International Journal of Biological Macromolecules 92 (2016) 523–532

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Molecular aspects of the interaction of spermidine and

␣-chymotrypsin
Sadegh Farhadian a , Behzad Shareghi a,∗ , Ali A. Saboury b,c , Ali Kazemi Babaheydari d ,
Fatame Raisi e , Ehsan heidari f
a
Department of Biology, Faculty of Science, University of Shahrekord, Shahrekord, P. O. Box.115, Iran
b
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
c
Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran
d
Department of Chimistry, Faculty of Science, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
e
Department of Biology, Faculty of Sciences, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
f
Young Researchers and Elites Club, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Polyamines such as spermidine are essential for survival. The purpose of the present study was to
Received 12 May 2016 investigate how spermidine could influence the conformation, thermal stability and the activity of ␣-
Received in revised form 18 July 2016 chymotrypsin. The influence of spermidine on the structure and stability of ␣-Chymotrypsin (␣-Chy)
Accepted 21 July 2016
explored using different spectroscopy method and molecular docking simulations. The stability and
Available online 22 July 2016
activity of ␣-Chy were increased in the presence of spermidine. Increasing of the ␣-Chy absorption
in the presence of spermidine was as a result of the formation of a spermidine – ␣-Chy complex. The
Keywords:
results of fluorescence spectroscopic measurements suggested that spermidine has a vigorous ability to
␣-Chymotrypsin
Spectroscopic techniques
quench the intrinsic fluorescence of ␣-Chy through the dynamic quenching procedure. Near and Far-UV
Dynamic quenching CD studies also confirmed the transfer of aromatic residues to a more flexible environment. The absorp-
Docking simulations tion increasing of ␣-Chy in the presence of spermidine was as a result of the formation of spermidine –
Thermal stability ␣-Chy complex. Molecular docking results also revealed the presence of one binding site with a negative
value for the Gibbs free energy of the binding of spermidine to ␣-Chy. Further, the docking study revealed
that van der Waals interactions and hydrogen bonds play a main role in stabilizing the complex.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Polyamine compounds, with regard to its being essential for
survival, have been found in both prokaryotes and eukaryotes.
Polyamine has an important role in many cellular activities, includ-
ing the immune system and translation, embryonic development,
cell cycle, cell death and cancer planned plays. Spermine (N-
(3- Aminopropyl) - 1,4- diaminobutane) is a low molecular weight
polyamine often presents in food, induces the maturation of small
intestine mucosa, pancreas, liver and spleen when orally ingested
by suckling Rodents [1–5]. Polyamines such as spermine interact
with proteins such as Hen egg white lysozyme [1].
Serine proteases such as S1 family are widely distributed in
live organism, performing many important functions, especially
in digestion [6,7]. ␣-Chymotrypsin (EC 3.4.21.1) is one of the pre-
∗ Corresponding author.
E-mail address: b shareghi@yahoo.com (B. Shareghi).
dominant members of this family (S1 serine proteases family) [8].
This protein has three chains with five inter- and intra-chain disul-
fide bonds. This enzyme is one of the valuable enzymes used for
understanding the mechanism of protein folding or unfolding. ␣-
Chymotrypsin (␣-Chy) is folded into two anti-parallel ␤-barrel
domains including a Greek key motif followed by an anti-parallel
hairpin motif [9].
One of the most important challenges in the use of the enzymes
are their stability and activity; Catalytic activity of enzymes
decrease when exposed to high temperature or organic solvents.
The stability of an enzyme is a difficult problem in protein chem-
istry due to the great number of factors involved and the lack of
experimental methods that would allow the evaluation of their
individual contributions [10]. To prevent protein denaturation, we
can add small organic molecules to the solution. Spermidine, as a
small molecular additive, is a candidate for the prevention of heat
denaturation and inactivation of ␣-chy. [11]. The structural and
functional information regarding ␣-chy in different liquids such as

