Research Journal of Chemical Sciences 
______
______________________________
______
____
 
ISSN 2231
-
606X
 
 
 
Vol. 
2
(
4
), 
76
-
78
, 
April 
(201
2
)
 
 
Res.J.Chem.Sci.
 
 
 
International Science Congress Association
 
 
 
 
    
76
 
Short Communication
 
New methods for 
Data Analysis 
of 
Isothermal Titration Calorimetry 
for 
studying binding of two n
-
alkyl 
Xanthates 
to 
Mushroom Tyrosinase
 
 
Rezaei Behbehani 
G.
1
, Barzegar L.
2
, Mehreshtiagh 
M.
1
, Mosavi M.
1
 
and 
Saboury 
A.A.
3
 
1
Department of Chemistry, Imam Khomeini International University, Qazvin, IRAN
 
2
Department of Chemistry, Faculty of science, Islamic Azad University, Takestan branch, Takestan, IRAN
 
3
Institute of 
Biochemistry and Biophysics, University of Tehran, Tehran, IRAN
 
 
Available online at: 
www.isca.in
 
(Received
 
6
th
 
March 
201
2
, revised
 
26
th
 
March 
201
2
, accepted
 
31
st
 
March
 
201
2
)
 
 
Abstract
 
A simple rapid direct isothermal titration calorimetry (ITC) method was applied to study the binding properties and structura
l 
changes of mushroom tyrosinase enzyme (MT) due to the interaction with  
two iso
-
alkyl dithiocarbonates (xanthates),
 
C
3
H
7
OCS
2
Na (I
) and C
4
H
9
OCS
2
Na (II) at 27 °C in phosphate buffer (10 mM) at pH 6.8. The extended solvation model provides 
more insights into this interaction for further understanding of the effect of iso
-
propyl and iso
-
butyl xanthate on the stability and 
the structural
 
changes of MT
.
 
The solvation parameters derived from the solvation model can be related to the changes in the 
stability of enzyme and type of inhibition.
 
ITC implies that there is a set of two binding sites for two new synthesized xanthates 
on MT with non
 
cooperativity in the binding process.
 
 
Keywords
: Mushroom tyrosinase
,
 
iso
-
propyl xanthate
,
 
i
so
-
butyl xanthate
,
 
the extended solvation theory
.
 
 
 
Introduction
 
Tyrosinase is a copper containing monooxygenase catalyzing 
the 
o
-
hydroxylation of monophenols to the corresponding 
catechols and the oxidation of catechols to the corresponding 
o
-
quinones
1
. mushroom tyrosinase is popular among researchers 
as it is commercially available and inexpensive
2
. In mushrooms 
as well as in fruits and vegetables, this enzyme is responsible for 
browning, a commercially undesirable phenomenon. Therefore, 
tyrosinase inhibitors have attracted interest recently due to this 
undesired browning in vegetables and fruits in
 
post
-
harvest 
handling. The di
-
copper centre of this enzyme has been the 
target of many inhibitors
3
. Xanthate compounds act as inhibitors 
of mushroom tyrosinase due to their ability to chelate copper 
ion. Lineweaver
-
Burk plots showed different patterns of 
mixed 
and competitive inhibition for iso
-
propyl and iso
-
butyl xanthate, 
respectively
4,5
.
 
We attempted to apply the extended solvation 
model to allow one to interpret the enzyme stability due to its 
binding with two new synthesized xanthates and to predict 
the 
inhibition type.
 
 
Material and Method
s
 
MT was obtained from 
s
igma, iso
-
propyl and iso
-
butyl xanthate 
was synthesized
5
. All other materials and reagents were of 
analytical grade, and solutions were made in 10 mM phosphate 
buffer using double
-
distilled w
ater.
 
 
The isothermal titration calorimetric experiments were 
performed with the four channel commercial microcalorimetric 
system, Thermal Activity Monitor 2277, Thermometric, 
Swe�en. The sa�ple cell containe� 1.8 �L MT (8.3 μM) an� 
phosphate buffer soluti
on (10 mM; pH 6.8) and the reference 
cell filled with phosphate buffer. The titration of MT with iso
-
propyl and iso
-
butyl xanthate involved 20 consecutive injections 
of the ligan� an� each injection inclu�e� 20 μL iso
-
propyl or 
iso
-
butyl xanthate. The calo
rimetric signal was gauged by a 
digital voltmeter that was part of a computerized recording 
system. The heat of injection was calculated by the 
‘Ther�o�etric Digita� 3’ software progra�. The 
microcalorimeter was frequently calibrated electrically during 
th
e course of the study. 
 
 
Results and Discussion
 
As we have shown before the heats of interactions between a 
protein and a ligand in the aqueous solvent systems, can be 
analyzed by the following equation
6
-
15
.
 
 
The obtained heats from extended solvation model (
equation
 
1) 
are in principle compatible with ITC enthalpies over the entire 
range of iso
-
propyl and iso
-
butyl xanthate, as shown in 
f
igure
-
1.
 
