Interaction of retinoic acid radical cation with lysozyme and antioxidants: Laser flash photolysis study in microemulsion

Article abstract of DOI:10.1111/php.12128

All-trans retinoic acid (ATRA) plays essential roles in the normal biological processes and the treatment of cancer and skin diseases. Considering its photosensitive property, many studies have been focused on the photochemistry of ATRA. In this study, we investigated the transient phenomena in the laser flash photolysis (LFP) of ATRA in microemulsion to further understand the photochemistry of ATRA. Results show that 355 nm LFP of ATRA in both acidic and alkaline conditions leads to the generation of retinoic acid cation radicals (ATRA^?+) via biphotonic processes. The employment of microemulsion system allows us to investigate the reaction of hydrophobic ATRA^?+ with molecules of different polarity. Therefore, we studied the reaction activity of ATRA^?+ to many hydrophobic and hydrophilic molecules. Results show that ATRA^?+ can efficiently interact with lysozyme, tyrosine, tryptophan and many antioxidants, such as curcumin (Cur), vitamin C (VC) and gallic acid (GA). The apparent rate constants of these reactions were measured and compared. These findings suggest that ATRA^?+ is a reactive transient product which may pose damage to lysozyme, and antioxidants, such as Cur, VC and GA, may inactivate ATRA^?+ by efficient quenching reactions. 355 nm laser flash photolysis of all-trans retinoic acid (ATRA) in microemulsion leads to the formation of retinoic acid cation radicals (ATRA^?+) via biphotonic processes. Deprotonated form of ATRA is more favorable for the formation of ATRA^?+. ATRA^?+ is proved to be reactive to lysozyme, tyrosine and tryptophan which is suggestive of its destructive effect on proteins. Meanwhile, some antioxidants, such as curcumin, gallic acid and vitamin C, can efficiently interact with ATRA^?+, which indicates that it may competitively protect proteins from the attack of ATRA^?+ by inactivating free radical.

Full text of DOI:10.1111/php.12128

Photochemistry and Photobiology, 2013, 89: 1064–1070
Interaction of Retinoic Acid Radical Cation with Lysozyme and
Antioxidants: Laser Flash Photolysis Study in Microemulsion
1
2
1
1
1
1
3
Kun Li , Mei Wang , Ting Wang , Dongmei Sun , Rongrong Zhu , Xiaoyu Sun , Xianzheng Wu*
1
and Shi-Long Wang*
1
Tenth People’s Hospital, School of Life Science and Technology, Tongji University, Shanghai, China
Department of Chemistry, Tongji University, Shanghai, China
Department of Emergency, Shanghai Tongji Hospital, Shanghai, China
2
3
Received 28 December 2012, accepted 25 June 2013, DOI: 10.1111/php.12128
•
+
ABSTRACT
of ATRA by peroxyl radicals and ATRA had efficient reaction
•
+
activity to ascorbate (17). The interaction of ATRA with ascor-
bate implies that ATRA is a reactive substance which may
pose oxidative damage to other biomolecules such as proteins.
Therefore, further studies are needed to investigate the reactivity
All-trans retinoic acid (ATRA) plays essential roles in the
normal biological processes and the treatment of cancer and
skin diseases. Considering its photosensitive property, many
studies have been focused on the photochemistry of ATRA.
In this study, we investigated the transient phenomena in the
laser flash photolysis (LFP) of ATRA in microemulsion to
further understand the photochemistry of ATRA. Results
show that 355 nm LFP of ATRA in both acidic and alkaline
conditions leads to the generation of retinoic acid cation radi-
•+
•+
of ATRA with other biomolecules.
