Photoinactivation of tyrosinase sensitized by folic acid photoproducts

Article abstract of DOI:10.1016/j.jphotobiol.2015.06.002

Tyrosinase catalyzes in mammals the first and rate-limiting step in the biosynthesis of the melanin, the main pigment of the skin. Pterins, heterocyclic compounds able to photoinduce oxidation of biomolecules, accumulate in the skin of patients suffering from vitiligo, where there is a lack of melanin. Folic acid (PteGlu) is a conjugated pterin widespread in biological systems. Aqueous solutions of tyrosinase were exposed to UV-A irradiation (350 nm) in the presence of PteGlu and its photoproducts (6-formylpterin and 6-carboxypterin). The reactions were followed by UV-Vis spectrophotometry, enzyme activity measurement, fluorescence spectroscopy and HPLC. In this work, we present data that demonstrate unequivocally that solutions of tyrosinase exposed to UV-A irradiation in the presence of PteGlu, undergo enzyme inactivation. However, PteGlu itself causes a negligible effect on the activity of the enzyme. In contrast, PteGlu photoproducts are efficient photosensitizers. The tyrosinase inactivation involves two different pathways: (i) a photosensitization process and (ii) the oxidation of the enzyme by the hydrogen peroxide produced during the photooxidation of PteGlu and its photoproduct. The former pathway affects both the active site and the tryptophan residues, whereas the latter affects only the active site. The biological implications of the results are discussed.

Full text of DOI:10.1016/j.jphotobiol.2015.06.002

Journal of Photochemistry and Photobiology B: Biology 149 (2015) 172–179
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Photoinactivation of tyrosinase sensitized by folic acid photoproducts
⇑
M. Laura Dántola , Beatriz N. Zurbano, Andrés H. Thomas
Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata,
CCT La Plata-CONICET, Casilla de Correo 16, Sucursal 4, 1900 La Plata, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Tyrosinase catalyzes in mammals the first and rate-limiting step in the biosynthesis of the melanin, the
main pigment of the skin. Pterins, heterocyclic compounds able to photoinduce oxidation of biomole-
cules, accumulate in the skin of patients suffering from vitiligo, where there is a lack of melanin. Folic acid
Received 3 February 2015
Received in revised form 11 May 2015
Accepted 3 June 2015
(PteGlu) is a conjugated pterin widespread in biological systems. Aqueous solutions of tyrosinase were
Available online 6 June 2015
exposed to UV-A irradiation (350 nm) in the presence of PteGlu and its photoproducts (6-formylpterin
and 6-carboxypterin). The reactions were followed by UV–Vis spectrophotometry, enzyme activity mea-
surement, fluorescence spectroscopy and HPLC. In this work, we present data that demonstrate unequiv-
ocally that solutions of tyrosinase exposed to UV-A irradiation in the presence of PteGlu, undergo enzyme
inactivation. However, PteGlu itself causes a negligible effect on the activity of the enzyme. In contrast,
PteGlu photoproducts are efficient photosensitizers. The tyrosinase inactivation involves two different
pathways: (i) a photosensitization process and (ii) the oxidation of the enzyme by the hydrogen peroxide
produced during the photooxidation of PteGlu and its photoproduct. The former pathway affects both the
active site and the tryptophan residues, whereas the latter affects only the active site. The biological
implications of the results are discussed.
Keywords:
Tyrosinase
Folic acid
Folic acid photoproducts
Photosensitized processes
Reactive oxygen species
UV-A radiation
Vitiligo
Ó 2015 Elsevier B.V. All rights reserved.
1
. Introduction
Pterins, a family of heterocyclic compounds, are present in bio-
hydrocarbon chain, and (ii) conjugated pterins, with larger sub-
stituents containing a p-aminobenzoic acid (PABA) moiety.
