Comparative Studies on the Interaction of Spermidine with Bovine Trypsin by Multispectroscopic and Docking Methods

Article abstract of DOI:10.1021/acs.jpcb.6b06648

The effect of spermidine on the kinetics, conformation, and dynamics of native trypsin was studied by steady-state thermal stability, intrinsic fluorescence, circular dichroism (CD), ultraviolet-visible (UV-vis) spectroscopy, and kinetic techniques, as well as molecular docking, at the temperatures of 298 and 308 K. The Stern-Volmer quenching constants (K_sv) for the trypsin-spermidine complex were obtained at two temperatures, revealing that spermidine quenched the intensity of trypsin through the static mode of the quenching mechanism. The corresponding thermodynamic parameters, Gibbs free-energy, enthalpy, and entropy changes, showed that the binding process was spontaneous. These values and the molecular docking technique revealed that the hydrogen bonding and van der Waals forces played a major role in stabilizing the complex. CD, absorption, and fluorescence results also indicated that spermidine binding had a partial effect on trypsin structure. Spermidine could also influence the activity of trypsin. Upon spermidine binding, the V_max value of the enzyme was increased and the k_cat/K_m values were enhanced slightly. The T_m of the trypsin-spermidine complex was enhanced probably due to the higher H-bond formation and lower surface hydrophobicity after spermidine modification, as confirmed by UV-vis spectroscopy and fluorescence spectra. UV absorption and CD studies also indicated that the binding of spermidine to trypsin had induced microenvironmental changes around the enzyme, leading to changes in its secondary structure.

Full text of DOI:10.1021/acs.jpcb.6b06648

Subscriber access provided by United Arab Emirates University | Libraries Deanship
Article
Comparative Studies on the Interaction of Spermidine With
Bovine Trypsin by Multi-Spectroscopic and Docking Methods
Lida Momeni, Behzad Shareghi, Ali Akbar Saboury, and Sadegh Farhadian
J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b06648 • Publication Date (Web): 19 Aug 2016
Downloaded from http://pubs.acs.org on August 21, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
The Journal of Physical Chemistry B is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 20
The Journal of Physical Chemistry
1
2
3
4
5
Comparative Studies on the Interaction of Spermidine with Bovine Trypsin by
Multi-Spectroscopic and Docking Methods
6
7
8
9
Lida Momeni a, Behzad Shareghi b,*, Ali Akbar Saboury c, d, Sadegh Farhadian b
a Department of Biology, Faculty of Science, University of Payam Noor, Iran
b Department of Biology, Faculty of Science, University of Shahrekord, Shahrekord, P. O. Box.115, Iran.
c Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
d Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran
* Corresponding author: Tel.: +989131093764, fax: +983832324419, Eꢀmail address: b_shareghi@yahoo.com
Abstract
The effect of spermidine on the enzyme kinetic, conformation and dynamics of native trypsin
was studied by steady state thermal stability, intrinsic fluorescence, circular dichroism, UVꢀ
Vis spectroscopy and kinetic techniques, as well as molecular docking, at the temperatures of
298 and 308 K. The SternꢀVolmer quenching constants (Ksv) for the trypsinꢀspermidine
complex were obtained at two temperatures, revealing that spermidine quenched the intensity
of trypsin through the static mode of the quenching mechanism. The corresponding
thermodynamic parameters, Gibbs free energy, enthalpy and entropy changes showed that the
binding process was spontaneous. These values and the molecular docking technique revealed
that the hydrogen bonding and van der Waals forces played a major role in stabilizing the
complex. Circular dichroism, absorption and fluorescence results also indicated that
spermidine binding had a partial effect on trypsin structure. Spermidine could also influence
the activity of trypsin. Upon spermidine binding, the Vmax value of the enzyme was increased,
and the kcat/Km values were enhanced slightly. Tm of trypsinꢀspermidine complex was
probably enhanced due to the higher Hꢀbond formation and the lower surface hydrophobicity
after spermidine modification, as confirmed in the UVꢀVis spectroscopy and the fluorescence
spectra. UV absorption and CD studies also indicated that the binding of spermidine to
trypsin had induced micro environmental changes around the enzyme, leading to changes in
its secondary structure.
Abbreviations: CD: Circular dichroism, BAEE: NαꢀbenzoylꢀLꢀarginine ethyl aster.
1.