http://dx.doi.org/10.1016/j.ijbiomac.2016.07.069
0141-8130/© 2016 Elsevier B.V. All rights reserved.
524 S. Farhadian et al. / International Journal of Biological Macromolecules 92 (2016) 523–532
polyamine can help understanding their activity and stability for
use in chemical and detergent industry.
The purpose of the present study was to investigate the binding
mechanism of spermidine and its effect on the activity and thermal
stability of ␣-Chy. Various methods including UV–vis spectroscopy,
thermal stability, fluorescence spectroscopy, circular dichroism
(CD) and kinetics, as well as molecular docking, were used to this
end. In this work, mechanisms and characteristics of the interaction
between spermidine and ␣-Chy were investigated systematically
by spectroscopic and molecular modeling methods for the first
time. We hope that this work would be helpful for contributing
to our understanding of the binding mechanism and dynamics of
spermidine at the molecular level.
2. Materials and methods
2.1. Materials
␣-Chymotrypsin (␣-Chy), spermidine trihydrochloride and N-
benzoyl-l-tyrosyl-ethyl ester (BTEE) were purchased from Sigma.
All ␣-Chy solutions were prepared at the pH 8. All solutions were
made in a 50 mM Tris–HCl buffer (pH 8.0).
2.2. Methods
2.2.1. UV–vis absorption spectra
The UV–visible absorption spectra were measured using an
Ultrospec 4000 Pharmacia UV–vis Spectrophotometer equipped
with a thermostatic cell holder. The concentration of ␣-Chy was
0.1 mg/ml. Absorbance value changes were recorded at 280 nm. All
studies were carried out in quartz cells containing 0.1 mg/ml ␣-Chy
and different concentrations of spermidine solution.
2.2.2. Thermal stability of ˛-Chy
The thermal stability of free protein and protein-spermidine
complexes was investigated by monitoring absorbance intensities
at different temperatures according to the two state equilibrium
model N ↔ U [12–14]. The UV–vis spectrum of ␣-Chy at 280 nm
was obtained with UV–visible spectrophotometer in the presence
and absence of various amounts of spermidine. All studies were
carried out in quartz cells including 0.1 mg/cm3 ␣-Chy and differ-
ent concentrations of spermidine. The scan rate was 1 ◦ C/min. The
absorption data were plotted as a function of temperature. By the
two-state model, the thermodynamic profile was predicted. The
melting temperature, Tm , which was defined as the temperature
at which the standard Gibbs free energy of protein conformation
(G◦ ) was zero, was determined as the transition midpoint of the
melting curve.
2.2.3. Fluorescence spectroscopy measurements
Steady-state fluorescence measurements studies were per-
formed using a Shimadzu RF-5301 fluorescence spectrophotometer
equipped with a temperature adjustable cell holder. The emis-
sion spectrum was measured from 290 to 450 nm in the presence
and absence of different concentrations of spermidine and at the
temperatures of 25 and 35 ◦ C, with the excitation wavelength of
280 nm. The slits were 3 and 5 nm for excitation and emission scans,
respectively. The solution was secured for 4 min before the scan to
Fig. 1. Structure of spermidine.
equilibrate the sample solution fully. All studies were carried out
in quartz cells containing 2 cm3 (0.1 mg/ml) ␣-Chy.
2.2.4. Circular dichroism
Circular dichroism (CD) spectra in the far-UV (190–260 nm) and
near-UV (260–320 nm) regions were obtained by an AVIV 215 spec-
tropolarimeter at 25 ◦ C, using a 1 mm-path cell for far-UV and a
10 mm-path cuvette for near-UV experiments. The protein concen-
tration was 8 ␮M (0.2 mg/ml) and 16 ␮M (0.4 mg/ml) in the far-UV
and near-UV regions, respectively. The spectra were recorded after
4 min incubation with spermidine to allow sample equilibration as
stated above. The protein secondary structure was determined by
the CDNN program, version 2.1.0.223, using a network trained with
33 complex spectra as the reference set [9]. The results of all CD
measurements were expressed as mean residue ellipticity ([␪]␭ )
in deg cm2 dmol−1 at a given wavelength ␭ (nm). According to
statistical methods implemented in the CD software of CDNN, sec-
ondary structure changes of ␣-Chy were determined in the absence
and presence of various concentrations of spermidine. It should be
noted that each observed ␪␭ of the protein was corrected for the
contribution of the solvent.
2.2.5. Enzymatic activity assay
The catalytic activity of 37.5 ␮g/ml ␣-Chy (E.C. 3.4.21.1; type
II from bovine pancreas, Sigma) was determined by UV–vis
spectrophotometer at 256 nm and 37 ◦ C, with the rate of BTEE (N-
Benzoyl-l-Tyrosine Ethyl Ester; Sigma) hydrolysis being in 50 mM
Tris/HCl and, pH 8, in the absence and presence of 0–0.5 mM sper-
midine for steady-state kinetics.
2.2.6. Molecular docking studies
In this study, the ␣-Chy structures (PDB code: 1YPH) were
obtained from RCSB Protein Data Bank (www.rcsb.org); they had
been solved at the resolutions of 1.34◦ A. The water and non-
protein molecules in the PDB files were removed. In the present
study, we chose spermidine as a ligand. The structure of Sper-
midine was drawn using the quantum chemistry software Gauss
view 5.0 (Fig. 1) [15]. In order to get the most stable Spermidine
conformations, the structure-optimizing calculation was carried
out at the 6–31G** level by employing the Becke three-parameter
Lee–Yang–Parr (B3LYP) hybrid density functional theory, and the
structures with the lowest energy were selected for the follow-
ing docking study. Docking simulations were carried out with the
prepared ligands.
Molecular docking of protein ␣-Chy and Spermidine model was
carried out using the AutoDock4.0 software package [16]. All the
torsion angles in the small-molecules were set free to perform
flexible docking. Polar hydrogen was added by using the Hydro-
gen module in AutoDock Tools (ADT) for Spermidine. After that,
Kollman united atom partial charges were assigned for the pro-
tein ␣-Chy. The empirical free energy function and Lamarckian
genetic algorithm (LGA) were used for docking with the following
settings: a maximum number of 25,000,000 energy evaluations, an
initial population of 150 randomly placed individuals, a maximum
number of 27,000 generations, a mutation rate of 0.02, a crossover
rate of 0.80, and an elitism value of 1. For the local search, the
so-called Solis and Wets algorithm was applied with a maximum
of 1000 iterations per search. The results were clustered accord-
ing to the root-mean-square deviation (RMSD) criterion. The best
docked conformations of Spermidine were selected as the initial
 S. Farhadian et al. / International Journal of Biological Macromolecules 92 (2016) 523–532 525