Folic acid, or pteroyl-L-glutamic acid (PteGlu), is a conjugated
logical systems in multiple forms and play different roles ranging
from pigments to enzymatic cofactors for numerous redox and
one-carbon transfer reactions [1,2]. The most common pterin
derivatives are 6-substituted compounds (Fig. 1). According to
the molecular weight and the functional groups of these sub-
stituents, pterins can be divided into two groups: (i) unconjugated
pterins, containing substituents with one carbon atom or a short
pterin widespread in biological systems, whose chemical structure
is composed of three moieties: a 6-methylpterin (Mep) residue, a
PABA residue, and a glutamic acid (Glu) residue (Fig. 1). In living
systems, PteGlu is present in multiple forms including molecules
attached to several glutamate residues and dihydro and tetrahydro
pterin derivatives. Mammals do not synthesize folates and there-
fore they have to get them from food. The main form of folates
in human plasma is 5-methyltetrahydrofolate (5CH
3 4
-H PteGlu)
[
3,4]. This compound is oxidized to corresponding dihydropterin
Abbreviations: A, absorbance of the reactant at the excitation wavelength; Aemi
,
derivatives at pH close to 7 [5], such as skin pH. It is know that
7,8-dihydrofolic acid is oxidized by reactive oxygen species, like
hydrogen peroxide (H
absorbance of the system at emission wavelengths; Aexc, absorbance of the system
a;V
n;p
at excitation;
q
,
absorbed photon flux density; Bip, biopterin; Cap,
corrected fluorescence intensities; DOPA,
-phenylalanine; PteGlu, folic acid; Fop, 6-formylpterin; Glu, glu-
1
6
3
-carboxypterin;
,4-dihydroxy-
I
Fcorr
,
2
O
2
) and singlet oxygen ( O
2
), yielding
L
PteGlu as one of the products [6,7]. Taking into account that in
some pathological conditions the concentration of reactive oxygen
species increases significantly, the oxidation of reduced folic acid
derivatives can be occurred, yielding a source of folic acid in the
cell.
In particular, folates are involved in DNA biosynthesis, repair,
DNA methylation and aminoacid metabolism [8]. Folate require-
ments increase in periods of rapid cell division and growth, and,
tamic acid; HPLC, high-performance liquid chromatography;
H
2
O
2
,
hydrogen
0
;V
0
peroxide;
interval; Mep, 6-methylpterin; 5CH
observed fluorescence intensities; PABA, p-aminobenzoic acid; PABA–Glu, p-ami-
nobenzoylglutamic acid; Ptr, pterin; v, rate of formation of
-dopachrome; 1
, total fluorescence intensities; Trp,
q
n;p
,
incident photon flux density;
q
,
incident photons per time
n;p
3 4 Fobs
-H PteGlu, 5-methyltetrahydrofolate; I ,
L
2
O ,
singlet oxygen; H
tryptophan; Tyr,
4
Bip, tetrahydrobiopterin; I
F
Åꢀ
L
-tyrosine; O
2
, superoxide anion; V, volume.
⇑
Corresponding author at: C. C. 16, Sucursal 4, B1904DPI La Plata, Argentina.
E-mail address: ldantola@inifta.unlp.edu.ar (M. Laura Dántola).
http://dx.doi.org/10.1016/j.jphotobiol.2015.06.002
011-1344/Ó 2015 Elsevier B.V. All rights reserved.
1

M. Laura Dántola et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 172–179
173
Scheme 1. Photooxidation of PteGlu in air-equilibrated aqueous solutions under
UV-A irradiation.
Folates participate in photobiological processes, e.g. the deriva-
tive 5,10-methenyltetrahydrofolic acid acts as photoantenna in
DNA photolyases and cryptochromes [19]. In addition, the pho-
todegradation of PteGlu has been discussed as one of the reasons
for the development of skin tanning in evolution because the
PteGlu depletion by natural exposure to sunlight can become
physiologically relevant [20–24]. PteGlu decomposes under UV-A
radiation in the presence of oxygen, yielding Fop, and p-aminoben-
zoylglutamic acid (PABA–Glu) as photoproducts (Scheme 1) [25–
(a)
3
0]. The rate of this process increases with irradiation time. The
data point to an ‘‘auto-photo-catalytic’’ effect, wherein Fop acts
as photosensitizer and photoinduces the oxidation of PteGlu [30].
In turn, Fop is transformed into Cap upon further photooxidation
(
Scheme 1). Reactive oxygen species, like H
2
O
2
and superoxide
Åꢀ
anion (O
2
) are formed during the photooxidation of PteGlu into
Fop, and in the reaction where Fop is converted into Cap [30].
Therefore under irradiation of skin affected by vitiligo Fop and
Cap are formed, very likely, due to the photolysis of both PteGlu
and H Bip. In fact, Cap has been isolated from the skin of patients
4
suffering from vitiligo [31].