Introduction
The history of a living cell provides information about the chemical environment and its
changes. This information shows how cells adapt themselves to various conditions. The
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 2 of 20
1
2
3
4
5
environmental changes are dependent on the concentration of dissolved solutes and several
1
Field Code Chan
Field Code Chan
factors such as pressure and temperature . The organic molecules such as polyamines can
6
7
8
9
protect the protein molecules, with varying protection of cells biological molecules against
thermal aggregation 2.
Polyamines such as spermidine (NH2(CH2)3NH(CH2)4NH2) are low molecular weight
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aliphatic polyamines that participate in cell growth, differentiation, proliferation processes
3,
4. Natural polyamines are positively charged at the
Field Code Chan
Field Code Chan
and nucleic acid biosynthesis
physiological pH. The cationic nature of polyamines enables them to connect with negatively
charged biomacromolecules such as proteins, phospholipids and nucleic acids like RNA,
2
Field Code Chan
Field Code Chan
tRNA and DNA . The distribution of charge is important to identify the molecules and the
hydrophobic polymethylene backbone of them confers structural flexibility 5. Polyamines are
also involved in the biology of protein aggregation and can be used to prevent the thermal
2
Field Code Chan
Field Code Chan
aggregation . The effects of polyamines in pulmonary cell physiology and pathophysiology,
6
carcinogenesis and tumor growth have been studied . Polyamine levels change in various
illnesses such as pancreatitis, cancer, Parkinson's disease, Alzheimer's disease, Inflammation,
2
Field Code Chan
Field Code Chan
etc. . The toxicity of spermidine has been studied in Wistar rats, showing decreased plasma
7
creatinine, calcium and inorganic phosphate . Spermidine and compounds resulting from its
metabolism may play the main roles in membrane dysfunctions occurring in the cystic
8
2, 9
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
fibrosis disease . The polyamines have kosmotropic properties
. Kosmotropes cause water
molecules to favorably interact, thereby stabilizing the intramolecular interactions in macromolecules
such as proteins 10.
Bovine trypsin (T8003, E.C. 3.4.21.4), a serine protease, is a digestive enzyme in the
pancreas and contains 223 amino acid residues in a single chain polypeptide. Trypsin has two
11
Field Code Chan
Field Code Chan
Field Code Chan
domains with six antiparallel strands making a βꢀsheet unit . The amino acid sequence of
this enzyme is linked through six disulfide bridges. The isoelectric point, pI, of trypsin is
around 10.5 11. Trypsin breaks peptide bonds at the cꢀterminal end of lysine, arginine and
ornithine residues widely applied in protein analysis, biotechnology and food industry, as
well as biomedicine 12.
The stability of the enzymatic activity of trypsin has been studied in different solvent
11, 12
Field Code Chan
Field Code Chan
media
to understand the biological processes. Literature survey shows that there has
been no work on the interaction of spermidine with trypsin. Spermidine, as a small molecular
additive, has been used to prevent heat denaturation and inactivation of trypsin. The large
number of hyper thermophiles polyamines can serve an important role in the modification of
13
Field Code Chan
hyperthermophilic enzymes at high temperatures . The present research reports the binding
ACS Paragon Plus Environment

Page 3 of 20
The Journal of Physical Chemistry
1
2
3
4
5
6
affinities of this polyamine to trypsin by employing fluorescence, absorption, CD, kinetic and
molecular docking techniques. The purpose of this study was to investigate the effect of
natural spermidine on the structure, stability and activity of trypsin.
7
8
9
2. Materials and methods
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.1 Methods
Bovine pancreatic trypsin (T 8003, molecular weight: 23.3 kDa), spermidine trihydrochloride
and NαꢀbenzoylꢀLꢀarginine ethyl aster (BAEE, catalogue no. B 4500) were obtained from
Sigma Aldrich Co. All solutions were made in a TrisꢀHCl buffer (50 mM, pH 8).
2.2 Materials
2.2.1 Thermal stability of trypsin
Stability of trypsin in the absence and presence of spermidine was investigated. According to
the method developed by Zhang et al. (1999), thermal stability of trypsin (0.1 mg/ml) was
14
Field Code Chan
estimated by incubating enzyme solutions in the buffer, for 10 min, at 280 nm and pH 8 .
The Pharmacia 4000 UVꢀVis spectrophotometer was applied to record the UVꢀVis spectrum
of trypsin.