Fig. 2. (a): UV–vis spectra of ␣-Chy in the presence and absence of spermidine (0–1 mM) at pH 8.0 and 298 K, and (b): absorbance at 280 nm in the presence and absence of
spermidine (0–1 mM).

active/binding conformations to evaluate the potential correlations
between the experimental activities and the predicted log Ki values.
The Thousand docking conformations for protein ␣-Chy and Sper-
midine were divided into groups according to a 0 −1.96 Å RMSD
criterion, using the Cluster module in ADT. Cluster conformation
analysis was used to compare the RMSD of the lowest energy con-
formations and their RMSD to one another and to group them into
families of similar conformations or clusters. Besides RMSD clus-
ter analysis, AutoDock could also binding free energy evaluation to
find the best binding mode. Energy items calculated by AutoDock
were characterized by intermolecular energy (consisting of van der
Walls energy, hydrogen bonding energy, desolvation energy, and
electrostatic energy), internal energy of ligand, and torsional free
energy. During all these interactions, the electrostatic interaction
between ligands and receptor was the most important, because in
most cases it could decide the binding strength and the location
of ligand, while the hydrophobic interaction of some groups could
affect the agonistic activity to a larger extent.
3. Results and discussion
3.1. Absorption spectroscopy
To obtain insight into the nature of ␣-Chy – spermidine inter-
action, the UV/Vis absorption spectra of ␣-Chy – spermidine were
recorded. The UV–vis absorption spectra of ␣-Chy (0.1 mg/ml) in
the absence and presence of a definite concentration of spermi-
dine are shown in Fig. 2. The main absorption peak waslocated at
260–300 nm [17,18]. With the addition of spermidine, the peaks of
␣-Chy showed hyperchromism (Fig. 2b). This was due to the forma-
tion of the ground state complex between ␣-chy and spermidine.
It was likely that this complex had a higher molar extinction coef-
ficient than that in the unabsorbed state, but it had the absorption
maximum at the same position [19,20]. The environment of tryp-
tophan residues was changed upon interaction with spermidine,
and the hydrophobicity of the microenvironment of tryptophan
residues was decreased [21,22].
3.2. Thermal stability of ˛-Chy
The protein stability is defined as the difference in the Gibbs
free energy (G◦ ) between the native (folded) and denatured
(unfolded) states [13]. We can represent the equilibrium between
these two states using a simple mechanism shown below:
F(folded) ↔ U(unfolded)
The net protein stability is defined as represented by Eq. (1)
[13,23]:
[U]
G◦ = G◦ U − G◦ F = −RT ln K = −RT ln (1)
[F]
where [U] and [F] and G◦ U and G◦ F denote the concentrations and
the free energies of the unfolded and folded states, respectively.
K is the equilibrium constant [24]. The thermodynamic parame-
ters of G◦ and Tm were estimated to evaluate thermal stability
of the protein. Fraction of the unfolded form, FU , was computed by
normalizing denaturation curves and utilizing Eq. (2) [25]:
(Y − YF )
Fu = (2)
(YU − YF )
In this equation, Y shows the observed variable parameter at a
given denaturant concentration. YF and YU are the variable char-
acteristics of the folded and unfolded states, respectively; also,
these values were acquired by the linear extrapolation of pre- and
post-transition regions [26]. The resulting changes in FU , in the
presence and absence of spermidine, were plotted versus temper-
ature (Fig. 3).
The equilibrium constant, K, was computed by utilizing the
values of FU . Computation of Kvalues between the folded and
unfolded states of protein at a given denaturant concentration can
be obtained by Eq. (3):
Fu Yf − Y
K= = (3)
1 − Fu Y − Yu
Gibbs free-energy changes (G◦ ) were computed from K values
using Eq. (4):
 F   
u Yf − Y
G◦ = −RTlnK = −RTln = −RTln (4)
1 − Fu Y − Yu
In this equation, R is the gas constant (1.987 cal/deg/mol) and T is
the absolute temperature [27]. The Gibbs freeenergy of the unfold-
ing, G◦ , as a function of temperature for ␣-Chy in the presence and
absence of spermidine, is shown in Fig. 4. These results can be used
to determine Tm at which G◦ = 0, Sm and Hm . The standard
entropy of transfer to unfolding (Sm ), can be obtained from the
526 S. Farhadian et al. / International Journal of Biological Macromolecules 92 (2016) 523–532