Several studies have demonstrated that pterin (Ptr), the parent
and unsubstituted compound of oxidized pterins (Fig. 1), is able to
photosensitize free amino acids, such as tryptophan (Trp) [32] and
Tyr [33], peptides [34] and proteins, such as bovine serum albumin
(b)
[
35,36]. A very recent study has demonstrated that tyrosinase is
inactivated by Ptr through a photosensitized process and suggested
that the process is initiated by an electron transfer reaction from
the enzyme to the triplet excited state of Ptr [37]. In 2003 it was
reported that PteGlu is able to photoinduce DNA damage via its
photolysis products [38]. More recently it was informed that pro-
tein damage takes place when albumin is exposed to UV-A radia-
tion in the presence of PteGlu [39,40].
Taking into account that in some skin disease, such as vitiligo,
the protection against UV radiation fails due to the lack of melanin,
the photodegradation of PteGlu in vivo may occur in the depig-
mented patches of patients with this disease. We chose this com-
pound because: it is more stable than most member of folate’s
family and efficiently metabolized into biologically active deriva-
tives, it is used in tablet form and in fortified foods for dietary sup-
plementation [8,41] and its photooxidation products are the same
Fig. 1. Molecular structure of PteGlu and unconjugated oxidized pterins, and their
corresponding absorption spectra in air-equilibrated aqueous solutions (pH 6.0). (a)
PteGlu; (b) Fop and Cap. UV–Vis absorption spectra of Ptr is almost identical to that
shown for Cap.
as such, it is imperative that pregnant women, for example, keep
folate concentrations at an appropriate level [9]. Folate deficiency
in pregnant women has clearly been shown to be related to neural
tube defects, such as spina bifida and anencephaly [10,11].
Recent research has demonstrated that folates regulate melanin
biosynthesis and, hence, is present in human skin, because it is
required for the novo production of tetrahydrobiopterin (H
in melanocytes and keratinocytes [12,13]. H Bip acts as a cofactor
for hydroxylation of -phenylalanine via phenylalanine hydroxilase
and regulates tyrosinase activity in melanosome [14]. The synthe-
sis of H Bip is highly activated in vitiligo, a skin disorder which is
4
Bip)
4
L
4
oxidized pterins produced during the photooxidation of H Bip.
Therefore, the main aim of this work was investigated the capabil-
ity of PteGlu and its photoproducts to photoinduced the inactiva-
tion of tyrosinase, the key enzyme in melanin biosynthesis.
4
characterized by the acquired loss of constitutional pigmentation
manifesting as white macules and patches. The photooxidation of
4
H Bip yield biopterin (Bip) [15], which under UV-A (315–
4
6
6
00 nm) irradiation leads to oxidation of the 6-substituent to yield
-formylpterin (Fop, Fig. 1), which, in turn, is photooxidized to
-carboxypterin (Cap, Fig. 1) [16,17].
2
. Materials and methods
2.1. General
Tyrosinase ((
L-tyrosine, L-dopa: oxygen oxidoreductase, EC
1
.14.18.1)) is a copper-containing glycoprotein that, in mammals,
Pterin derivatives (PteGlu, Fop, Cap and Ptr) were purchased
catalyzes the first and rate-limiting step in melanin biosynthesis,
the hydroxylation of -tyrosine (Tyr) to 3,4-dihydroxy-
phenylalanine (DOPA) and the subsequent oxidation of DOPA to
-dopaquinone [18]. This compound, in turn, undergoes fast oxida-
tion and rearrangement to yield -dopachrome.
from Schircks Laboratories and used without further purification.
Tyrosinase from mushroom (lyophilized powder, P1000 unit/mg
solid), catalase from bovine liver (lyophilized powder,
10,700 unit/mg solid) and other chemicals were provided by
L
L-
L
L
2 2
Sigma Chemical Co. H O (30%) was from Merck.

1
74
M. Laura Dántola et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 172–179
The pH measurements were performed using a pH-meter
2 4
reaction was performed at 37 °C in KH PO buffer 20 mM at pH
PHM220 (Radiometer Copenhagen) combined with a pH electrode
pHC2011-8 (Radiometer Analytical). The pH of the aqueous solu-
tions was adjusted by adding drops of HCl and NaOH solutions
from a micropipette. The concentration of the acid and the base
used for this purpose ranged from 0.1 M to 2 M. When necessary
the samples were incubated using a low temperature bath/circula-
tor R1 (Grant Instruments).
6.5 using -tyrosine 0.15 mg/mL (0.83 mM) as the substrate. The
L
conversion of an inactive form of the catalytic site of the enzyme
into an active form gives rise to a lag period before the reaction
reaches maximal rate, which is a characteristic of the overall activ-
ity of the tyrosinase (oxidation of
L-tyrosine into L-dopachrome)
[45]. Therefore the enzyme activity (rate of formation of
ꢀ1
L
-dopachrome (v), lM min ) was determined in the linear phase
by measuring the slope of the curve of absorbance at 475 nm vs.
time after the addition of the substrate.