Where 
x
'
B
 
and 
x
'
A
 
can be defined as follows:
 
 
x
B 
is equal to the total ligand concentrations divided by the 
maximum concentration of the ligand upon saturation of all 
enzyme. The obtained result of p=1 shows that ligand bind at 
each site independently. 
L
A
 
and 
L
B
 
are the relative of unbound 
and
 
bound xanthate contributions to the enthalpies of dilution in 
the absence of tyrosinase.
 
Research Journal of Chemical Sciences 
______
_
_
_______________________________
______________
_
____
 
ISSN 2231
-
606X
 
 
Vol. 
2
(
4
), 
76
-
78
, 
April 
(201
2
)
 
  
Res.J.Chem.Sci
 
International Science Congress Association
 
 
77
 
 
 
δ
θ
A 
and 
δ
θ
B
 
values reflect the tyrosinase structural changes due 
to the interaction with xanthates
 
in the low and high 
concentrations in infinite dilution of tyrosinase, respectively. 
The negative values of 
δ
θ
A 
and 
δ
θ
B
 
show that tyrosinase is 
substantially destabilized by iso
-
propyl and iso
-
butyl xanthate at 
27 
°C.
 
It is worth noting that, the approxim
ately identical values 
of 
δ
θ
A 
and 
δ
θ
B
 
for iso
-
propyl xanthate (
t
able
-
 
1) can be attributed 
to the mixed inhibition and the different 
δ
θ
A 
and 
δ
θ
B
 
values for 
iso
-
butyl (
t
able
-
1) can be related to the competitive inhibition, 
which are in good agreement with p
revious results
3,4
.
 
 
For a set of identical and independent binding sites, the number 
of binding sites and dissociation equilibrium constant can be 
calculated by:
 
 
 
q
max
 
represents the heat value upon saturation of all 
biomacromolecule.
 
.
 
q
 
represents the heat value 
at a certain ligand and biomolecule concentration. 
M
0
 
and L
0
 
are 
total concentrations of enzyme and ligand, respectively. The 
�olar enthalpy of bin�ing for each bin�ing site (∆
H
°) wi
ll be 
 
The standard Gibbs free energy, 
∆
G
°, can be 
calculated from association constant 
 
as follows:
 
∆
G 
= 
-
R T 
ln 
K
a
   
(5)
 
 
The negative values of ∆
G
° suggest that the binding processes 
of MT to
 
I and II 
proceed spontaneously, which are both 
enthalpy and entropy driven. 
∆
S
° is directly calculated from 
∆
G
° an� ∆
H
° according to 
equation 
6:
 
 
All calculated thermodynamic parameters are summarised in
 
t
able
-
1.
 
 
Conclusion
 
Considering the massive number of different formulations in the 
healthcare and cosmetic market which contain tyrosinase 
controlling substances, our focalization on the thermodynamic 
parameters of the interaction between MT and iso
-
propyl and 
iso
-
butyl xant
hate as inhibitors, shows that
 
there is a set of two 
binding sites with non cooperativity for both xanthates on MT 
and the binding processes of both ligands are spontaneous, 
which are both enthalpy and entropy driven. The agreement 
between the experimental
 
heats and the calculated results via 
Eq. 1, support the extended solvation model.
 
The discovered 
binding parameters from solvation model indicate that the 
stability of tyrosinase is decreased as a result of its interaction 
with iso
-
propyl and iso
-
butyl xa
nthate
. The recovered
 
δ
θ
A 
and 
δ
θ
B
 
values can be used to predict the mode of inhibition.
 
 
 
Acknowledgement
 
Financial support from the Universities of Tehran and Imam 
Khomeini (Qazvin) are gratefully acknowledged.
 
  
 
 
Table
-
1
 
Binding parameters for 
xanthates+MT
 
interactions
 
recovered from Eqs. 1, 4, 5 and 6. 
p
=1 indicates that the binding is non
-
cooperative in two binding sites. The negative values of 
δ
θ
A 
and 
δ
θ
B
 
show that xanthates destabilize the MT structure. The 
binding process for MT inhibition is both enth
alpy and entropy
-
driven
 
 
parameters
 
I
 
II
 
p
 
1±0.01
 
1±0.01
 
g
 
2±0.02
 
2±0.02
 
K
a 
/ M
-
1
 
9.07
10
4
±24
 
1.26
10
5
±12
 
Δ
H
°/ kJ.mol
-
1
 
-
18.70±0.06
 
-
19.30±0.07
 
Δ
G
°/ kJ.mol
-
1
 
-
28.47±0.12
 
-
29.28±0.14
 
Δ
S
°/  kJ.mol
-
1
.K
-
1
 
0.03±0.01
 
0.03±0.01
 
 
-
4.99±0.02
 
-
4.47±0.06
 
 
-
4.23±0.02
 
-
6.58±0.08
 
 
Research Journal of Chemical Sciences 
______
_
_
_______________________________
______________
_
____
 
ISSN 2231
-
606X
 
 
Vol. 
2
(
4
), 
76
-
78
, 
April 
(201
2
)
 
  
Res.J.Chem.Sci
 
International Science Congress Association
 
 
78
 
Figure
-
1
 
Comparison between the experimental heats, q, for the interaction between mushroom tyrosinase and iso
-
propyl xanthate 
(
ï‚¡
)
, 
iso
-
butyl xanthate (

) at 27 °C and calculated data (lines) via 
equation 
1.
 
 
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