Fig. 3. The fraction unfolded of ␣-Chy in various concentrations of spermidine at pH 8.0.

3.3. Circular dichroism spectra

3.3.1. Far-UV CD
Far CD spectroscopy is one of the most widely utilized tech-
niques for detecting the secondary structure changes of ␣-Chy
when interacting with ligands, thereby making it possible to quan-
tify conformational modifications in the 3D structure [18]. The
analysis of far-UV CD spectra can be used to assess the content of
different secondary structure elements in proteins. The CD spec-
trum of ␣-Chy in the buffer have a minimum at ≈202–205 nm
(depending on pH value) and no positive band [6,28]. By using it, the
fractions contents of different secondary structures of ␣-Chy in the
absence and presence of spermidine were calculated using the soft-
ware package CDNN. The far-UV CD spectra of ␣-Chy and ␣-Chy –
spermidine mixture are shown in Fig. 5. ␣-Chy was folded into two
domains with very little ␣-helix content and wild regions of anti-
Fig. 4. The effect of spermidine on G◦ of ␣-Chyin different concentrations of sper- parallel ˇ-sheets, along with two interchain and three intrachain
midineat pH 8.0.
disulphide bonds [29]. Spectral deconvolution of ␣-Chy spectrum
in the buffer showed the low decrease of ␣- helix and the increase
of ˇ structures. In the conditions used here, the far-UV CD spectrum
slope denaturation curves at Tm . The standard enthalpy of transfer
of ␣-Chy showed a negative band in the 205 nm region (Fig. 5).
to unfolding (Hm ) can be obtained from Hm = Tm Sm .
The data proposed that the secondary structure change and the
Our results also suggested that by increasing spermidine con-
unfolding of the protein skeleton by spermidine were low. The
centrations, the curves were shifted to the upper temperatures
conformational changes here implied that the exposure of some
(Fig. 4). ␣-ChyTm at variable concentrations of spermidine can be
hydrophobic regions previously buried was increased and that
seen in Table 1. As shown, by increasing spermidine concentrations,
spermidine bound the amino acid residues of the main polypeptide
Tm of ␣-Chy is increased. Since the stabilizing effect was observed in
chain of the protein and ruined their hydrogen bonding networks
a small range of spermidine concentration, it was likely to be caused
[30]. All experiments discussed above provided good evidence
by their preferential binding to the native protein structure. Alter-
showing that the conformational change of ␣-Chy was caused by
natively, the stabilizing effect of spermidine could be mediated by
the interaction between spermidine and the protein.
their impacts on the solvent structure, as mentioned in the last
section. The results clearly showed the change of Tm values, which
corresponded to the transition of ␣-Chyto the unfolded state, as a 3.3.2. Near-UV CD
function of the concentration of spermidine. It could be seen that Near-UV CD was also used,In order to assure that the observed
spermidine increased Tm of ␣-Chy (Fig. 4). effects were related to conformational changes. This technique can
be very useful to study changes caused by external factors on the
tertiary structures [31]. The near-UV CD spectra of the enzyme indi-
Table 1 cated that the ␣-Chy had a particular tertiary structure in each of
Tm changes of ␣-Chy at variable concentrations of spermidine. the solvents with different concentrations of spermidine.
Concentration (mM) Tm (K) Sm (J/mol K) Hm (kJ/mol) The near-UV CD spectrum of ␣-Chy (Fig. 6) in the buffer dis-
0.0 315 ± 0.0577 608.75 ± 22.93 191.756 ± 20.137
played the contributions of Tyr and Trp residues responsible for
0. 1 324 ± 0.1288 1268 ± 68.12 410.832 ± 49.87 peaks and shoulders between 270 and 305 nm, and those of Phe
0.2 324 ± 0.1527 1271 ± 82.01 411.804 ± 52.61 residues, which strongly contributed to bands in the 258–270 nm
0.3 323.7 ± 0.1155 1212 ± 59.39 392.324 ± 43.21 region [32]. Therefore, the disappearance of these bands suggested
0.7 322.9 ± 0.4618 1139.7 ± 103.81 368.009 ± 91.51
the enhanced flexibility of peptide chains around aromatic residues
 S. Farhadian et al. / International Journal of Biological Macromolecules 92 (2016) 523–532 527