2
2
.2. Steady-state irradiation
.2.1. Irradiation setup
Air equilibrated aqueous solutions containing a given pterin
2.3.4. Fluorescence spectroscopy
Fluorescence measurements were performed on air-
derivative and tyrosinase were irradiated in quartz cells (1.0 cm
optical path length) at room temperature using a Rayonet RPR
lamp (Southern N. E. Ultraviolet Co.) with emission centered at
equilibrated aqueous solutions of pterin derivatives and tyrosinase
using a Single-Photon-Counting equipment FL3 TCSPC-SP (Horiba
Jobin Yvon). The equipment has been previously described in detail
3
50 nm (bandwidth ꢁ20 nm). To regulate the intensity of the inci-
[
46]. Briefly, in steady-state measurements the sample solution in
dent light, the photolyses were performed at two different dis-
tances between the lamp and the sample (4 and 20 mm).
a quartz cell was irradiated with a 450 W Xenon source through an
excitation monochromator. The fluorescence, after passing through
an emission monochromator, was registered at 90° with respect to
the incident beam using a room-temperature R928P detector. The
2.2.2. Actinometry
Aberchrome 540 (Aberchromics Ltd.) was used as an actinome-
F
total fluorescence intensities (I ) were calculated by integration of
ter for the measurement of the incident photon flux density (q0
at the excitation wavelength, which is the amount of incident pho-
tons per time interval (q ) and divided by the volume (V) of the
;V
n;p
)
the fluorescence band centered at ca. 340 nm. The tyrosinase fluo-
rescence intensity was corrected for inner filter effect using the fol-
lowing equation [47]
0
n;p
sample [42]. Aberchrome 540 is the anhydride form of the
Fcorr ¼ IFobs 10ðAexcþAemiÞ=2
I
ð2Þ
(
E)-a-(2,5-dimethyl-3-furylethylidene)(isopropylidene)succinic
acid which, under irradiation in the spectral range 316–366 nm
where IFcorr and IFobs are the corrected and observed fluorescence
intensities, respectively, and Aexc and Aemi are the absorbance of
the system at excitation and emission wavelengths, respectively.
leads to a cyclized form. The method for the determination of
0
;V
q
has been described in detail elsewhere [43].
n;p
Taking into account the distance from the irradiation source to
0
;V
0;V;1
the sample, two different values of q were measured (qn;p = 1.7
n;p
3. Results and discussion
ꢀ5
ꢀ1 ꢀ1
0;V;2
n;p
ꢀ6
(
±0.2) ꢂ 10 Einstein L
s
and
q
= 3.4
(±0.4) ꢂ 10
ꢀ
1 ꢀ1
3.1. Evaluation of the photoinactivation of tyrosinase by PteGlu
Einstein L
s , for 4 and 20 mm, respectively). Values of the
a;V
0;V
absorbed photon flux density (qn;p ) were calculated from qn;p
To check the photochemical behavior of PteGlu, we carried out
according to the Lambert–Beer law:
photolysis in the absence of substrates and the results observed
were compatible with those reported in the literature [30].
Photooxidation of PteGlu under UV-A radiation may be divided
into three stages (Fig. S1. Supporting material): (i) in the first
phase, which follows a zero-order kinetics, PABA–Glu and Fop
are photogenerated; (ii) in the second phase, Fop photoinduces
the photooxidation of PteGlu and its degradation process is accel-
erated; (iii) in the third phase the degradation of Fop to Cap is the
dominating process. This result indicated that the photodegrada-
tion of the sensitizer itself could not be avoided in the photolysis
experiments performed in the presence of tyrosinase. Therefore,
the irradiated samples were analyzed by HPLC to evaluate the
PteGlu photooxidation.
a;V
n;p
¼ qn;p ð1 ꢀ 10ꢀA
0;V
q
Þ
ð1Þ
where A is the absorbance of the reactant at the excitation
wavelength.
2
2
.3. Analysis of irradiated solutions
.3.1. UV–Vis analysis
Electronic absorption spectra were recorded on a Shimadzu
UV-1800 spectrophotometer. Measurements were made using
quartz cells of 1 cm optical pathlength. The absorption spectra of
the solutions were recorded at regular intervals of irradiation time.