2.2.2 Fluorescence spectra analysis
Intrinsic fluorescence spectra of trypsin were recorded by exciting 280 nm, with scanning
being between 300 and 450 nm, at 298 and 308 K. All samples were prepared at 0.1 mg/ml
trypsin concentration in various concentrations of spermidine and equilibrated for 10 min. A
Shimadzu RFꢀ5301 fluorescence spectrophotometer was used to record the intrinsic
fluorescence of enzyme. The slits were 3 and 5 nm by width for excitation and emission
scans, respectively. The SternꢀVolmer equation was used to obtain the binding parameters15ꢀ
Field Code Chan
17
.
2.2.3 UV absorption spectra analysis
A Pharmacia 4000 spectrophotometer was applied to obtain the UVꢀVis absorbance spectra
in the range of 260ꢀ310 nm. Trypsin at 0.1 mg/ml and similar volumes of spermidine (1ꢀ4
mM) were equilibrated at 310 K for 15 min and then the enzyme spectra were recorded at
λ280.
2.2.4 Circular dichroism spectra analysis
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 4 of 20
1
2
3
4
5
6
7
8
9
The CD spectra were obtained with a spectropolarimeter model AVIV 215 (USA). The
suspension TrisꢀHCl buffer, spermidine and trypsin were preꢀequilibrated at the room
temperature for 15 min. The secondary structure of trypsin was monitoring by FarꢀUV (200ꢀ
260 nm), (trypsin concentration: 0.8 mg/ml, 1.0 cm path length cuvette) and the tertiary
structure of enzyme was studied by NearꢀUV CD (260ꢀ300 nm), (trypsin concentration: 1.6
mg/ml, 1.0 mm path length cuvette). Each spectrum was performed twice and averaged. The
observed ellipticity results in degree (θobs) were expressed as the mean residue ellipticity,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[θ]λ, in the degree cm2dmolꢀ1 15
.
Field Code Chan
2.2.5 Enzymatic activity assay
To evaluate the effects of spermidine on the trypsin kinetic, the enzyme activity assays were
performed. The activity of trypsin was assayed by monitoring its ability in hydrolyzing
BAEE. Various concentrations of spermidine were incubated with trypsin (25 ꢁg/ml) and the
reaction mixture was used to monitor the change in absorbance at 253 nm and 310 K after the
addition of BAEE. The unit enzymatic activity (U) represented the hydrolysis of 1 ꢁmol of
NαꢀbenzoylꢀLꢀarginine ethyl aster (BAEE) to NꢀbenzoylꢀLꢀarginine ethanol per minute at 310
K 18. Kinetic parameters were determined from the equation of LineweaverꢀBurk.
Field Code Chan
2.2.6 Molecular Docking studies
The molecular docking AutoDock 4.0 software package was used to assess the manner of
binding between spermidine and trypsin 19. The molecular structure of trypsin (PDB ID
Field Code Chan
Field Code Chan
2PTN) was downloaded from RCSB Protein Data Bank (PDB). The threeꢀdimensional
20
structure of spermidine was optimized by HyperChem program
molecular mechanics method via MM+ force field (Fig. 1).
and minimized by the
All water molecules were omitted from the molecular structure of enzyme. Furthermore, the
Kollman charges and the essential hydrogen atoms were added into trypsin and Gasteiger
charges were defined. To confirm the protocol of docking, bound spermidine coordinates in
the molecular structure were omitted and all rotatable bonds were determined. The grid maps
were generated by the program Auto Grid. The grid box sizes were 87 × 87 × 87 A°, with the
spacing of 10 A°. For the ligand, random torsions, and orientations, random starting positions
were applied. The pseudoꢀSolis and Wets methods and the Lamarckian genetic algorithm
were used for minimization by default parameters 21, 22_ENREF_21. Docking energy in the
largest cluster was selected as the representative binding pose 21.
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
ACS Paragon Plus Environment

Page 5 of 20
The Journal of Physical Chemistry
1
2
3
4
5
3. Results and Discussion
3.1 Thermal analysis of trypsin
6
7
Thermal denaturation experiments were performed by UVꢀVis spectrophotometer and the
8
9
midpoint of the thermal transition (Tm) of the enzyme was obtained from the thermal stability
curve. The fraction of the unfolded state of protein, fu, was computed by Eq. 2 23, 24
:
Field Code Chan
Field Code Chan
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢂꢃꢂ
ꢄ
ꢀ =
(2)
ꢁ
ꢂ ꢃꢂ
ꢅ
ꢄ
In this equation, Y is the experimental variable parameter at a certain spermidine
concentration, and Yf and Yu refer to the intensity of the folded and unfolded trypsin,
respectively. The curve of trypsin unfolding is shown in the presence and absence of
spermidine in Fig. 2A. As can be seen, with the addition of spermidine, the curves were
moved to the higher temperature, showing the more stability of trypsin. The Gibbs free
energy, ꢁG°, could be determined by Eq. 3 23, 25ꢀ27
:
Field Code Chan
Field Code Chan
ꢂꢃꢂ
ꢌ
ꢅ
ꢄ
∆ꢆ° = −ꢇꢈꢉꢊ ꢋꢍꢃꢌ ꢎ = −ꢇꢈꢉꢊ [ꢂ ꢃꢂ
]
(3)
ꢅ
ꢅ
ꢄ
, where R is the constant of gas and T is the absolute temperature. Fig. 2B shows the free
energy of denaturation, ꢁG°, in the presence and absence of spermidine. According to Fig.
