Inhibition kinetics of chlorobenzaldehyde thiosemicarbazones on mushroom tyrosinase

Article abstract of DOI:10.1021/jf1033625

2-Chlorobenzaldehyde thiosemicarbazone (2-Cl-BT) and 4-chlorobenzaldehyde thiosemicarbazone (4-Cl-BT) were synthesized, and their inhibitory kinetics on the activity of mushroom tyrosinase were investigated. Results showed that these compounds exhibited s

Full text of DOI:10.1021/jf1033625

J. Agric. Food Chem. 2010, 58, 12537–12540 12537
DOI:10.1021/jf1033625
Inhibition Kinetics of Chlorobenzaldehyde
Thiosemicarbazones on Mushroom Tyrosinase
†
,‡
†,‡
†
†
Z
HI-CONG
L
I
,
L
IANG-HUA
C
HEN
,
X
IAO-JIE
Y
U
, YONG-HUA
H ,
*
U
,†,§
§
,
K
ANG-KANG
S
ONG, XING-WANG
Z
HOU,* AND
Q
ING-X
I
CHEN
†
Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life
§
Sciences, Xiamen University, Xiamen 361005, China, Key Laboratory for Chemical Biology of Fujian
Province, Xiamen University, Xiamen 361005, China, and Department of Biochemistry and Pharmacology,
Zhongshan School of Medicine, Sun Yat-sen University, Key Laboratory of Tropical Disease Control,
‡
Ministry of Education, Guangzhou, 510080, China. These authors contributed equally to this work
2
-Chlorobenzaldehyde thiosemicarbazone (2-Cl-BT) and 4-chlorobenzaldehyde thiosemicarbazone
(4-Cl-BT) were synthesized, and their inhibitory kinetics on the activity of mushroom tyrosinase were
investigated. Results showed that these compounds exhibited significant inhibitory potency on both
monophenolase activity and diphenolase activity of tyrosinase. For the monophenolase activity, both
compounds could decrease the steady-state activity of the enzyme sharply, without any influence on
the lag period. The IC50 values of them were estimated to be 15.4 μM and 6.7 μM, respectively. For
the diphenolase activity, both compounds belonged to reversible inhibitors, but their mechanisms
were different: 2-Cl-BT was a noncompetitive type inhibitor, while 4-Cl-BT was a mixed-type
inhibitor. Their inhibition constants were determined and compared.
KEYWORDS: Mushroom tyrosinase; monophenolase activity; diphenolase activity; chlorobenzaldehyde
thiosemicarbazones; inhibition kinetics; synthesis
INTRODUCTION
with inhibitory potency ranging from slight to significant, have
been reported (15-17). Several thiourea derivatives were re-
ported to have moderate antityrosinase activity, and their in-
hibitory mechanisms were elucidated (18). Xue et al. reported the
Tyrosinase (EC 1.14.18.1), a type-3 dicopper multifunctional
oxidase widely distributed in nature, plays a crucial role in
melanogenesis. It catalyzes the key step of the formation of
melanin, the hydroxylation of monophenol to o-diphenol and
the oxidation of diphenol to o-quinones (1, 2). Melanogenesis is
an important physiological process that results in the production
of melanin pigment, which is of importance in the prevention of
UV-induced skin injuries (3). However, excessive accumulations
of epidermal pigmentation can cause various hyperpigmentation
disorders, such as melasma, age spots, and sites of actinic
damage (4). Therefore, the regulation of melanin synthesis via the
inhibition of tyrosinase is a current research topic in the context of
preventing hyperpigmentation. Also, tyrosinase participates in
the undesired enzymatic browning progress in plants, which may
drastically decrease the quality and value of such food pro-
ducts (5). Furthermore, tyrosinase distributed in insects is
involved in the host defense, wound healing, molting and
sclerotization process of insects (6, 7). Thus, tyrosinase inhibi-
tors are of great concern to scientists in clinical medicinal (8-10)
and cosmetic research (11) in relation to hyperpigmentation,
food preservation technology (12) and alternative insect
control (13).
3D-QSAR and molecular docking studies of benzylaldehyde
thiosemicarbazone derivatives as phenoloxidase inhibitors; re-
sults showed that these thiosemicarbazone compounds possess
much higher inhibitory activity (19, 20). Also our previous work
showed that trans-cinnamaldehyde thiosemicarbazone exhibits
significant tyrosinase inhibitory activity (21).
In this study, 2-chlorobenzaldehyde thiosemicarbazone
(2-Cl-BT) and 4-chlorobenzaldehyde thiosemicarbazone (4-Cl-BT)
were synthesized, and their inhibitory kinetics on the activity of
mushroom tyrosinase were investigated.