To ascertain if PteGlu is able to photoinduce the inactivation of
the enzyme, air-equilibrated aqueous solutions (1 mM KH PO , pH
2
.3.2. High-performance liquid chromatography (HPLC)
Chromatographic analysis was performed using a Prominence
2
4
6.0) containing PteGlu and tyrosinase were irradiated at 350 nm
during different periods of time. After the irradiation, the samples
were analyzed by UV–Vis spectrophotometry, enzyme activity
measurement, fluorescence spectroscopy and HPLC. Under these
experimental conditions only PteGlu was excited (Fig. 1), whereas
tyrosinase did not absorb radiation. The analysis of the treated
solution showed that the activity of tyrosinase decreased signifi-
cantly when the enzyme was exposed to UV-A radiation in the
presence of PteGlu.
instrument from Shimadzu (solvent delivery module LC-20AT,
on-line degasser DGU-20A5, auto sampler SIL-20A HT, column
oven CTO-10AS VP, photodiode array detector SPD-M20A). A
Synergi Polar-RP column (150 ꢂ 4.6 mm, 4
used for product separation. Solutions containing 7% of methanol
and 93% of 10 mM NH OAc aqueous solution (pH 7.0) were used
lm, Phenomenex) was
4
as the mobile phase. HPLC runs were monitored by UV–visible
spectroscopy at different wavelengths.
In control experiments, tyrosinase solutions were exposed to
UV-A radiation in the absence of PteGlu and no inactivation of
the enzyme was detected, thus excluding the possibility that spu-
rious effects of direct light absorption by the enzyme could affect
its activity. In another set of control experiments, tyrosinase
2
.3.3. Enzyme activity
The tyrosinase activity was assayed according to the method of
Pomerantz [44]. Briefly, the formation of
sured spectrophotometrically at 475 nm (e = 3600 M cm ). The
L-dopachrome was mea-
ꢀ1
ꢀ1

M. Laura Dántola et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 172–179
175
inactivation was not observed in a solution of PteGlu with tyrosi-
nase that was kept in the dark, thus excluding the possibility of
thermal reactions. The same result was also observed in dark con-
trol experiments carried out with Fop, Cap and Ptr instead of
PteGlu.
The comparison of the kinetic profiles of PteGlu photodegrada-
tion and tyrosinase photoinactivation indicated that in the first
four minutes of irradiation, the concentration of PteGlu did not
change significantly, whereas no lost of the enzyme activity was
registered (Fig. 2). It can be assumed that, in this time range, no
photoproduct of PteGlu was generated. Therefore, PteGlu itself pre-
sented a negligible capability to photoinactivate the enzyme.
After five min of irradiation, an important increase on the rate
of photodegradation of PteGlu and on the rates of formation of
Fop and Cap were observed (Fig. 2a). Therefore, a significant pro-
portion of light was absorbed by Fop and Cap. Simultaneously, a
considerable decrease of the enzyme activity was observed
comparison of the rates of photoinactivation registered using the
different pterin derivatives revealed that, in fact, Fop is a photosen-
sitizer much more efficient than PteGlu (Fig. 3), which explains
why the rate of tyrosinase photoinactivation increased with irradi-
ation time when Fop is accumulated during the photooxidation of
PteGlu. Cap and Ptr, in turn, have the same capability to generate
inactivation of tyrosinase, and are less efficient than Fop (Fig. 3).
The comparison of the kinetic profiles of Fop degradation and
enzyme activity in Fig. 3a, indicated that after 10 min of irradia-
tion, almost 70% of the initial Fop was consumed to form Cap, with
a linear decrease in the rate of enzyme inactivation. Taking into
account that Cap is less efficient photosensitizer than Fop, it is
expected that the rate of enzyme photoinactivation should be
lower when the Fop concentration decreases as a function of irra-
diation time. In order to elucidate the reason of the kinetic behav-
ior of the enzyme inactivation when Fop is used as photosensitizer,
and taking into account that under UV-A radiation Fop generates
(
Fig. 2b). Consequently, these results suggest that, in contrast to
one molecule of H
(Scheme 1) [48], it was appropriate to explore the stability of
tyrosinase in the presence of H (see next section).
2 2
O for each molecule of Fop consumed
PteGlu, Fop and Cap photoinduce the inactivation of the enzyme.