2B, the slope denaturation curves at Tm was ꢁS°m and ꢁH°m was computed from ꢁH°m = Tm
ꢁS°m (thermodynamic parameters are listed in Table 1). As indicated in Table 1, Tm, ∆Hºm,
∆Sºm and trypsin stability were increased with the addition of the concentration of
spermidine. It could also be seen that spermidine was effective on the further stability of
trypsin because it increased Tm of trypsin. A previous study had also indicated a similar effect
upon the interaction of chymotrypsin with spermine 25.
Field Code Chan
3.2 Fluorescence measurement
Information regarding the binding between protein and small molecules can be provided by
fluorescence measurements28, 29. Three fluorophores of Trp, Tyr and Phe lead to the intrinsic
fluorescence of a protein30_ENREF_30. If Tyr is ionized or is close to a carbonyl group, a Trp
residue, or an amino group, its fluorescence is quenched and Phe has a little quantum yield;
thus, alterations in the emission spectra of tryptophan are common in response to enzyme
conformational transitions, substrate binding or denaturizing. Trypsin contains four
tryptophan (Trp) residues 11, 31. Thus, Trp (indole group of Trp) can be used as some intrinsic
fluorophore 28, 32_ENREF_33. The fluorescence spectra of trypsin in the absence and presence
of various concentrations of spermidine at 298 and 308 K in the 50 mM TrisꢀHCl buffer (pH
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 6 of 20
1
2
3
4
5
6
7
8
9
8) are illustrated in Fig. 3. When trypsin was excited at 280 nm, it showed a strong emission
peak at 332 nm. Fig. 3 shows that the addition of different concentrations of spermidine
caused a gradual decrease in the fluorescence intensity of trypsin. It also suggested that the
intrinsic fluorescence of trypsin was quenched by spermidine. After the conjugation of
spermidine to trypsin, which was in line with the exposure of hydrophobic amino acids to the
environment, it was prevented 12. As shown, the conformational change reflected in the
reduction of fluorescence intensity could result from the change of interactions between
spermidine and all residues of trypsin, which contributed to the increase of surface
hydrophilicity 12.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Field Code Chan
Field Code Chan
3.3 Mode of fluorescence quenching
Molecular interaction can quench the quantum yield of fluorophore; this includes molecular
rearrangements, excited state reactions, energy transfer ground state formation and collisional
33,
Field Code Chan
Field Code Chan
quenching
34. There are two quenching mechanisms: dynamic quenching and static
quenching. Diffusive collisions between fluorophore and the quencher cause the dynamic
quenching. Since higher temperatures increase the diffusion coefficients, the bimolecular
quenching constants are expected to rise with increasing the temperature. Static quenching
refers to the formation of the ground state complex between fluorophore and the quencher 35,
36. The increased temperature can result in the reduced stability of complexes, thus lowering
the values of the static quenching constants. To analyze the mechanism responsible for the
quenching of fluorescence intensity of trypsin by spermidine, we recorded the emission
spectra of the spermidineꢀtrypsin complex at two temperatures and analyzed the data using
Field Code Chan
Field Code Chan
the SternꢀVolmer equation shown below (Eq. 4) 15, 16, 28, 37ꢀ40
:
Field Code Chan
Field Code Chan
Field Code Chan
Field Code Chan
ꢏ
ꢐ
[ ꢕ
= 1 + ꢑꢒꢓ ꢔ = 1 + ꢖꢗꢘꢙ[ꢔꢕ
(4)
ꢏ
, where F0 and F refer to the fluorescence intensities of trypsin in the presence and absence of
spermidine, respectively. Ksv is the SternꢀVolmer quenching constant, [Q] is the spermidine
concentration, ꢘꢙ is taken as 10ꢀ8 for trypsin, and kq is the quenching rate constant for trypsin
41. As can be seen in Fig. 4, the plot of F0/F vs. [Q] showed a good linearity within the
comparative concentrations at two temperatures, indicating that the quenching type was
single static or dynamic quenching. The Ksv values for the spermidineꢀtrypsin complex were
obtained from the slopes of these plots and recorded at 298 and 308 K, as shown in Table 2.