ATRA is an amphiphilic molecule and its amphiphilic property
makes biological membranes one of the most likely sites for the
distribution of ATRA. The molecule of ATRA consists of two
parts, the hydrophobic part which binds efficiently to the hydro-
carbon chain region of the lipid membranes, and the other
hydrophilic carboxylic group in the head group region which is
anchored in the surface of the lipid bilayer via nonspecific electro-
static interactions as well as hydrogen bonding with lipid head
groups (29,30). The distribution of ATRA in biomembrane actu-
ally is heterogeneous. Therefore, heterogeneous system is more
suitable than homogenous system to mimic membrane structure.
In R ꢀo z_ anowska’s studies, a heterogeneous system, Triton X-100
micelle was used as reaction system which facilitated the interac-
•
+
cals (ATRA ) via biphotonic processes. The employment of
microemulsion system allows us to investigate the reaction of
hydrophobic ATRA with molecules of different polarity.
•
+
•
+
Therefore, we studied the reaction activity of ATRA to
many hydrophobic and hydrophilic molecules. Results show
•+
that ATRA can efficiently interact with lysozyme, tyrosine,
tryptophan and many antioxidants, such as curcumin (Cur),
vitamin C (VC) and gallic acid (GA). The apparent rate con-
stants of these reactions were measured and compared. These
•+
tion of hydrophobic ATRA with water-soluble ascorbate (17).
Meanwhile, micelle system was also employed in the LFP study
of retinol and pulse radiolysis study of retinyl polyenes
•
+
findings suggest that ATRA is a reactive transient product
which may pose damage to lysozyme, and antioxidants, such
as Cur, VC and GA, may inactivate ATRA by efficient
quenching reactions.
•
+
(
12,14,20). Their works inspired us to refer to another heteroge-
neous system, microemulsion which is a thermodynamically and
mechanically stable and optically transparent system, and can be
self-assembly to form after simple mixing of oil phase, water
phase, surfactant and cosurfactant in term of appropriate propor-
tion. The oil and water phases of microemulsions facilitate simul-
taneous dissolution of hydrophilic and hydrophobic molecules in
one transparent system which provides an easily preparing system
for laser flash photolysis study to investigate the reactions between
hydrophilic molecules and hydrophobic molecules. This study
aimed to investigate the photochemical and photophysical proper-
ties of ATRA in microemulsions by 355 nm LFP. Meanwhile, the
interaction of ATRA with lysozyme and many antioxidants was
also investigated to evaluate the reaction activity of ATRA to
biomolecules. In this study, the employment of microemulsion
system facilitates our LFP studies on the reactions between hydro-
phobic ATRA and molecules with different polarity.
INTRODUCTION
All-trans retinoic acid (ATRA) is one kind of retinoids and plays
an important role in different biological processes, such as embry-
onic development, cellular differentiation (1,2). Besides, it was
widely used in the treatment of cancer and some kinds of skin
diseases (3–7). However, the use of retinoids, in particular topical,
can cause skin photosensitization or phototoxicity (8–11).
Many studies have been made to investigate the transient phe-
nomena in the laser flash photolysis and pulse radiolysis studies
of retinoids (12–28). Previous studies on ATRA in methanol
showed that 347 nm laser flash photolysis (LFP) of ATRA led
•+
•+
3
to the production of triplet state of ATRA ( ATRA*) via photo-
excitation and retinoic acid cation radicals (ATRA ) via
photoionization (16). Pulse radiolysis study by R ꢀo z_ anowska
•
+
•+
•
+
et al. showed that ATRA could also be generated via oxidation
MATERIALS AND METHODS
*
Corresponding authors email: wuxianzheng1962@yahoo.com.cn (Xianzheng Wu);
wsl@tongji.edu.cn (Shi-Long Wang)
2013 The American Society of Photobiology
ATRA, lysozyme (Lyso), tert-butyl alcohol (t-BuOH) and b-carotene
(b-car) were purchased from Sigma. Diphenylamine (DPA), N, N-dime-
©
1
064

Photochemistry and Photobiology, 2013, 89 1065
thylaniline (DMA), curcumin (Cur), gallic acid (GA), cyclohexane,
-butanol, H PO , NaH PO , Na HPO and Na PO were purchased
Evaluation of the degree of ionization of ATRA in microemulsion.