MATERIALS AND METHODS
Reagents. Mushroom tyrosinase (EC 1.14.18.1) was purchased from
Sigma (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO),
L-tyrosine
(
L
-Tyr) and -3,4-dihydroxyphenylalanine ( -DOPA) were the products of
L
L
Aldrich (St. Louis, MO, USA). All other reagents were local and of
analytical grade. The water used was redistilled and ion-free.
Synthesis. Compounds were prepared by a simple one-step reaction of
corresponding chloride substituted benzylaldehyde with thiosemicarbazide in
an acidic solution of ethanol, as previous described (21). A mixture of the
corresponding benzylaldehyde (10 mM) with thiosemicarbazide (10 mM)
in 40 mL of ethanol with 2 mL of acetic acid solution was refluxed until
the reaction was complete and then cooled to room temperature. The
precipitates were collected and washed with cold ethanol. The products
were purified by recrystallization from 50% ethanol and were identified by
Many efforts have been addressed to screening efficient and
safe tyrosinase inhibitors from natural materials and synthetic
methods (14). To date, numerous novel tyrosinase inhibitors,
*
Corresponding authors. E-mail: chenqx@xmu.edu.cn (Q.-X.C.),
zhouxw2@mail.sysu.edu.cn (X.-W.Z.).
©
2010 American Chemical Society
Published on Web 11/09/2010
pubs.acs.org/JAFC

12538 J. Agric. Food Chem., Vol. 58, No. 23, 2010
Li et al.
Figure 1. Chemical structure of 2-Cl-BT (a) and 4-Cl-BT (b).
1
ESI-MS and H NMR analysis. ESI-MS data were performed on
1
ESQUIRE-LC ion trap mass spectrometry. H NMR data were acquired
on a 400 MHz NMR spectrometer (Bruker AV400). The purity was
confirmed by elemental analysis.
Enzyme Activity Assay. Monophenolase and diphenolase activities
of mushroom tyrosinase were determined as previously reported (22) with
modification, by measuring the rate of dopachrome formation at 475 nm
Figure 2. Inhibition effects of 2-Cl-BT on monophenolase activity of
mushroom tyrosinase. (a) Progress curves for the oxidation of L-Tyr by
the enzyme. The concentrations of inhibitor for curves 0-7 were 0, 5, 10,
15, 20, 30, and 40 μM, respectively. (b) Effects of 2-Cl-BT on the steady-
state rates of monophenolase. (c) Effects of 2-Cl-BT on the lag time of
mushroom tyrosinase.
-1
-1
(
ε=3700 M cm ). In this investigation,
L
-Tyr was used as substrate for
-DOPA was used as substrate for
diphenolase activity assay. The activity assay used 300 μL of reaction
medium containing 2 mM -Tyr or 0.5 mM -DOPA in 50 mM Na HPO
NaH PO buffer (pH 6.8). The final concentration of tyrosinase was
3.33 μg/mL for monophenolase activity and 6.67 μg/mL for diphenolase
3
monophenolase activity assay and
L
L
L
2
4
-
2
4
1
activity. The reaction was controlled at a constant temperature of 30 °C. The
assay was performed by SPECTRAMAX M2e from Molecule Device Co.
Effects of Inhibitors on the Enzyme Activity. Inhibitors were first
dissolved in DMSO and used for the test after a 30-fold dilution. In this
assay, 10 μL of DMSO solution with different concentrations of inhibitors
was first mixed with 240 μL of substrate solution (contained 0.5 mM
L-
DOPA or 2 mM -Tyr in 50 mM Na HPO -NaH PO buffer, pH 6.8);
L
2
4
2
4
then, a portion of 50 μL of enzyme solutionwas added to this blend and the
residual activity was determined. The final concentration of DMSO in the
test solution was 3.33%. Controls, without inhibitor but containing 3.33%
DMSO, were routinely carried out (23). The measurement was performed
in triplicate for each concentration and averaged before further calcula-
tion. The extent of inhibition by the addition of the sample was expressed
as the percentage necessary for 50% inhibition (IC50).
Figure 3. Determination of the inhibitory mechanism of 2-Cl-BT on mush-
room tyrosinase. The concentrations of inhibitor for curves 0-5 were 0,
0.3, 0.6, 0.9, and 1.5 μM, respectively.
Determination of the Inhibition Type and the Inhibition Constant.
The inhibition type was assayed by the Lineweaver-Burk plot, and the
inhibition constant was determined by the second plots of the apparent
m m m
K /V or 1/V versus the concentration of the inhibitor (23).
the steady-state rate decreased distinctly and dose-dependently.
The inhibitory effects of 4-Cl-BT on the oxidation of L-Tyr follow
RESULTS AND DISCUSSION
Chemical Synthesis of 2-Cl-BT and 4-Cl-BT. 2-Cl-BT recrys-
tallized as a light yellow powder. Yield: 92.5%. H NMR
the same mechanism. The concentration leading to 50% loss of
1
enzyme activity (IC ) was determined to be 15.4 μM and 6.7 μM,
50
(
(
DMSO-d , TMS, 400 MHz): δ (ppm) 11.20 (1H, s, HN), 7.91
respectively.