2 2
O
3
.2. Evaluation of the photoinactivation of tyrosinase by PteGlu
2 2
3.3. Evaluation of the inactivation of tyrosinase by H O
photoproducts
The study of the stability of tyrosinase in the presence of H
very important from a biomedical point of view because patients
with vitiligo accumulate high levels (mM) of H in their epider-
mis [49,50]. In addition, it has been suggested that photooxidation
of pterins is one of the H sources in this depigmentation disor-
der [31]. Schallreuter et al. have studied the direct effect of H on
the tyrosinase reaction. The results showed that the enzyme was
activated by low concentrations of H (<300 M), while at con-
centrations in the range 500–5000 M the tyrosinase was inacti-
vated [51]. Taking into account this report, since in the
photooxidation of Fop, one molecule of H is produced per mole-
cule of Fop consumed, the H formed in our experiments would
not be able to produce the inactivation of tyrosinase. Therefore, to
assess the participation of H in the photoinactivation of tyrosi-
nase by Fop, air equilibrated aqueous solutions of tyrosinase and
(pH 6.0) were incubated at 20 °C in the dark for different
2 2
O is
To confirm that PteGlu photoproducts are able to photoinduced
the inactivation of tyrosinase, the rate of enzyme inactivation was
determined in experiments carried out under the same conditions
as those used in the experiment of Fig. 2 (pH, absorbance at the
irradiation wavelength (350 nm), irradiation source, optical geom-
etry, etc), but changing the pterin derivatives used as a photosen-
sitizer. After the irradiation, the samples were analyzed by UV–Vis
spectrophotometry, enzyme activity measurement, and HPLC. The
2 2
O
2 2
O
2 2
O
2
O
2
l
l
2 2
O
(
a)
2 2
O
60
2 2
O
2 2
H O
PteGlu
Fop
times and then the activity of the enzyme was determined.
Surprisingly, the data showed that tyrosinase was deactivated by
4
2
0
0
Cap
low H
2
O
2
concentrations (Fig. 4). These results, in disagreement
pro-
with those reported by Wood et al. [51], suggest that the H
2 2
O
duced during the photooxidation of PteGlu and its photoproducts
is enough to cause, at least, a partial inactivation of the enzyme.
2 2
To further investigate the contribution of H O in the deactiva-
0
5
4
3
2
1
0
tion of the enzyme during the photosensitized process, air equili-
brated aqueous solutions of tyrosinase and Fop were exposed to
UV-A radiation in the absence and in the presence of catalase, an
(
b)
0
246810
enzyme that catalyzes specifically the decomposition of H
2 2
O into
H
2
O and O . Fop was chosen for this study because, according to
2
the results shown so far, this derivative is the main sensitizer
responsible for the photoinactivation of the enzyme. The data
obtained clearly showed that the photoinactivation of tyrosinase
was much more efficient in the absence than in the presence of
catalase (Fig. 5), thus suggesting the participation of H
To discard potential interferences in the experiment shown in
Fig. 5 and then to confirm the participation of H in the inactiva-
2 2
O .
2 2
O
tion of tyrosinase upon irradiation in the presence of Fop, several
controls were carried out. In first place, to determine if catalase
is able to react with Fop under excitation, air equilibrated aqueous
solution of the photosensitizer and catalase were irradiated with
UV-A radiation. Concentration profiles showed that the rates of
Fop photooxidation did not change in the presence of catalase
0
2
4
6
8
10
time (min)
Fig. 2. Photolysis of air-equilibrated aqueous solution of PteGlu and tyrosinase
exposed to UV-A radiation. Time evolution of: (a) PteGlu, Fop and Cap concentra-
tions; (b) tyrosinase activity (v, rate of formation of
PteGlu] = 70 M, [tyrosinase] = 28 g/mL, 1 mM KH PO
(
data not shown). In another control experiment, solutions of
L
-dopachrome).
;V;1
[
0
l
0
l
2
4
, pH = 6.0, q0
.
tyrosinase without photosensitizer were irradiated in the presence
n;p

1
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M. Laura Dántola et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 172–179
5
4
3
2
1
5
4
3
2
1
5
4
3
2
1
(
a)
(b)
(c)
0
1
00
100
80
100
80
60
40
20
Fop
Cap
Ptr
Cap
Ptr
Ptr
8
6
4
2
0
0
0
0
0
60
40
20
0
5
10
15
20
25 0
5
10
15
20
25 0
5
10
15
20
25
time (min)
time (min)
time (min)
Fig. 3. Evolution of tyrosinase activity (v), and concentrations of pterins in air-equilibrated aqueous solution containing tyrosinase and a given pterin derivative exposed to
;V;2
UV-A radiation as a function of the elapsed irradiation time (kexc = 350 nm, [tyrosinase]
(350 nm) = 0.50, [Fop] = 44 M); (b) Cap (A (350 nm) = 0.50, [Cap] = 65 M); (c) Ptr (A
0
= 23
(350 nm) = 0.50, [Ptr]
2 4
lg/mL, 1 mM KH PO
n;p
, pH = 6.0, q0 ). The photosensitizers used were: (a) Fop
(A
0
0
l
0
0
l
0
0
= 104 lM).