1
3
4
2
4
2
4
3
4
According to the Lambert–Beer law, the optical density (OD) of ATRA
ꢀ
from Sinopharm Chemical Reagent Co. Ltd (China). Sodium dodecyl
sulfonate (SDS), vitamin E (VE), tyrosine (Tyr), tryptophan (Trp) and
ascorbate (VC) were purchased from Sangon Biotech (Shanghai) Co.
Ltd. (China). All reagents are analytic grade reagent and used without
further purification. Water was purified in a Millipore Milli-Q unit. The
pH value of solution was adjusted by phosphate solution (0.04 M) with
at a fixed wavelength where both ATRA and ATRA have absorbance
can be presented as follows:
ꢀ
OD ¼ e1L½ATRA ꢁ þ e2L½ATRAꢁ
ꢀ
¼
¼
Lðe1 ꢀ e2Þ½ATRA ꢁ þ ae2L ð½ATRAꢁ þ ½ATRAꢁ ¼ aÞ
different pH values. The solutions were saturated with high-purity N
≥99.99%), N O, or O (≥99.5%) for different purposes by bubbling for
2
ꢀ
½
ATRA ꢁ
(
2
2
aLðe1 ꢀ e2Þ
þ ae2 L
ꢀ
at least for 20 min prior to the experiments. All experiments were per-
formed in a 1 cm quartz cuvette at room temperature. The structures of
some substances used in this study are presented in Scheme 1.
½ATRA ꢁ þ ½ATRAꢁ
ð1Þ
Preparation of microemulsion. The microemulsion of this study is oil in
water (o/w) which was composed of emulsifier 30% (v/v), cyclohexane
ꢀ
In Eq. (1), [ATRA ] is the concentration of deprotonated form of
ꢀ
(
oil) 10% (v/v) and water 60% (v/v). The emulsifier was prepared by
ATRA (ATRA ) and [ATRA] is the concentration of protonated form of
ꢀ
mixing SDS (surfactant) 25% (m/m), 1-butanol (cosurfactant) 50% (m/m)
and water 25% (m/m). ATRA was dissolved in the oil phase (cyclohex-
ane) of microemulsion. The preparation of microemulsion used in this
study has been described in our previous laser flash photolysis study of
Coenzyme Q10 in microemulsion (31).
Absorption spectroscopy. Absorption spectra of ATRA in microemul-
sion were measured as a function of pH using a UV–Vis spectrometer
ATRA. e
1
and e
2
are the molar absorption coefficients of ATRA and
ATRA at the fixed wavelength respectively. L, a, e
1
2
and e are constants
in the condition of this study. This equation indicates that OD is linearly
ꢀ
correlated with the fraction of ATRA in different pH values. The isos-
ꢀ
bestic point for ATRA and ATRA is the wavelength where e
1
is equal
2
to e and the OD is theoretically not influenced by pH. Then, Eq. (2) can
be derived from Henderson–Hasselbalch equation:
(
Varian Cary 50 Probe). The solvent pH was varied by 0.04 M phosphate
ꢀ
solution with different pH, and the solution pH was measured with a
calibrated pH meter (Mettler).
OD ꢀ ae2 L
½ATRA ꢁ
1
¼
¼
ð2Þ
ꢀ
1 þ 10ðpKaꢀpHÞ
aLðe1 ꢀ e2Þ ½ATRA ꢁ þ ½ATRAꢁ
1 2
In Eq. (2), e is not equal to e . By fitting OD values at the fixed
wavelength (except for the isosbestic point) in different pH conditions
a
and corresponding pH values using Eq. (2), pK for ATRA in micro-
emulsion was obtained.
