Visualization of tyrosinase activity in melanoma cells by a BODIPY-based fluorescent probe

Article abstract of DOI:10.1039/c1cc15061h

We have presented a fluorescent probe 1 that exhibits a fluorescence turn-on signal upon reaction with tyrosinase, and we show that it is readily employed for the assessment of tyrosinase activity and tyrosinase inhibitor activity in buffered aqueous solu

Full text of DOI:10.1039/c1cc15061h

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COMMUNICATION
Visualization of tyrosinase activity in melanoma cells by a
BODIPY-based fluorescent probew
Tae-Il Kim,a Jihye Park,a Seonhwa Park,b Yongdoo Choi*b and Youngmi Kim*a
Received 15th August 2011, Accepted 30th September 2011
DOI: 10.1039/c1cc15061h
We have presented a fluorescent probe 1 that exhibits a fluorescence
turn-on signal upon reaction with tyrosinase, and we show that it
is readily employed for the assessment of tyrosinase activity and
tyrosinase inhibitor activity in buffered aqueous solution, and
further utilized for the visualization of endogenous tyrosinase
activity in living melanoma cells.
function in a turn-off mode in which the tyrosinase-catalyzed
oxidation product, the quinone moiety, quenches fluorescence of
the fluorophores or QDs via electron transfer. For practical
applications, however, it is preferable to perform a bioassay in
the turn-on mode because this mode is much more sensitive and
better suited for bioimaging intracellular tyrosinase activity in
living systems.
4,4-Difluoro-4-borata-3a-azonia-4a-aza-s-indacene (BODIPY)
derivatives are well-known fluorophores with many valuable
properties, including elevated (photo)chemical stability, relatively
high fluorescence quantum yields and absorption coefficients,
and versatility in terms of chemical derivatization.6 In addition,
the fluorescence properties of BODIPY derivatives can be
systematically modulated by controlling the relative free energy
change of an intramolecular photoinduced electron transfer
(PeT) process (DGeT).7 Several research groups have used the
PeT-dependent fluorescence off/on switching mechanism to
successfully design meso-modified BODIPY dyes as fluorescence
probes for redox active molecules and conditions (e.g., nitric oxide
and nitrative stress),8 pH,9 and various metal ions (e.g., Fe3+).10
In these, fluorescence enhancement is achieved by perturbing
the highest occupied molecular orbital (HOMO) energy levels
of the meso-substituents upon interaction with target analytes.
We utilized this finding to construct a tyrosinase-selective
fluorescence ‘‘turn-on’’ probe, which functions despite the
strong quenching of fluorescence by the tyrosinase-generated
oxidation product. Herein, we report a PeT-controlled fluorescence
turn-on probe for monitoring tyrosinase activity in aqueous
media and in living cells.
Tyrosinase, which is widespread in plants and animal tissues,
is a copper-containing enzyme that catalyzes the hydroxylation
of phenol derivatives, such as tyrosine or tyramine, to the
respective catechol derivatives (i.e., L-DOPA or dopamine,
respectively), followed by further oxidation to the corresponding
o-quinone products.1 The enzyme, specifically found in melano-
cytes, plays an essential role in the biosynthesis of melanin, the
pigment that gives skin color, protects DNA in skin cells from
ultraviolet (UV) radiation, and removes reactive oxygen species
(ROS).1 In humans, disruption of tyrosinase is responsible for
severe skin diseases such as oculocutaneous albinism type I,2 and
elevated expression of this enzyme is found in melanoma cells.3
In addition, tyrosinase might contribute to dopamine neurotoxicity
and neurodegeneration associated with Parkinson’s disease.4
Consequently, a sensitive and selective assessment of this
biochemical marker would be highly valuable not only for
understanding its role in biological and pathological processes
but also for providing effective diagnostic approaches and/or
therapeutic targets in biomedical research.