Fig. 5. Far-UV CD spectra of ␣-Chy in the presence and absence of spermidine at pH 8.0. The Y-axis is the mean-residue Elipticity with the unit of degree cm2 dmol−1 .

Fig. 6. Near-UV CD spectra of ␣-Chy in the presence and absence of spermidine at pH 8.0. The Y-axis is the mean-residue ellipticity with the unit of degree cm2 dmol−1 .

due to the partial unfolding of the proteins. The CD spectrum in
the near-UV region was very complicated, since the microenviron-
ment for each amino acid in a protein was different. Nevertheless,
the asymmetry of the microenvironments was lost when a protein
was unfolded, and the corresponding decrease in the near-UV CD
signals reflected the degree of tertiary structure loss around the
aromatic chromophores [28].
The results of ␣-Chy structure in spermidine showed that max-
ima at 280–300 nm could be best assigned to Trp residues. The
intensity of this band was decreased when the aromatic residues
were away from each other, that is, the native structure of the pro-
tein was not preserved in the spermidine solution. The ellipticity
values in the near UV-CD region were changed with a decrease in
the peak intensity in the presence of spermidine. When spermidine
was added to the solution of the ␣-Chy, a gradual negative decline in
the molar ellipticity values was observed. This difference in ellip-
ticity can be attributed to the unfolding of the ␣-Chy. Therefore,
these changes suggested the enhanced flexibility of peptide chain
around aromatic residues due to the partial unfolding of ␣-Chy.
The analysis of near CD results has been brought in Fig. 5. As can be
seen, ␣-Chy showed the change in the tertiary structure at differ-
ent concentrations of spermidine. After 4 min of incubation with
spermidine, changes occurred in the 270–305 nm regions, thereby
indicating a conformational change that led to a more flexible envi-
ronment near the aromatic residues.
The intrinsic fluorescence only monitored the changes in some
of the Trp residues, while the near-UV CD had the contributions of
all aromatic residues, not only tryptophan’s [32]. It is reasonable to
assume that the spectral changes and thermal stability observed by
intrinsic fluorescence, UV–vis and near-UV CD were related to the
changes in ␣-Chy conformation upon binding to spermidine.
3.4. The effect of spermidine concentration on the fluorescence
quenching
The intrinsic fluorescence of ␣-Chy is mainly contributed by the
tryptophan residue alone [33,34]. The change in the intrinsic fluo-
rescence intensity of ␣-Chy is mainly due to the Trp residue when
small molecules are bound to ␣-Chy. Additionally, the changes in
the emission spectra from Trp are also related to the protein con-
formational changes, subunit association apart from anion binding,
or direct denaturation [28]. ␣-Chy is a globular protein containing
528 S. Farhadian et al. / International Journal of Biological Macromolecules 92 (2016) 523–532

Fig. 7. a) Fluorescence quenching of ␣-Chy, ␭exi = 280 nm, and ␭emi = 290–450 in the presence of different concentrations of spermidine at the pH 8.0 and 298 K, and b)
Fluorescence intensity in 333 nm and in the presence of different concentrations of spermidine at pH 8.0 and 298 K.