COOH
Laser flash photolysis. Laser flash photolysis (LFP) experiments were
carried out using Nd:YAG laser of 355 nm light pulses with a duration
of 5 ns and a laser energy range 0–65 mJ per pulse used as the pump
light source. A xenon lamp was employed as analyzing 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 oscilloscope was transferred to a
personal computer for data treatment. The intensity of laser pulse was
measured with a laser energy meter (Coherent EPM1000).
ATRA
COOH
H N CH
COOH
H2N CH
2
CH2
CH2
N
ATRA is an amphiphilic molecule and it cannot be guaranteed that
ATRA only distribute in the interface of heterogeneous microemulsion.
Therefore, it is hard to confirm the accurate ATRA concentrations in
different phases and which phase the excited ATRA may distribute in,
which makes it difficult for us to measure the accurate reaction rate
OH
Tyr
H
Trp
HO
OH
OCH3
•+
constants of the reactions of ATRA with the chosen compounds. Mean-
while, unlike in homogeneous system, the free diffusion ability of
•+
ATRA may be limited because of its limited distribution in heteroge-
H CO
3
neous microemulsion. Herein, “apparent rate constant” was used to repre-
O
O
•+
sent the apparent and average reactivity of ATRA to the chosen
Cur
compounds in the condition of this study. Apparent bimolecular rate con-
•+
R
stant (k ) for the reaction of ATRA with the chosen compound in mi-
croemulsion was roughly estimated from the plot of the observed
pseudo-first-order rate constant (kobs) for the decay of ATRA at 590 nm
O
•+
vs reductant concentration ([R]) using the following equation:
HO
k
obs = k
0
R
+ k [R]
VE
•
+
Here, k
0
(intercept) is the apparent rate constant for the decay of ATRA
in the absence of reductant.
β−car
RESULTS AND DISCUSSION
Influence of pH on the absorption of ATRA in
microemulsion
HO
HO
HO
CH3
H C
3
N
H
N
COOH
As shown in Fig. 1B, the maximum absorption wavelength
kmax) of ATRA varies between the constant values 355 and
35 nm, as a function of the pH values from 2 to 13. The
(
3
GA
DMA
DPA
pH-dependent kmax of ATRA in microemulsion agrees well with
Scheme 1. The structures of molecules used in this study.
the case for ATRA in lipid bilayers and membrane (29,30,32).

1
066 Kun Li et al.
The blueshift of kmax observed when the pH was adjusted from
ATRA in photoreaction. The kinetic decay at 590 nm is not influ-
enced by N O and O . The insensitivity of this intermediate to
O and O agrees with retinyl polyene radical cations obtained
by pulse radiolysis (13). Moreover, the generation of ATRA
low to high values is attributed to the deprotonation process of
2
2
ꢀ
ATRA. From Fig. 1A, the isosbestic point for ATRA and
N
2
2
•
+
ATRA can be roughly evaluated at 352 nm. Eq. (2) indicates
ꢀ
that OD is linearly correlated with the percentage of ATRA at
with maximum absorption around 590 nm was confirmed previ-
ously by laser flash photolysis study and pulse radiolysis study
(13,16,17). Therefore, the absorption band centered at 590 nm
different pH values in the condition of this study. And the pK
a
for ATRA in microemulsion was evaluated as 6.98.
•
+
herein is assigned as the absorption of ATRA .
Characterization of photoionization of ATRA in
microemulsion
ꢀ
þ O2 ! Oꢂꢀ2
e
ð3Þ
ð4Þ
ð5Þ
aq
The UV–Vis spectra show that microemulsion system itself has
negligible absorption at 355 nm compared with that of ATRA
ꢀ
ꢂ
ꢀ
eaq þ N2O þ H2O ! HO þ OH þ N2
(
Fig. 1A). Therefore, 355 nm laser pulses are mainly absorbed
by ATRA in microemulsion.