Fluorescent probes have recently attracted considerable
attention owing to their simple and rapid implementation, as
well as their high sensitivity and high spatial resolution. How-
ever, there are only a few fluorescent probes that can detect
catalytic activity of tyrosinase, with the exception of quantum
dots (QDs)-, cyanine dye-, and oligo(phenylenevinylene)-based
fluorescent probes.5 Although these probes may be used to
monitor tyrosinase activity, a general problem is that they all
Scheme 1 illustrates our design for tyrosinase assays, in
which we consider substrate specificity of the enzyme and the
turn-on signaling mechanism. Studies have reported that
tyrosinase is able to use mono-, di-, and trihydroxyphenols
a Department of Chemistry, 126 Jukjeon-dong, Yongin-si,
Gyeonggi-do, 448-701, Korea. E-mail: youngmi@dankook.ac.kr;
Fax: +82 31-8005-3148; Tel: +82 31-8005-3156
b Molecular Imaging & Therapy Branch, National Cancer Center,
323 Ilsan-ro, Goyang-si, Gyeonggi-do 410-769, Korea.
E-mail: ydchoi@ncc.re.kr; Fax: +82 31-920-2529;
Tel: +82 31-920-2512
w Electronic supplementary information (ESI) available: Experimental
details, additional characterization studies, and additional information
about experiments performed in living cells. See DOI: 10.1039/
c1cc15061h
Scheme 1 Proposed fluorescence turn-on assay for tyrosinase by
probe 1.
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as substrates but has greater affinity for dihydroxyphenols,
especially for catechol;11 therefore, we incorporated a catechol
moiety into the BODIPY chromophore to provide the fluorogenic
substrate 1. For the turn-on signaling mechanism, we harnessed
the PeT process from the pendant catechol moiety to the
excited fluorophore to quench the emission of the probe in
aqueous media. Before the reaction of probe 1 with tyrosinase,
the HOMO energy level of the catechol moiety is higher than
that of the BODIPY unit in polar aqueous solution; hence, the
fluorescence of probe 1 is quenched through a reductive PeT
process.7 The tyrosinase-catalyzed reaction will trigger the
oxidation of the catechol unit of probe 1 to the corresponding
benzoquinone; the HOMO energy level of the benzoquinone
moiety is lower than that of the BODIPY skeleton. Such a
transformation will suppress the PeT process and result in
enhanced fluorescence intensity. This provides a method to
selectively monitor tyrosinase activity, wherein fluorescence
intensity depends upon the enzyme activity.
Fig. 2 (A) Increase in fluorescence intensity of probe 1 (1 mM) at
515 nm (lex 465 nm) upon incubation with different amounts of
tyrosinase (from bottom to top: 0 to 90 nM) in the presence of O2.
The reaction was conducted in PBS buffer (50 mM, pH 6.3, 0.2%
DMSO, 37 1C). F0 and F correspond to the fluorescence intensity of
probe 1 in the absence and presence of tyrosinase, respectively. (B)
Inhibition assay of tyrosinase activity by kojic acid, benzaldehyde,
anisaldehyde, and benzoic acid: relative fluorescence intensity of the
reaction with probe 1 as a function of concentration of inhibitors.
Tyrosinase (90 nM) was incubated with an inhibitor for 20 min at
25 1C before addition of probe 1 (1 mM). After 300 min of incubation
at 37 1C, the emission intensity at 515 nm was recorded (lex 465 nm).
Each point represents the average value of three measurements.
Probe 1 was prepared according to Scheme S1 (see ESIw).
UV-Vis absorption and emission spectra of 1 were measured in
an aqueous buffered solution (50 mM PBS, pH 6.3) containing
0.2% DMSO as a co-solvent. Probe 1 shows an absorption
maximum at 496 nm (e = 8.8 Â 104 MÀ1ÁcmÀ1) and an
emission maximum at 512 nm with a low fluorescence quantum
yield (FF) of approximately 0.001. This is similar to the
observations by Burdette et al., and the low quantum yield
is attributed to an efficient PeT process in aqueous solution.10
To confirm our hypothesis, the expected quinone product 1-Q
was synthesized independently, purified, and fully characterized.
Conversion to the quinone is accompanied with slight bathochromic
shifts in the absorption (labs of 498 nm) and emission (lem of
515 nm) maxima. Moreover, 1-Q exhibits an enhanced fluores-
cence quantum yield (FF of 0.018) in an aqueous buffered
solution, as anticipated. This result suggests that the PeT
process of probe 1 will be attenuated upon tyrosinase-catalyzed
oxidation, resulting in enhanced fluorescence intensity.
