fig. 1. Absorption spectrum of lysozyme, salicylic acid and riboavin in neutral

aqueous solution.

energy of the light was absorbed by RF. As shown in lane 2 (Fig. 2A),
the light controlled in our experiment cannot cause damage to lyso.
Compared with lane 1, without irradiation RF also caused a little
damage to lyso, which was probably because of the effect of natural
light. However, under the irradiation RF showed obvious damage
to lyso. In the SDSPAGE study, the appearance of the bands more
than 26 kDa indicated that the damage induced by 3 RF* caused the
cleavage of lysozyme and then the produced peptide fragments can
further react with the dimer or other fragments to produce the band
of higher aggregates.
In order to evaluate the protective effect of SA on RF-sensitized
lyso photooxidation, different concentrations of SA were added and
the result was shown in Fig. 2B. Obviously, the intensity of aggregation band was reduced with the increase of SA concentration.
Densitometric analysis of the aggregation bands is presented in
Fig. 2C. The control group was the irradiated mixture of lyso and
RF without SA. The extent of the protein damage was evaluated
according to the intensity of the rst aggregation band of each sample (the dimer 26.0 KDa). The damage extent of positive control was
set as 100%, and all the other samples were compared to the positive
control via their band intensities. Values were presented as means
of three independent experiments, and for each experiment, the
standard errors were less than 10%. In the presence of SA, aggregation of protein was efciently alleviated. The band intensity of
the dimer was reduced by 53% and the band of higher aggregates
nearly disappeared when the concentration of SA was increased to
0.5 mM. Conspicuously, the RF-induced photo-oxidization to lyso
characterized by protein fragmentation and cross-linking can be
alleviated by SA.
3.2. Reaction between triplet riboavin and salicylic acid
To gain insight into the mechanism of antioxidant activities of SA
on riboavin-mediated photooxidation of lysozyme, the reaction
between SA and 3 RF* was investigated by LFP.
Under the irradiation of UV or visible light, triplet state of
riboavin (3 RF*) can be generated. The absorption of salicylic acid
shows that SA has no absorption at 355 nm (Fig. 1B). So the laser
energy was mainly absorbed by RF. In this condition, RF was initially exited by 355 nm laser to generate 3 RF*. The absorption peaks
at 300 nm, 380 nm, 520 nm and 680 nm should be assigned to the
typical absorption of 3 RF* (shown in Fig. 4). This was also in good
agreement with the reported absorption of triplet state of riboavin
and FMN [36,37].

K. Li et al. / Spectrochimica Acta Part A 79 (2011) 15

Fig. 2. (A) SDSPAGE proles of the RF-sensitized lyso photooxidation. Lane 1: lyso (0.25 mM); Lane 2: lyso (0.25 mM) + irradiation; Lane 3: lyso (0.25 mM) + RF (0.1 mM); Lane
4: lyso (0.25 mM) + RF (0.1 mM) + irradiation. (B) SDSPAGE proles of the solutions of lyso (0.25 mM) and RF (0.1 mM) containing different concentrations of SA (00.75 mM)
irradiated for 40 min. (C) The densitometry analysis of the bands of dimerized protein obtained from gels shown in panel B (n = 3).

The absorption of 3 RF* at 680 nm was further conrmed by O2

and N2 O. The kinetic decay curve at 680 nm was obtained from
355 nm LFP of 0.1 mM RF neutral aqueous solution saturated with
N2 , O2 and N2 O, respectively (Fig. 3). The decay at 680 nm was
quenched quickly by O2 , but hardly inuenced by N2 O with t-BuOH.
Therefore, the absorption at 680 nm was assigned to 3 RF* rather
than hydrated electron.
As shown in Fig. 4, following the decay of 3 RF*, a maximum
absorption at 320 nm and a broad absorption around 510 nm

Fig. 4. Transient absorption spectra obtained from 355 nm LFP of 0.2 mM RF and
0.5 mM SA neutral aqueous solution saturated with N2 recorded at: 0.1 s () and
5 s ( ). Inset: transient time proles at (a) 510 nm; (b) 680 nm; (c) the buildup
trace of 510 nm obtained by subtracting trace (b) from trace (a).

Fig. 3. The kinetic decay curve observed at 680 nm from 355 nm LFP of (a) N2 saturated, (b) N2 O-saturated with 0.2 M t-BuOH, or (c) O2 -saturated 0.1 mM RF
neutral aqueous solution, respectively.

appeared. These two absorption bands agree very well with the
absorption of reduced neutral avin radicals (RFH or FADH )
reported previously by pulse radiolysis or laser photolysis experiments [38,39]. Therefore, it could be conrmed that SA was
oxidized by 3 RF* via electron transfer from SA to 3 RF* to pro-

K. Li et al. / Spectrochimica Acta Part A 79 (2011) 15

Fig. 5. Transient time proles at 680 nm from 355 nm LFP of aqueous solution
containing 0.2 mM riboavin deoxygenated with N2 at pH 7.0 in the presence of
different concentrations of SA. (a): Without SA; (b): 0.05 mM SA; (c): 0.4 mM SA.
Inset: dependence of the observed decay constant for 3 RF* at 680 nm (kobs ) on the
SA concentrations.

duce RF anion radical (RF ) and salicylic cation radical (SAH+ ).