55 nm LFP of N -saturated ATRA in microemulsion gives a
ꢂ
ꢂ
HO þ tꢀBuOH ! tꢀBuOHðꢀ HÞ þ H O
2
3
2
broad absorption band above 630 nm, a strong absorption band
centered at 590 nm and a strong bleaching around 350 nm
The kinetic curve at 420 nm includes a generation process
ꢀ
which can be eliminated by eaq scavengers N
cating that the subsequent reaction of eaq accounts for the gen-
2
O (Fig. 2B), indi-
ꢀ
(
Fig. 2A). The kinetic decay profile observed at 720 nm can be
ꢀ
efficiently removed by typical hydrated electrons (e ) scavengers
eration. The decay process at 420 nm which is insensitive to
aq
(
Inset of Fig. 2A), such as O and N O with addition of t-BuOH
2 2
N O can be accelerated by O , suggesting that formation of trip-
2
2
ꢀ
3
Eqs. (3–5), which suggests that e is generated immediately after
3
microemulsion can be confirmed by the appearance of e . The
appearance of bleaching at 350 nm suggests the participation of
let state of ATRA ( ATRA*) occurs after 355 nm laser pulses,
aq
ꢀ
55 nm laser pulse (33). Therefore, photoionization of ATRA in
for O
2
is not only an eaq scavenger but also a triplet state
ꢀ
quencher. It is reasonable because the characteristic absorption of
aq
3
ATRA* was reported around 440 nm (16,23).
ꢀ
Figure 1. (A) UV–Vis absorption spectra of ATRA in microemulsion at pH 5.3, 7.3 and 9.6 respectively. The isosbestic point for ATRA and ATRA
was roughly evaluated from the intersection of the three absorption spectra. (B) kmax for ATRA as a function of pH in microemulsion (●).
Figure 2. (A) Transient absorption spectra obtained by 355 nm LFP of 0.06 mM ATRA in N
3
2
-saturated microemulsion at pH 7.4, recorded at 0.1, 0.5,
and 8 ls after laser pulse. Inset: Kinetic decay curves recorded at 720 nm obtained from 355 nm LFP of 0.06 mM ATRA at pH 7.4 in N -saturated,
O-saturated and O -saturated microemulsion. (B) Kinetic decay curves recorded at 420 nm obtained from 355 nm LFP of 0.06 mM ATRA at pH 7.4
-saturated, N O-saturated and O -saturated microemulsion.
2
N
2
2
in N
2
2
2

Photochemistry and Photobiology, 2013, 89 1067
•
+
Influence of pH on the formation of ATRA in
microemulsion
ability and may pose oxidative damage to other biomolecules.
•
+
To evaluate the potential of ATRA to damage biomolecules,
•
+
we herein investigated the interactions of ATRA with Lyso and
two amino acids. Lyso was chosen as a model protein because
It was found that DOD (the maximum optical density of decay
•
+
curves) at 590 nm, i.e. the yield of ATRA , is very sensitive to
the pH of microemulsion (Fig. 3A). Therefore, we investigated
the influence of pH on DOD at 590 nm. Figure 3B shows that
DOD and the fraction of ATRA have the same variation ten-
dency as the pH was changed, which indicated that the yield of
ATRA is positively dependent on the degree of deprotonation
of ATRA in microemulsion.
To determine whether the formation of ATRA by 355 nm
irradiation is a one- or a two-photon process, the DOD of
of its extensive use in the studies of photodamage of proteins
•+
(
35–40). Result shows that the decay of ATRA at 590 nm is
accelerated by the presence of Lyso at pH 7.4, indicating that
ꢀ
•+
ATRA can interact with Lyso (Fig. 5). The apparent rate
constant was measured (Table 1).
•
+
Tyr and Trp, the aromatic amino acid residues of proteins, are
prime targets for oxidation by various forms of reactive oxidative
species (ROS) (41). Previous reports indicated that Lyso could
be attacked by ROS via the interaction of ROS with its Tyr and
•
+
•
+
ATRA at 590 nm in both alkaline and acidic microemulsions
was measured as a function of relative laser intensity (16,34).