As can be seen from Table 2, there was an inverse correlation between Ksv and temperature,
so the results suggested that static quenching mechanism was the dominant mechanism.
Field Code Chan
ACS Paragon Plus Environment

Page 7 of 20
The Journal of Physical Chemistry
1
2
3
4
5
Therefore, the quenching rate constant, kq, was greater than 2.0 × 1010 Mꢀ1sꢀ1. The static
quenching confirmed the quenching mechanism in this study (Fig. 4) 17.
Field Code Chan
6
7
8
9
3.4 Analysis of binding and its thermodynamic properties
The binding constant (Ka) can be derived from Eq. 5 in the static quenching mechanism 17, 28
Field Code Chan
Field Code Chan
:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢉꢚꢛ ꢏ ꢃꢏ = ꢉꢚꢛꢑꢜ + ꢊ log[ꢔꢕ
(5)
ꢐ
ꢏ
Ka and n could result from the values of the slope from the curve of log F0ꢀF/F vs. log [Q]
(Fig. 5). According to Table 3, the values of n were determined as the binding site, indicating
that there was approximately one binding site on trypsin for spermidine. The results showed
an inverse correlation with temperature, thereby indicating that the interaction between
trypsin and spermidine was weakened by increasing the temperature.
3.5 Analysis of thermodynamic parameters
The intermolecular acting forces between biomolecule and small molecular substrates may be
electrostatic interactions, hydrogen bonds, hydrophobic force, van der Waals interactions, etc.
The thermodynamic parameters of the reaction provide significant evidence confirming the
17
Field Code Chan
Field Code Chan
binding force. . The values of enthalpy change (∆Hº), entropy change (∆Sº) and the freeꢀ
energy change (∆Gº) can be evaluated by the van’t Hoff Equation (Eq. 6) 42:
∆ꢠ°
ꢉꢊꢑꢜ = − ∆ꢝ°
+
(6)
ꢞꢟ
ꢞ
and thermodynamic equation (7):
∆ꢆ° = −ꢇꢈꢉꢊꢑꢜ
(7)
, where Ka is the LineweaverꢀBurk binding constant, and R is the gas constant. Table 3 shows
the thermodynamic parameters. The negative values of ∆Gº revealed that the interaction
process was spontaneous. According to the views of Ross and Subramanian if ∆Hº > 0, ∆Sº >
0, the hydrophobic interaction is the main force in binding; if ∆Hº < 0, ∆Sº < 0, van der
Waals interaction and hydrogen bonds play the main role in the reaction; if ∆Hº ≈ 0, ∆Sº > 0,
the specific electrostatic interactions are characterized 42, 43. As shown in Table 3, the
negative values of ∆S° and ∆H° indicated that the van der Waals force and hydrogen bonding
force were the main binding forces.
Field Code Chan
Field Code Chan
3.6 UV Absorption spectroscopy
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 8 of 20
1
2
3
4
5
6
7
8
9
Absorption spectroscopy is often used to explore the conformational change and understand
the complex formation44. The absorption spectral changes of trypsin in the presence and
absence of spermidine are displayed in Fig. 6. The absorption of trypsin was increased upon
the addition of spermidine and a slight hyperchromic effect was monitored in the absorption
peak at 280 nm. The wavelength of trypsin intensity did not have any blue or red shift. The
results showed that the spermidine interacted with trypsin and the environment around the
tryptophan residues was changed. So, it confirmed that the probable quenching mechanism of
the fluorescence of trypsin by spermidine was a static one. These results were consistent with
the fluorescence results 44.
Field Code Chan
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Field Code Chan
3.7 Circular dichroism spectra
CD spectroscopy is a convenient tool for detecting the structural change in a protein when
interacting with ligands. Analysis of FarꢀUV CD spectra (200ꢀ260 nm) and NearꢀUV CD
spectra (260ꢀ320) was used to explore the secondary and tertiary structure in proteins. Farꢀ
UV CD spectra reflected light absorption of peptide bond and characterized the secondary
structure of a peptide 45. Fig. 7A shows FarꢀUV CD spectra of trypsinꢀspermidine complexes.