(5 mM) was incubated with tyrosinase (90 nM), the fluores-
cence signal at 515 nm increased with the incubation time
(Fig. 1B). As shown in Fig. 2A, the results of kinetic analysis
clearly indicate that the enzyme-catalyzed reaction is time-
dependent. In addition, the change in fluorescence intensity is
directly proportional to the amount of enzyme added over the
range tested (see ESIw); higher concentrations of tyrosinase
result in a greater signal-to-noise ratio. Tyrosinase could be
assayed with a sensitivity limit as low as 0.5 nM of tyrosinase.
Control experiments revealed little change in the fluorescence
intensity of probe 1 in the absence of tyrosinase or O2 from the
reaction mixture during the same period of time (see ESIw).
These results indicate that the tyrosinase-catalyzed oxidation
of the catechol unit leads to the increase in fluorescence
intensity of probe 1. Complementary LC-MS analysis of the
assay solution confirmed that the tyrosinase-induced reaction
generated the corresponding quinone product, 1-Q ([M+H] =
355.1, see ESIw). The nonfluorescent nature of probe 1 and the
significant fluorescence turn-on response of tyrosinase-treated
probe 1 are clearly visualized (Fig. 1B inset). We then deter-
mined kinetic parameters for the enzymatic oxidation reaction
of probe 1 using Lineweaver–Burk analysis. The kcat and Km
values of probe 1 (kcat = 0.01 sÀ1, Km = 5.3 mM) were higher
than those of L-DOPA, a commercially available tyrosinase
substrate (kcat = 0.007 sÀ1, Km = 1.7 mM). These data
indicate that enzymatic efficiency for probe 1, as estimated
by a kcat/Km of 2.0 mMÀ1ÁsÀ1, is lower than that of L-DOPA
(kcat/Km = 4.0 mMÀ1ÁsÀ1).
To examine whether probe 1 can be used to measure
tyrosinase activity, we conducted enzymatic assays with probe
1 as the tyrosinase substrate in the presence of O2 under
optimized assay conditions (50 mM PBS buffer solution,
0.2% DMSO, pH 6.3, 37 1C).12 Fig. 1 shows the time-
dependent changes in absorption and fluorescence spectra of
probe 1 upon the reaction with tyrosinase/O2. When probe 1
Next, to investigate the utility of probe 1 for screening
tyrosinase inhibitors, tyrosinase activity was tested in the
presence of common tyrosinase inhibitors, kojic acid, benzal-
dehyde, anisaldehyde, and benzoic acid at pH 6.3 (Fig. 2B).11
Tyrosinase was pre-incubated with each inhibitor before being
added to probe 1, and the enzyme reaction was monitored by
measuring fluorescence intensity at 515 nm. The enhancement
of fluorescence intensity of probe 1 was inhibited in a dose-
dependent manner by each inhibitor. In particular, addition of
100 mM kojic acid entirely blocked the fluorescence increase,
Fig. 1 Time-dependent absorption (A) and emission (B) spectra of
probe 1 (5 mM) upon treatment with tyrosinase (90 nM)/O2 at 37 1C in
a PBS buffer solution (50 mM, pH 6.3, 0.2% DMSO). The spectra
were obtained every 30 min (0–300 min). The emission spectra were
obtained after excitation at 465 nm. Inset shows photographs, taken
under UV light illumination (365 nm), of probe 1 (5 mM) before (left)
and after (right) addition of tyrosinase (90 nM)/O2 and incubation at
37 1C for 300 min.
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regions in a spotted pattern. These are in good agreement with
the results of Ballotti et al. that a-MSH induces the accumulation
of melanosomes at the dendrite tips of melanocytes.14 Treat-
ment of B16F10 melanoma cells with the tyrosinase inhibitor
kojic acid (KA; 100 mM) during a-MSH stimulation resulted
in a reduced fluorescent signal in the cells (Fig. 3C). In
addition, we investigated tyrosinase activity in melanoma cells
treated with miconazole (MIC), which significantly suppresses
tyrosinase activity and tyrosinase protein expression in the
cells (see ESIw).15 Following addition of 30 mM MIC to the
a-MSH-stimulated B16F10 cells, negligible fluorescence intensity
at both peripheral and overall intracellular regions was
observed (Fig. 3D). These results clearly demonstrate that probe
1 can be used to visualize the tyrosinase-induced oxidation in
melanoma cells. Cell viability assays confirm that 5 mM probe 1
shows low cytotoxicity to B16F10 cells even upon treatment of
cells for as long as 24 h (see ESIw).