Fig. 8. a) Fluorescence quenching of ␣-Chy, ␭exi = 280 nm, and ␭emi = 290–450 in the presence of different concentrations of spermidine at pH of 8.0 and 308 K, and b)
Fluorescence intensity in 333 nm and in the presence of different concentrations of spermidine at pH 8.0 and 308 K.

eight tryptophan residues, and its intrinsic fluorescence is almost
contributed by the Trp residues. The interaction of spermidine with
␣-Chy at different temperature conditions was evaluated by mon-
itoring the intrinsic fluorescence intensity changes of ␣-Chy upon
the addition of spermidine.␣-Chy in solution was excited at 280 and
emission spectra were recorded in the range of 290–450 nm (the
emission maximum of 333 nm) at 25 and 35 ◦ C, in the presence
and absence of different concentrations of spermidine, as shown
in Figs. 7 and 8. The fluorescence intensity of ␣-Chy was decreased
gradually, but a red or blue shift was not observed with the addition
of spermidine, when the concentration of ␣-Chy was fixed.These
observations can be attributed to a strong binding of spermidine
to ␣-Chy.Additionally, it implied that the conformational changes
were induced in ␣-Chy by spermidine under the conditions [9]. The
chromospheres of ␣-Chy (Trp-27, 29, 141, 172, 207, 215, and 237)
were possibly placed in a more hydrophilic environment, or the
polarity of the environment was increased. Moreover, the intrinsic
fluorescence spectrum of the N-acetyl tryptophan demonstrated
no significant shift (data not shown) in the presence of spermidine
[9,32]. Therefore, a change in the fluorescence of ␣-Chy due to the
binding of spermidine to ␣-Chy would affect the microenvironment
around Trp.
The changes in fluorescence intensity on ␣-Chy spectrum can
be due to a local effect on some Trp residues when binding to the
spermidine. In general, the fluorescence intensity is increased as the
polarity of the environment is decreased [32]. When spermidine
interacts with ␣-Chy, some Trp residues cause no change in the
accessible area, but other Trp residues contribute to a slight increase
in ASA. These Trp residues are more exposed to the polar solvent
and so, they cause the decrease in fluorescence intensity.
3.5. Mode of fluorescence quenching
fluorescence quenching is Any process which decreases the
fluorescence intensity of a protein [35]. Different molecular interac-
tions can eventuate in quenching, including excited state reactions,
molecular rearrangements, energy transfer, ground-state complex
formation and collisional quenching [35,36]. By measuring the
intrinsic fluorescence quenching of ␣-Chy, the accessibility of the
quenchers to the fluorophore groups of ␣-Chy can be estimated.
This can help us to predict the binding mechanisms of spermidine
to ␣-Chy. Any change in the environment and structure of protein
can be investigated by the fluorescence measurements [21]. Fluo-
rescence quenching proceeds via different mechanisms generally
classified as dynamic and static quenching. Quenching mechanism
is described by the Stern–Volmer equation [37–45] (Eq. (5)):
F0
= 1 + Ksv [Q] = 1 + kq 0 [Q] (5)
F
where F0 and F are the fluorescence intensities of ␣-Chy in the
absence and presence of spermidine (quencher), respectively; [Q]
is the total concentration of the quencher (spermidine); KSV is the
Stern–Volmer quenching constant, which is determined by the lin-
 S. Farhadian et al. / International Journal of Biological Macromolecules 92 (2016) 523–532
Fig. 9. Stern–Volmer plots for the quenching of ␣-Chy by spermidine at 25 and 35 ◦ C,
␭exi = 280 nm, ␭emi = 290–450 nm, and pH = 8.0.
Table 2
Stern–Volmer quenching constants of the complex of ␣-Chy with spermidine at the
pH 8.
T(K) KSV (L mol−1 ) kq (L mol−1 S−1 )
308 57.59 × 10−2 19.45 × 107
298 13.15 × 10−2 4.44 × 107
ear regression of Stern–Volmer equation; also, KSV is described by
the following equation [46] (Eq. (6)).
Ksv = kq 0
where kq is the quenching rate constant of the biological macro-
molecule and ␶0 is the fluorescence average lifetime of the
fluorophore which has been reported to be 2.96 × 10−9 s for the
Trp residues of ␣-Chy at the neutral pH [18,47]. Eq. (5) was applied
to determine Ksv by the linear regression of a plot of F0 /F against
the concentration of spermidine. Quenching data are usually pre-
sented as plots of F0 /F versus [Q]. A plot of F0 /F versus [Q] yields an