•+
Trp residues (35,36). The ability of ATRA to react with Tyr
and Trp was evaluated in this study. Results showed that
•
+
The results suggest that formation of ATRA is biphotonic pro-
cesses in both alkaline and acidic microemulsion (Fig. 4). Previ-
ous study showed that LFP (347 nm) of ATRA in methanol led
•+
ATRA can also react with Tyr and Trp. The apparent rate con-
•+
stants for the reactions of ATRA with Tyr and Trp were also
measured, which were found to be close to the apparent rate con-
•
+
to biphotonic photoionization with the formation of ATRA
16). In alkaline and neutral conditions, eaq was observed, and
thus photoionization of ATRA can be confirmed to be responsi-
ble for the formation of ATRA . However, photoionization of
•+
stant for the reaction of ATRA with Lyso (Table 1). Therefore,
ꢀ
(
•+
it is inferred that ATRA may interact with Lyso by reacting
ꢀ
with the Tyr and Trp residues.
•
ꢀ
ATRA in microemulsion was not confirmed because eaq cannot
be observed in acidic condition.
•+
Reactions of ATRA with antioxidants
•
+
Pulse radiolysis study on the interaction of ATRA with VC
•
+
Reactions of ATRA with lysozyme and amino acids
•+
showed that VC reacts effectively with ATRA (17). VC has a
•+
•+
Previous study demonstrated that ATRA had efficient reaction
activity to ascorbate (17). It indicates that ATRA has oxidative
higher reaction rate than Lyso to react with ATRA (Table 1).
•
+
And thus it may competitively protect Lyso from the attack of
Figure 3. (A) Influence of pH on kinetic decay curves recorded at 590 nm obtained from 355 nm LFP of 0.06 mM ATRA in O
2
-saturated microemul-
•+
sion. (B) Dependence of DOD at 590 nm for ATRA on pH values in O
titration curve derived from Eq. (2) when pK was set as 6.98.
2
-saturated microemulsion (▲). The short dot line (…) is the theoretical pH
a
Figure 4. (A) Variation of DOD on relative laser intensity immediately after 355 nm LFP of 0.06 mM ATRA at pH 9.26 in O
2
-saturated microemul-
sion. (B) Variation of DOD on laser intensity immediately after 355 nm LFP of O -saturated 0.06 mM ATRA in microemulsion at pH 4.53.
2

1
068 Kun Li et al.
•
+
•+
10 ꢀ1 ꢀ1
M
ATRA . To find efficient antioxidants to scavenge ATRA , we
2.1 9 10
s
in hexane via electron transfer (16). How-
•
+
•+
then investigated the reactions of ATRA with several hydrophilic
and hydrophobic antioxidants, including VC, GA, Cur, VE and
b-car, and corresponding apparent rate constants were measured
and listed in Table 1. Results show that Cur is the most efficient
antioxidant to interact with ATRA in the investigated antioxi-
dants. Generally, hydrophilic antioxidants are more favorable than
hydrophobic antioxidants to quench ATRA in microemulsion.
ATRA was found to be quenched by b-car with bimolecular
rate constants of 1 9 10
ever, the interaction of ATRA with b-car (0.05 mM) in micro-
emulsion was not confirmed by our obtained data. The effect of
solvent polarity on the kmax of radical cation of retinyl polyenes
suggested that there existed a strong ion–dipole interaction
between the ground state of radical ions and polar solvent (13).
In microemulsion, the polar part of ATRA may be anchored
via ion–dipole interaction in the oil/water interface where it is
difficult for highly hydrophobic molecule to reach. It may
•
+
•
+
•
+
•
+
1
0
ꢀ1 ꢀ1
•+
M
s
in methanol and
explain why the interaction of b-car with ATRA cannot be
observed in microemulsion. VE has a comparatively polar
phenolic hydroxyl group that can reach the oil/water interface,
•
+
Table 1. Apparent rate constants for reactions of ATRA with lysozyme
•
+
and thus shows a higher reaction activity to ATRA than b-car.
and amino acids and reductants at neutral pH in microemulsion. (Unit:
ꢀ
1 ꢀ1
•+
M
s
).