1
1
10
00
100
8
6
4
2
0
0
0
0
0
9
8
7
0
0
0
60
[
[
[
[
[
H O ]= 0μM
2 2
5
4
0
0
H O ]= 10μM
2
2
H O ]= 20μM
2
2
H O ]= 50μM
2
2
H O ]= 100μM
30
2
2
0
2
4
6
8
0
20 40 60 80 100 120 140 160
time (min)
time (min)
Fig. 5. Determination of the activity of tyrosinase as a function of irradiation
time in the presence of catalase and absence of Fop (j), in the presence of
Fop and absence of catalase (d) and in the presence of Fop and catalase
Fig. 4. Determination of the activity of tyrosinase as a function of incubation time
at different concentrations of H ([tyrosinase] = 23 g/mL, T = 20 °C, 20 mM
KH PO pH = 6.0). For comparative purposes, the data are expressed as the
2
O
2
0
l
2
4
,
(.) ([tyrosinase]
0
= 50
l
2 4
g/mL, 1 mM KH PO , kexc = 350 nm, pH = 6.0, catalase
percentage of the initial enzyme activity.
0;V;1
(
5800 U/mL), [Fop]
0
= 44
lM,
q
). For comparative purposes, the data are
n;p
expressed as the percentage of the initial enzyme activity.
and in the absence of catalase. No decrease of the activity of tyrosi-
nase was registered in the presence of catalase, thus excluding the
possibility that this enzyme directly affects the activity of tyrosi-
nase (Fig. 5).
this reactive oxygen species reacts with the enzyme causing its
inactivation.
The results presented in this section clearly indicated that pho-
toinactivation of tyrosinase in our reaction system takes place
through two different types of pathways: (i) photosensitized reac-
3.4. Effect on the tryptophan residues
Taking into account the photosensitization of Trp free [32] and
in polypeptide chains [34] by Ptr previously reported, and that Trp
is particularly susceptible to a variety of oxidizing agents [52], it is
straightforward to expect that Trp residues of tyrosinase were
tions and (ii) the oxidation of the enzyme by H
is produced in the photooxidation of PteGlu into Fop, and
in the subsequent photooxidation of Fop into Cap [30] and then
2 2
O . In the latter,
2 2
H O

M. Laura Dántola et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 172–179
177
1
0
0
0
0
0
0
.0
.9
.8
.7
.6
.5
.4
1
.0
6
5
4
3
2
1
0
t=0
t=0.5
t=2
t=6
0.8
0.6
0.4
v/vt=0
0
I /I
F
F
3
00 320 340 360
λ (nm)
0
.2
0.0
0
2
4
6
8
10
0
5
10
15
20
25
30
time (min)
time (min)
0
0
F
Fig. 6. Time evolution of the relative total fluorescence intensity (I
F
=I ) of an
Fig. 7. Evolution of the relative total fluorescence intensity (I =I ) and relative
F
F
aqueous solution of tyrosinase irradiated at 350 nm in the absence of photosen-
sitizer (d) and in the presence of Fop ((A0(350 nm) = 0.50, [Fop] = 44 M)) (.) or Cap
0(350 nm) = 0.50, [Cap] = 65 M) (j). [Tyrosinase] = 50 g/mL, pH = 6.0, q
tyrosinase activity (v/vt=0) of an aqueous solution of tyrosinase in the presence of
H O as a function of incubation time. [Tyrosinase]0 = 33 g/mL, [H O ] = 95.0 M,
20 mM KH PO , pH = 6.0, T = 20 °C, k = 280 nm, optical path length 0.4 cm.
2 4 exc
0
l
2
2
l
2 2 0
l
0
;V;1
(A
0
l
0
l
.
n;p
Corrected fluorescence spectra were obtained by excitation at 280 nm and recorded
between 300 and 380 nm, T = 20 °C, optical path length 0.4 cm. Inset: Corrected
fluorescence spectra registered using Fop as a photosensitizer. The irradiation time
(
min) appears above each spectrum.