Fig. 3 Relative confocal fluorescence images of B16F10 melanoma
cells treated under different conditions with probe 1. (A) Melanoma
cells were incubated with 2.5 mM probe 1 for 30 min at 37 1C and then
imaged. (B) Melanoma cells were stimulated with 100 nM a-melano-
cyte-stimulating hormone (MSH) for 48 h and then incubated with 2.5 mM
probe 1 for 30 min at 37 1C followed by imaging. Either 100 mM kojic
acid (KA) (C) or 30 mM miconazole (MIC) (D) was co-incubated with
cells during a-MSH stimulation, while all other conditions were the
same. Top row of panels, fluorescence images; middle, bright field
images; bottom, merged images. lex = 488 nm; lem = 505–550 nm.
In summary, we have shown that our BODIPY-based
fluorescent probe 1 can be used for the selective detection of
tyrosinase activity in buffered aqueous solution. Probe 1 is also
suitable for screening potential inhibitors of tyrosinase, as
well as for bioimaging intracellular tyrosinase activity in
living cells.
This research was supported by a grant from the Fundamental
R&D Program for Core Technology of Materials that was
funded by the Ministry of Knowledge Economy, Republic of
Korea, and by a Korea Research Foundation (KRF) grant
funded by the Korean government (MEST) (No. 2010-0011160).
confirming that enzymatic activity of tyrosinase is required to
oxidize probe 1. The IC50 was calculated to be 18 mM for kojic
acid, 850 mM for benzaldehyde, 460 mM for anisaldehyde, and
750 mM for benzoic acid; these values are in good agreement
with previously reported values.11,13 These results highlight the
use of probe 1 for tyrosinase activity assays as well as for
screening potential tyrosinase inhibitors.
Notes and references
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To evaluate the practical utility of probe 1 for the detection
of tyrosinase activity, photo- and chemical stabilities of 1 and
1-Q were investigated under the assay conditions employed.
Photostability studies of 1 and 1-Q were performed by
continuous irradiation of 1 and 1-Q using a 150 W steady-state
Xe lamp as the light source. The photoinduced bleaching was
quantified by monitoring fluorescence intensity as a function of
irradiation time (see ESIw). The fluorescence intensity of 1 and
1-Q remained stable after irradiation at 465 nm for 2.5 h. In
addition to photostability, the chemical stability of 1 and 1-Q
was studied under aerobic conditions, and the results indicate
negligible changes in fluorescence spectra of both compounds in
aqueous buffers for periods as long as 5 h (see ESIw). These
properties suggest that probe 1 may be suitable for the investigation
of such biological events.
7 H. Sunahara, Y. Urano, H. Kojima and T. Nagano, J. Am. Chem.
Soc., 2007, 129, 5597.
8 Y. Gabe, Y. Urano, K. Kikuchi, H. Kojima and T. Nagano, J. Am.
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12 The maximum fluorescence response was observed at pH 6.3. It
was consistent with the optimum pH of tyrosinase activity reported
in the literature: F. Gauillard, F. Richard-Forget and J. Nicolas,
Anal. Biochem., 1993, 215, 59.
13 Y.-J. Kim and H. Uyama, Cell. Mol. Life Sci., 2005, 62, 1707.
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G. Valony, K. Bille, J. P. Ortonne and R. Ballotti, FASEB J., 2004,
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We then evaluated the applicability of probe 1 to living cells.
Bioimaging of tyrosinase activity was carried out in B16F10
cells, a melanoma cell line that expresses tyrosinase activity,
with and without treatment with a-melanocyte-stimulating
hormone (a-MSH), which upregulates tyrosinase expression
in these cells.14 Confocal fluorescence microscopic imaging of
a-MSH-stimulated B16F10 cells that were incubated with
probe 1 showed a significant increase in green fluorescence,
indicative of tyrosinase activity (Fig. 3B); cells that were not
treated with a-MSH exhibited less intense intracellular fluores-
cence (Fig. 3A). In particular, substantial fluorescent responses
in a-MSH-stimulated cells were acquired at peripheral cell
15 Y.-J. Mun, S.-W. Lee, H.-W. Jeong, K.-G. Lee, J.-H. Kim and
W.-H. Woo, Biol. Pharm. Bull., 2004, 27, 806.
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12642 Chem. Commun., 2011, 47, 12640–12642
This journal is The Royal Society of Chemistry 2011