ATRA
can be generated via not only photoinduced
ꢀ
1 ꢀ1
processes but also oxidation of ATRA by oxidative transient
species, such as peroxyl radicals and hydroxyl radicals (16,17).
Generation of ATRA is unavoidable in its application as drugs
Apparent rate constants (M
s )
8
•+
Lyso
(1.85 ꢃ 0.09) 9 10
(2.73 ꢃ 0.65) 9 10
(1.04 ꢃ 0.03) 9 10
(3.42 ꢃ 0.14) 9 10
(1.72 ꢃ 0.09) 9 10
(1.39 ꢃ 0.17) 9 10
(2.42 ꢃ 0.13) 9 10
(1.67 ꢃ 0.02) 9 10
(3.94 ꢃ 0.29) 9 10
—
8
Tyrosine
Tryptophan
VC
GA
Cur
DMA
DPA
VE
because peroxyl radicals and hydroxyl radicals are also generated
8
8
8
9
9
9
6
•+
in the biological systems. Although the interactions of ATRA
with Lyso and amino acids are suggestive of the potential
•
+
damage of ATRA to proteins, our studies on the reactions of
•+
ATRA
with antioxidants indicated rapid deactivation of
•+
ATRA by antioxidants is possible.
b-car
•
+
Reactions of ATRA with organic amines
These apparent rate constants were roughly evaluated and only used as a
reference to compare the reactivity of ATRA to the compounds used in
this study.
•+
Organic amines, strong nucleophilic agents, were proved to be
effective quenchers for radical cations of retinyl polyene and reti-
noids (12,13). The most possible mechanism for the quenching
reaction was proposed to be nucleophilic attack or electron trans-
fer (12). In this study, two organic amines, DPA and DMA were
•
+
used to quench ATRA and the apparent rate constants are
nearly diffusion-controlled rate (Table 1). The characteristic
•
+
absorption bands for radical cation of DPA (DPA ) and radical
•
+
cation of DMA (DMA ) were reported around 680 and 465 nm
respectively (42,43). In spite of the effective interaction of
•
+
•+
ATRA with DMA, the formation process of DMA cannot be
observed in its corresponding characteristic absorption spectra
•
+
most because of the strong absorption of ATRA . However, the
characteristic absorption band around 465 nm can be observed
after the absorption of ATRA decayed back to the baseline
Fig. 6A). The kinetic decay curve at 470 nm evidently included
•
+
Figure 5. Kinetic decay profiles observed at 590 nm from 355 nm LFP
of 0.05 mM ATRA in microemulsion at pH 7.4 in the presence of Lyso
0.08, 0.12, 0.20 and 0.24 mM). Inset: Dependence of kobs for ATRA at
90 nm on Lyso concentrations.
(
•
+
two decay processes: a fast decay process and a slow decay pro-
cess (Fig. 6B). By subtracting the decay curve at 590 nm from
(
5
Figure 6. (A) Transient absorption spectra obtained by 355 nm LFP of 0.06 mM ATRA in O
2
-saturated microemulsion at pH 7.4 with 4 mM DMA,
recorded at 0.5 ls (-■-) and 1.5 ls (-●-) after laser pulse. (B) Kinetic decay profiles observed at 470 nm (-■-) and 590 nm (-●-) obtained by 355 nm
LFP of 0.06 mM ATRA in O
at 590 nm from 470 nm.
2
-saturated microemulsion at pH 7.4 with 1 mM DMA, and the decay curve (-▲-) obtained by subtracting the decay curve

Photochemistry and Photobiology, 2013, 89 1069
4
70 nm, a formation process can be observed which should be
14. El-Agamey, A. and S. Fukuzumi (2011) Laser flash photolysis study
on the retinol radical cation in polar solvents. Org. Biomol. Chem. 9,
6437–6446.