1.0
affected by photosensitization with PteGlu and its photoproducts.
Therefore, this point was investigated, taking advantage of the flu-
orescence properties of this amino acid [47], i.e. it is the only amino
acid residue that absorbs at 295 nm and presents an intense emis-
sion band centered between 310 and 360 nm, depending on the
protein local environment [47].
In tyrosinase the Trp residues are buried in hydrophobic envi-
ronment when the protein is in the native state [53]. This enzyme
contains also Tyr residues which act as intrinsic fluorescence
probes, as well. In contrast to excitation at 295 nm, when excita-
tion wavelength of 280 nm is used, both Tyr and Trp residues are
excited. The comparison of fluorescence spectra of tyrosinase
obtained by excitation at 280 and 295 nm showed that there are
not changes in the shape and emission maximum of both spectra
0
0
0
0
0
.8
.6
.4
.2
.0
v/vt=0
0
F
I /I
F
0
5
10
15
20
time (min)
0
F
Fig. 8. Evolution of the relative total fluorescence intensity (I
relative tyrosinase activity (v/vt=0) (d) of an aqueous solution of tyrosinase and Cap
as a function of the irradiation time. [Tyrosinase] = 50 g/mL, [Cap ] = 60.0 M,
F
=I ) (.) and the
(
data not shown), thus indicating that emission fluorescence
comes mainly from Trp in tyrosinase, under this experimental
conditions.
0
l
0
l
0;V;1
1
2 4
mM KH PO , pH = 6.0, qn;p .
Therefore, to determine if the photosensitization process
altered the Trp residues of the enzyme, emission spectra of
air-equilibrated aqueous solutions containing tyrosinase and a
given pterin derivative were recorded after different irradiation
times. The typical emission band of Trp could be adequately regis-
tered when Fop and Cap were used as photosensitizers, since these
pterin derivatives fluoresce with a broad band centered at approx-
imately 450 nm [54]. In contrast, Trp emission could not be prop-
erly recorded when PteGlu was used as a photosensitizer because
PABA–Glu, one of the products of the PteGlu photodegradation
To determine if the H
tyrosinase, air equilibrated aqueous solutions of tyrosinase and
(pH 6.0) were incubated at 20 °C in the dark. After the incu-
bation, the samples were analyzed by enzyme activity measure-
ment and fluorescence spectroscopy. As shown in Fig. 7, Trp
emission did not change in 30 min of incubation. For comparative
purposes, the corresponding plot of activity as a function of time
was included in Fig. 7. This result indicated that the alteration to
Trp residues during the photosensitization process is only due to
the reaction of excited state of pterin derivatives with the enzyme
2 2
O is able to oxidize the Trp residues of
2 2
H O
(Scheme 1), absorbs at the excitation wavelength and fluoresces
with a broad and intense band centered at ca 350 nm.
2 2
and, H O does not contribute.
As shown in Fig. 6, Trp emission decreased significantly as a
function of time when tyrosinase was irradiated in the presence
of Fop or Cap. This result suggests that, Trp residues are affected
in the photosensitization process. The comparison of the Trp emis-
sion decrease registered using Fop or Cap as sensitizer revealed
that, Fop is more efficient than Cap to produce an alteration in
the Trp residues of tyrosinase.
As a control experiment, tyrosinase aqueous solutions were
exposed to UV-A radiation during different periods of time in the
absence of pterin derivatives. No changes in the total fluorescence
intensity were detected, even after more than 10 min of irradia-
tion, thus excluding the possibility that spurious effects of direct
light absorption by the protein could affect its fluorescence (Fig. 6).
Taking into account the data shown so far, it is clear that the
loss of enzyme activity is due to two different pathways: a photo-
sensitized process and the reaction with H O . On the other hand,
2 2
the Trp residues are affected only by the photosensitization. To
compare rate of photoinactivation by photosensitization itself with
the Trp alteration, additional experiments were carried out using
Cap as a photosensitizer. This pterin derivative was chosen for this
purpose because its photodegradation is negligible in the time
2 2
window used in our experiments and, consequently, no H O pro-
duction is possible. Fig. 8 shows that the rate of photoinactivation
is higher than the rate of Trp photoalteration, indicating that the
active site of the enzyme is affected earlier than the amino acid
residues.

1
78
M. Laura Dántola et al. / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 172–179
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Appendix A. Supplementary material
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Lorente, A.H. Thomas, Mechanism of photooxidation of folic acid sensitized
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