6
1H, s, CHdN), 8.04, 7.74 (2H, s, NH ), 9.48-7.19 (4H, m,
Effects of 2-Cl-BT and 4-Cl-BT on the Diphenolase Activity of
Mushroom Tyrosinase. We probed the effects of 2-Cl-BT and
4-Cl-BT on the activity of mushroom tyrosinase for the oxidation
2
þ
C H ). ESI-MS: m/z (100%) (M þ Na , methanol) 236.0; (M þ
6
þ
4
H , methanol) 214.2. Anal. Calcd: C, 44.97; H, 3.77; N, 19.66.
Found: C, 45.22; H, 3.799; N, 19.56. 4-Cl-BT recrystallized as a
of
L
-DOPA. When the diphenolase activity of tyrosinase was
1
light yellow powder, too. Yield: 88.1%. H NMR (DMSO-d ,
assayed by using
L-DOPA as substrate, the reaction course
6
TMS, 400 MHz): δ (ppm) 11.42 (1H, s, NH), 7.95 (1H, s,
CHdN), 8.02, 7.40 (2H, s, NH ), 8.21, 8.04 (4H, d, C H ).
immediately reached a steady-state rate. Both compounds can
inhibit the diphenolase activity of tyrosinase in a dose-dependent
manner. With increasing concentrations of inhibitors, the remain-
ing enzyme activity decreased exponentially. The inhibitor con-
centration leading to 50% activity lost (IC ) was estimated to be
2
þ
6
4
þ
ESI-MS: m/z (100%) (M þ Na , methanol) 236.0; (M þ H ,
methanol) 214.2. Anal. Calcd: C, 44.97; H, 3.97; N, 19.66. Found:
C, 45.00; H, 3.769; N, 19.79. (See Figure 1 for the structure.)
Effects of 2-Cl-BT and 4-Cl-BT on the Monophenolase Activity
of Mushroom Tyrosinase. The inhibitory effects of the different
50
1.22 μM and 1.82 μM, respectively.
The Inhibition Mechanism of 2-Cl-BT and 4-Cl-BT on the
Diphenolase Activity of Mushroom Tyrosinase Was Reversible.
The inhibition mechanism on mushroom tyrosinase by 2-Cl-BT
concentrations of 2-Cl-BT on the oxidation of
enzyme were studied. From the progress curve of the oxidation
of -Tyr without inhibitor, an apparent lag period, the character-
L-Tyr by the
L
and 4-Cl-BT for the oxidation of L-DOPA was investigated. Both
istic of monophenolase activity was observed (Figure 2a, curve 1).
The system reached a constant rate(the steady-state rate) after the
lag period, which was estimated by extrapolating the curve to the
abscissa (24). With increasing inhibitor concentration, the kinetic
inhibitors behaved in the same manner. Figure 3 showed the
relationship between enzyme activity and its concentration in the
presence of 2-Cl-BT. The plots of the remaining enzyme activity
versus the concentrations of enzyme at different inhibitor con-
centrations gave a family of straight lines, which all passed
through the origin. Increase of inhibitor concentration resulted
in descent of the slope of the line, indicating that the presence of
inhibitor did not reduce the amount of enzyme, but just resulted
course of the oxidation of -Tyr is shown in Figure 2a, curves 2-7.
L
The lag time and the steady-state rate were determined, and the
data are shown in Figure 2b and Figure2c, respectively. The lag
time did not change with increasing inhibitor concentration, but

Article
J. Agric. Food Chem., Vol. 58, No. 23, 2010 12539
Table 1. Inhibition Constants of 2-Cl-BT and 4-Cl-BT with Mushroom
Tyrosinase
2
-chlorobenzaldehyde
thiosemicarbazone
4-chlorobenzaldehyde
thiosemicarbazone
constants
IC50
monophenolase
diphenolase
15.4 μM
1.22 μM
reversible
6.7 μM
1.82 μM
reversible
inhibition
mechanism
inhibition type
noncompetitive
1.20 μM
1.20 μM
mixed type
1.25 μM
2.49 μM
K
K
I
IS
Figure 4. Determination of the inhibitory type and inhibition constants of
The results show that 2-Cl-BT and 4-Cl-BT could inhibit both the
diphenolase activity and the monophenolase activity of mush-
room tyrosinase. For diphenolase activity, the inhibition was
reversible, while the inhibition types were determined to be of
noncompetitive and competitive-uncompetitive mixed type,
respectively. As shown in Table 1, the IC50 values of 2-Cl-BT
and 4-Cl-BT for the diphenolase activity were similar. Kinetic
2
-Cl-BT. (a) Lineweaver-Burk plots for inhibition of 2-Cl-BT on mushroom
tyrosinase. The concentrations of inhibitor for curves 0-5 were 0, 0.3, 0.6,
.9, and 1.2 μM, respectively. (b) The plot of slope versus the concentra-
tion of 2-Cl-BT for determining the inhibition constants K and K .