intercept of one on the y-axis and a slope equal to KSV . As shown in
Fig. 9. Stern–Volmer plots were plotted based on Eq. (6). The solid
lines in Fig. 9 show the lines of the best fit of the experimental data
to the Stern–Volmer equation. A linear Stern–Volmer plot is usu-
ally indicative of a single class of fluorophores, all equally accessible
to the quencher. Recognition of Static and dynamic quenching can
be evaluated by temperature and viscosity, or, preferably, by flu-
orescence lifetime measurements. Higher temperatures result in
faster diffusion and hence, larger amounts of collisional quenching.
Higher temperatures will also typically result in the dissocia-
tion of weakly bound complexes and hence, smaller amounts of
static quenching. The increase of KSV with temperature indicated
dynamic quenching. In Table 2, the binding constant was obtained
from the Stern–Volmer method and listed for spermidine with ␣-
Chy. The kq , the quenching rate constant, was less than 2.0×1010 L
mol−1 s−1 . This also indicated that the quenching of ␣-Chy fluores-
cence by spermidine was a dynamic quenching (Fig. 9).
3.6. Enzymatic activity of ˛-chymotrypsine
The enzymes structure and conformational dynamics are influ-
enced by temperature. But too low or high temperatures denature
proteins, thereby deactivating their function too. Moreover, co-
solvents can stabilize or destabilize the conformation of proteins
and therefore, influence their enzymatic activity. Addition of sper-
midine to ␣-Chy solution stabilizes the enzyme by an increase in
the melting temperature, Tm . To ascertain whether the spermidine
529
Fig. 10. Line Weaver-Burk plot of ␣-Chy at various spermidine concentrations (mM)
at 37 ◦ C, and pH8.0.
can cause any alternation in the enzyme activity of the folding
R2 transition state of ␣-Chy, we further performed enzyme activity
experiments. We tested the ␣-Chy activity in the presence of sper-
0.9898
0.9714
midine using UV–vis spectrophotometer. The obtained results have
been represented and displayed in Fig. 10. The enzymatic activities
of ␣-Chy in the presence and absence of spermidine were mea-
sured at 37 ◦ C and pH 8.0 (Fig. 10, the double reciprocal plot of
line weaver-Burk). It can be observed from Fig. 10 that the activ-
ity of ␣-Chy was increased with increasing the concentration of
(6) spermidine. As illustrated in Fig. 10 and Table 3, as the concen-
tration of spermidine was increased, the maximum rate of ␣-Chy
(Vmax ) was increased too. Spermidine is a nonessential activator
for ␣-Chy [48]. The binding of spermidine may change the micro-
environment of the catalytic triad-His57, Asp102, and Ser195 for
the intruding molecule in the vicinity of them from the overall
perspective. Consequently, spermidine can influence the enzyme
activity.
3.7. Molecular modeling study
The complementary application of molecular modeling by com-
puter methods has been employed to improve the understanding of
the interaction between drugs and biomolecules, offering a molec-
ular level explanation with the ability to estimate the participation
of specific chemical groups and their interactions in complex sta-
bilization [49].
3.7.1. Molecular modeling study
Protein–ligand docking study was performed to identify the
binding sites in which spermidine was located. The above study
provided information regarding the binding of spermidine to ␣-
chy; Molecular modeling could be a suitable way to predict the
exact binding sites in which we carried out the docking calcula-
tions using AutoDock 4.0. The most possible binding mode between
spermidine and ␣-chy is shown in Fig. 11. The energy informa-
tion is listed in Table 4, and the interaction modes and H-bond and
hydrophobic interactions of the Spermidine and protein ␣-chyare
depicted in Figs. 11 and 12. The binding energy was calculated by
adding the final intermolecular energy and torsional free energy
used to find the best binding pose of spermidine at the ␣-Chy.
Final intermolecular energy, hydrogen bond, total internal energy,
binding energies and amino acid interactions in the docked com-
plexes of proteins are shown in Table 4. Our study revealed that
all the spermidine docked against ␣-Chy showed a binding energy
530 S. Farhadian et al. / International Journal of Biological Macromolecules 92 (2016) 523–532

Table 3
Parameters of ␣-chy activity in the presence of spermidine.