Under our experimental conditions (pH = 7), the neutral radical
RFH (pKa = 8.3) [38] and neutral radical (SA ) were then formed
by protonation of RF and deprotonation of SAH+ (pKa = 2.95)
[32]. Considering that the absorption of RFH was overlapped by
that of 3 RF*, the buildup trace at 510 nm was obtained by subtraction of decay curve at 680 nm from decay curve at 510 nm (inset
of Fig. 4). The result showed that the formation of RFH was synchronic with the decay of 3 RF* at 680 nm. However, the absorption
of SA at 390 nm [32] was hardly observed probably because of its a
low absorption coefcient and the effect of bleaching. The possible
reactions in the system were shown as below (Eqs. (1) and (2)):
h

RF3 RF
3 RF +SA
3 RF +SA

(1)

RF + SAH

RFH + SA

(2)

The quenching of 3 RF* by SA was observed by monitoring the

decay of 3 RF* at 680 nm. The decay at 680 nm was obviously accelerated in the presence of SA (Fig. 5). The observed decay constants
for 3 RF* at 680 nm (kobs ) increased with a rate roughly proportional
to the concentration of SA (inset of Fig. 4). The reaction rate constant was obtained using the equation kobs = k0 + kq [SA]. From plots
of kobs vs. SA concentrations [SA] (0.51 mM), the rate constant (kq )
was determined to be 2.25 109 M1 s1 .
3.3. Mechanism of antioxidant activities of salicylic acid
In this work, RF absorbed UVA light and turned into 3 RF*. 3 RF*
can directly oxidize the tryptophan (TrpH) and tyrosine (TyrOH)
residues in lyso via electron transfer from amino acids to 3 RF* giving corresponding amino acid radicals. Under the aerobic condition,
3 RF* can be quenched by O via energy transfer (to generate 1 O )
2
2
and electron transfer (to generate O2 ) [37]. 1 O2 is a strong oxidizing agent and can induce further oxidation of protein. Riboavin
sensitized photochemical damage to lysozyme is highly selective
and dependent on the degree of amino acid residues exposure to
the solvent [6]. Lyso has six Trp residues, four of which are exposed
to the solvent. TrpH residues are reported more vulnerable to the
3 RF* and 1 O than Tyr residues in proteins [41]. It was proposed
2
that TrpH was oxidized initially to form Trp radicals (Trp ). Tyrosyl
radicals (TyrO ) were then generated via long-range intramolecu-

Scheme 1. The possible mechanism of antioxidant activities of salicylic acid on

riboavin-mediated photooxidation of lysozyme.

lar electron transfer from TyrOH to Trp within the protein [40].
The resultant TyrO in lyso could form dityrosine through dimerization which was the main reason of lyso aggregation. In a word,
both 3 RF* and 1 O2 were responsible for the photo-damage of lyso
induced by RF in the presence of O2 .
When SA was added, SA could quench 3 RF* in competition with
O2 and lyso. The reaction rate constants of 3 RF* with lyso and
O2 are 9.1 108 M1 s1 and 4 108 M1 s1 , respectively [7,37].
The rate constant of scavenging 3 RF* by SA was determined as
2.25 109 M1 s1 , which suggested that SA showed a little predominance over lyso and O2 in the competitive reaction with 3 RF*.
Therefore, both 3 RF* and 1 O2 could be reduced in this competitive
reaction and the oxidation of lyso by 3 RF* and 1 O2 was thereby
alleviated. Previous reports showed that antioxidant could repair
damaged protein by electron transfer from antioxidant to amino
acid radicals and quench 1 O2 via charge transfer reaction mechanism [17,20,42,43]. Therefore, two probable mechanisms could be
supposed that SA protected lyso against riboavin-mediated photooxidation by trapping 1 O2 and repairing amino acid radicals in
the lyso. The possible mechanism for the protective effect of SA is
summarized in Scheme 1.
4. Conclusion
The anti-oxidative properties of SA in riboavin-mediated photooxidation of lyso were studied by using steady state analysis
method together with laser ash photolysis in this work. The
results showed that SA could protect lysozyme from photooxidation induced by RF. Kinetic results suggested that SA could
efciently quench 3 RF*. In addition, in the presence of O2 SA could
inhibit the generation of 1 O2 in the competitive reaction with 3 RF*,
and thereby reduced the oxidative pressure induced by ROS. All
these results suggested that SA might be considered as a potential
photo-antioxidant. As mentioned above, in the body aspirin mainly
turned into SA. So an inference could be made that the antioxidant
properties of aspirin in vivo, at least partly, resulted from SA. Meanwhile, the anti-oxidative properties of SA in protein photooxidation
reminded us that the application of SA in cosmetic industry might
be associated with its protection of skin from photooxidation.
Acknowledgements
The work was supported by the 973 project of the Ministry
of Science and Technology (No. 2010CB912604, 2010CB933901),

K. Li et al. / Spectrochimica Acta Part A 79 (2011) 15

Genetically modied organisms breeding major projects (No.

2009ZX08011-032B), Shanghai key laboratory of cell signaling and
diseases (Grant No 09DZ2260100) and Doctoral Program of Higher
Education of China (Grant No.20090072120019).
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