0
I
IS
study reveals that both compounds exhibit similar K , which
I
means the binding affinity to free enzymes is about the same,
while their affinity to enzyme-substrate complexes is different.
For monophenolase activity, both compounds could inhibit the
steady-state activity drastically, without changing the lag time.
There are two copper ions in the active centerof tyrosinase, and
a lipophilic long-narrow gorge exists close to the active center
(25, 26). According to the 3D structure of tyrosinase, the copper
ions were of great importance for tyrosinase activity, and slight
change in the dicopper center may lead to activity loss. Benzy-
laldehyde thiosemicarbazone is a kind of Schiff base compound
that exhibits strong affinity to copper ions. Thus, when mixed
with tyrosinase and its substrate, a benzylaldehyde thiosemicar-
bazone derivative may form a complex with tyrosinase molecule
by its sulfur atom and nitrogen atom though a hydrogen bond
and a coordinate bond. This tight complex could make the free
oxygen molecule decrease its reaction ability and even unable to take
part in the hydroxylation with monophenols and in the oxidation
with o-diphenols, as the free oxygen molecule was surrounded
closely by two copper ions from tyrosinase and two hydrogen atoms
from two benzylaldehyde thiosemicarbazone molecules. Therefore
tyrosinase would lose its catalyzing ability (27).
Figure 5. Determination of the inhibitory type and inhibition constants of
-Cl-BT. (a) Lineweaver-Burk plots for inhibition of 4-Cl-BT on mushroom
tyrosinase. The concentrations of inhibitor for curves 0-4 were 0, 0.5, 1.0, and
.0 μM, respectively. (b) The plot of slope versus the concentration of 4-Cl-BT
for determining the inhibition constants K. (c) The plot of intercept versus the
4
2
I
concentration of 4-Cl-BT for determining the inhibition constants KIS
.
in the inhibition of enzyme activity. Both compounds were
reversible tyrosinase inhibitors.
The Inhibition Type of 2-Cl-BT on the Diphenolase Activity of
Mushroom Tyrosinase Was Noncompetitive. The inhibitory type
This paper investigated the kinetics of 2-Cl-BT and 4-Cl-BT on
the activity of mushroom tyrosinase. Results showed that both
compounds had a strong inhibitory effect on the monophenolase
activity and diphenolase activity of the enzyme. Compared with
2-Cl-BT on the diphenolase activity, during the oxidation of L-
DOPA, was determined from Lineweaver-Burk double recipro-
cal plots. In the presence of 2-Cl-BT, the kinetics of the enzyme is
shown in Figure 4. The plots of 1/v versus 1/[S] gave a family of
straight lines with different slopes which intersected one another
in the X-axis, indicating that 2-Cl-BT is a noncompetitive inhi-
bitor. The equilibrium constants for inhibitor binding with the
free enzyme and the enzyme-substrate complex, K and K ,
the most used antityrosinase agents, arbutin (IC =30 mM) and
50
kojic acid (IC50 =23 μM), both compounds showed much stronger
activities (IC50 =1.22 μM and 1.82 μM, respectively). Such tyrosi-
nase inhibitors may have broad application in cosmetic, medicinal,
food preservation and insect control area.
I
IS
were obtained from the secondary plot (Figure 4b) as 1.20 μM and
ABBREVIATIONS USED
1.20 μM.
The Inhibition Type of 4-Cl-BT on the Diphenolase Activity of
DMSO, dimethyl sulfoxide;
L-DOPA, L-3,4-dihydroxyphenyla-
Mushroom Tyrosinase Was of Mixed Type. In contrast, the kinetics
of the enzyme in the presence of 4-Cl-BT is shown in Figure 5.
Results showed 4-Cl-BT was of competitive-uncompetitive
mixed type inhibitor, for the Lineweaver-Burk double reciprocal
plots yield a group of lines that intercept in the second quadrant.
The inhibitor constants (K and K ) were estimated to be 1.25 μM
lanine; -Tyr, -tyrosine; IC , the inhibitor concentrations leading
L
L
50
to 50% activity lost; K , equilibrium constant of the inhibitor
I
combining with the free enzyme; K , equilibrium constant of the
IS
inhibitor combining with the enzyme-substrate complex.
I
IS
LITERATURE CITED
and 2.49 μM, respectively. The inhibitor constants are summar-
ized in Table 1 for comparison.
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Received for review August 30, 2010. Revised manuscript received
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