[spermidine] mM Km (mM) Vmax (mM s−1 ) Kcat (s−1 ) Kcat /Km (mM−1 s−1 )

0 1.0233 0.0394 26.2666 × 10 3

25.66 × 103
0.1 1.4082 0.0603 40.2000 × 103 28.54 × 103
0.3 2.089 0.0956 63.7333 × 103 30.50 × 103
0.5 3.0624 0.1463 97.5333 × 103 31.85 × 103

Table 4
Docked Results with interacting residues after 100 runs of docking.

Lowest Binding Energy Estimated Inhibition Final Intermolecular vdWa + Hbond + desolv Electrostatic Energy Interaction bonds
(kCal/mol) Constant, Ki Energy (kCal/mol) Energy (kCal/mol) (kCal/mol)
Hydrogen-Bonding Hydrophobic- Bonding

−5.95 93.88 −8.63 −3.91 −4.72 Glu20 Glu21,Glu57,Val9, Leu10

a
van der Waals.

Fig. 11. H-bond and hydrophobic analysis of docking.

Fig. 12. Docked structure of Spermidine in the protein ␣-Chy binding.

 S. Farhadian et al. / International Journal of Biological Macromolecules 92 (2016) 523–532 531

Table 5 all cases, molecular docking and molecular dynamic simulations

ASA for the residues of ␣-chy and spermidine − ␣-chy complex in Å2 .
results are in agreement with spectroscopic results. Our findings
Residues ASA for ␣-chy ASA for complex ASA Change of ASA (%) suggested that the thermal stability of ␣-Chy was increased. Flu-
Trp27 6.12 5.32 0.8 −13.07 orescence intensity changes showed dynamic quenching during
Trp29 1.05 0.5 0.55 +47.61 spermidine binding. Near-UV and Far-UV CD studies also demon-
Trp51 3.02 11.42 8.4 +278.14 strated changes in ␣-Chy structure. The results of the kinetic study
Trp141 16.64 13.52 3.12 −19.45 showed that the activity of ␣-Chywas increased. Molecular dock-
Trp172 33.42 38.3 4.88 −14.60
ing results also revealed the presence of one binding site with a
Trp207 26.13 15.17 10.96 −41.94
Trp215 64.56 13.59 50.97 −78.94 negative value for the G◦ of the binding of spermidine to ␣-Chy.
Trp237 54.29 42.79 11.05 −20.35 The experimental results indicated that the microenvironments
of the tryptophan residue of ␣-Chy changed by spermidine and,
the quenching mechanism of ␣-Chy by spermidine was a dynamic
Table 6
ASA for the active site residues of ␣-chy and spermidine – ␣-chy complex in Å2 . quenching procedure. The stability of ␣-Chy during the simula-
tion was evaluated by the RMSD of ␣-Chy relative to the starting
Residues ASA for ␣-chy ASA for complex ASA Change of ASA (%)
structure. The RMSD of protein in the presence of spermidine was
His57 69.16 5.45 63.71 −92.11 stable over time and the structure of protein reached a conforma-
Asp102 0. 7 1.29 0.59 +84.28 tional steady state. The first major finding was that spermidine was
Ser195 24.96 20.97 3.99 −15.98
bound to ␣-Chy, leading to the increase of the thermal stability of
Ser214 3.34 1.19 2.15 −64.37
Gly193 33.06 20.99 12.07 −36.50 ␣-Chy and more biocompatiblity for the ␣-Chy structure. The sec-
ond major finding was that the activity of ␣-Chy was increased
.
in the presence of spermidine. Molecular docking combined with
spectroscopy is very pertinent in obtaining credible results of bind-
of −8.63 kJ/mol.The docking was performed to analyze the effect ing studies and this combination is also helpful in determining the
of Spermidine with protein ␣-Chy. The binding energy was calcu- mechanism of interaction. Hence, we believe that this study has
lated by adding the final intermolecular energy and torsional free highlighted a method that could offer a new research approach in
energy to find the best binding pose of Spermidine at the ␣-Chy. biological systems.
The final Intermolecular energy, van der Walls energy, total internal
energy, binding energies and amino acid interactions in the docked
Acknowledgement
complexes of proteins were calculated. Our study revealed that all
the Spermidine docked against ␣-Chy showed binding energy of
The work was financially supported by Shahrekord University,
G = −8.63 kJ/mol. In this docking study, electrostatic energy and
Iran.
the hydrophobic amino acids Glu21, Glu57, Val9, Leu10 with sper-
midine had links played an important role. Using fluorescent study
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