•
+
assigned to the formation of DMA (Fig. 6B). The similar phe-
•
+
nomena were also observed for the reaction of ATRA with
DPA (data not shown). Therefore, it can be inferred that electron
transfer should be responsible for the efficient quenching reac-
1
5. Sahyun, M. R. V. and N. Serpone (1998) Photophysics of all-trans-
retinoic acid (ATRA) chemisorbed to nanoparticulate TiO : evidence
2
for TiO * to ATRA energy transfer and reverse electron transfer sen-
2
•+
sitization. J. Photochem. Photobiol., A 115, 231–238.
6. Lo, K. K. N., E. J. Land and T. G. Truscott (1982) Primary interme-
tions of ATRA by DMA and DPA.
1
1
1
diates in the pulse irradiation of reinoids. Photochem. Photobiol. 36,
1
39–145.
CONCLUSION
7. R ꢀo z_ anowska, M., A. Cantrell, R. Edge, E. J. Land, T. Sarna and
T. G. Truscott (2005) Pulse radiolysis study of the interaction of reti-
noids with peroxyl radicals. Free Radic. Biol. Med. 39, 1399–1405.
8. Raghavan, N. V., P. K. Das and K. Bobrowski (1981) Transient phe-
nomena in the pulse radiolysis of retinyl polyenes.1. Radical anions.
J. Am. Chem. Soc. 103, 4569–4573.
We investigated the photochemistry of ATRA in microemulsion
and characterized the properties of reaction products. 355 nm
LFP of ATRA in both acidic and alkalic condition leads to the
generation of ATRA via biphotonic processes. It was found
that ATRA can efficiently react with Lyso and Tyr and Trp,
which is suggestive of the possibility of ATRA to damage pro-
teins. Many antioxidants, such as VC, GA and Cur, were proved
to be efficient quenchers for ATRA .
•
+
•
+
19. Bobrowski, K. and P. K. Das (1982) Transient phenomena in the
pulse radiolysis of retinyl polyenes. 2. protonation kinetics. J. Am.
Chem. Soc. 104, 1704–1709.
•
+
2
2
2
0. Bobrowski, K. and P. K. Das (1985) Transient phenomena in the pulse
radiolysis of retinyl polyenes. 4. environmental effects on absorption
maximum of retinal radical anion. J. Phys. Chem. 89, 5733–5738.
1. Bobrowski, K. and P. K. Das (1986) Transient phenomena in the
pulse radiolysis of retinyl polyenes. 5. association of radical cations
with parent molecules. J. Phys. Chem. 90, 927–931.
2. Bhattacharyya, K., K. Bobrowski, S. Rajadurai and P. K. Das (1988)
Transient phenomena in the pulse radiolysis of retinyl polyenes. 7.
radical anions of vitamin A and its derivatives. Photochem. Photo-
biol. 47, 73–83.
•+
Acknowledgements—This work was financially supported by the 973
Program (grant 2010CB912604 and 2010CB933901), the International
S&T Cooperation Program of China (grant 0102011DFA32980), the
National Natural Science Foundation of China (grant, 81271694), the
Science and Technology Commission of Shanghai Municipality (grant
11411951500 and 12nm0502200), 2012 and the Fundamental Research
2
3. Takemura, T., K. Chihara, R. S. Becker, P. K. Das and G. L. Hug
Funds for the Central Universities (grant 2000219085).
(
1980) Visual Pigments. 11. Spectroscopy and photophysics of reti-
noic acids and all-trans-methyl retinoate. J. Am. Chem. Soc. 102,
604–2609.
2
2
4. McGarry, P. F., J. Cheh, B. Ruiz-Silva, S. H. Hu, J. Wang, K. Nak-
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