A global minimum of about 208 nm was, as shown in Fig. 7A, related to the αꢀHelix
conformation in the molecular structure of trypsin (Fig. 7A). The negative peak was changed
with the addition of spermidine; thus small alternations occurred in the trypsin secondary
structure. The fraction contents of different secondary structures of trypsin in the absence and
presence of spermidine could be calculated using the software package CDNN (Table 4). The
increasing inclination of the αꢀHelix (from 5.40% to 6.28%) and the deceasing inclination of
βꢀsheet by adding spermidine could be observed. The results suggested that the secondary
structure of enzyme was changed by spermidine. NearꢀUV CD spectra of trypsin are shown
in Fig. 7B. The intensity of trypsin spectra was reduced by adding spermidine; this was due to
the intramolecular vibrations resulting in the change in the asymmetry of the environment of
amino acid residues 11. The band intensity was decreased because the aromatic residues were
farther from each other. These results established that the tertiary structure of enzyme was
altered by spermidine, which was matched with the earlier results of fluorescence and
absorption spectroscopy.
Field Code Chan
Field Code Chan
3.8 The effects of spermidine on trypsin activity
ACS Paragon Plus Environment

Page 9 of 20
The Journal of Physical Chemistry
1
2
3
4
5
6
7
8
9
To identify the effects of spermidine on the enzyme kinetic, at first, the catalytic activity was
studied in the absence and presence of various concentrations of spermidine (0ꢀ5 mM) and
substrate (BAEE) at the temperature of 310 K and pH 8.0. As illustrated in Fig. 8 and Table
5, there was a direct correlation between the trypsin activity and the concentration of
spermidine. Overall, the binding of spermidine could change the microꢀenvironment of the
catalytic triad for the intruding molecules in their vicinity; thus the enzyme activity could be
increased.11. With the addition of the spermidine, the affinity of the trypsin for BAEE was
reduced (Km was increased). The term kcat/Km refers to the catalytic efficiency and a high
value shows that the limiting factor for the overall reaction is the frequency of collisions of
trypsin with BAEE molecules. According to the results, Tm of trypsin and the enzyme activity
were increased with the addition of spermidine concentrations. As a result, spermidine could
be considered as an activator and stabilizer for trypsin. Kumar and Venkatesu have also
suggested that polyamines are good enhancers for the native conformation of chymotrypsin
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Field Code Chan
46
Field Code Chan
.
3.9 Molecular Docking studies
The above study provided information on the forces between trypsin and spermidine and the
number of binding sites. By AutoDock 4.0 and molecular docking, we could predict the exact
binding sites. The total internal energy, hydrogen bond, binding energies, final intermolecular
energy and amino acid interactions in the docked spermidineꢀtrypsin complexes are listed in
Table 6. The interaction modes of spermidine and trypsin are illustrated in Figs. 9 and 10,
showing a close interaction between the ligand and protein. According to the results, we
propose that the key mechanisms of the binding reaction are considered to be hydrogen
bonds, van der Waals forces and hydrophobic bonds. By fluorescent technique and molecular
docking, binding spermidine to trypsin by van der Waals interaction and hydrogen bonds can
be suggested, thereby leading to the static quenching of the fluorescence of trypsin. So, the
docking technique shows the same result, both confirming each other and the final
conclusion.
4. Conclusion
The interaction between spermidine and trypsin in the TrisꢀHCl buffer solution at pH 8 was
investigated for the first time by fluorescence, absorption, thermal stability, CD, kinetics and
molecular docking methods. The reduction in the intensity of fluorescence and the increase in
the intensity of UV spectroscopy demonstrated the combination of spermidine and trypsin
prevented the contact of the hydrophobic amino acids to the environment and reduced the
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 10 of 20
1
2
3
4
5
6
7
8
9
surface hydrophobicity. Thermodynamic parameters and molecular docking proposed the
contribution of van der Waals forces and hydrogen bonds in stabilizing the spermidineꢀ
trypsin complex. Absorption and CD studies showed the alterations in the secondary structure
of trypsin upon binding to spermidine. The higher thermal stability of spermidineꢀtrypsin
complex might be relevant to the conformational change induced by the higher hydrogen
bonds formation and the lower surface hydrophobicity after modification. The results
suggested that polyꢀmethylene skeletons of polyamine and its primary and secondary amines
contributed to this effect. It seemed that these effects of polyamines were due to their
kosmotropic properties.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Acknowledgement
The work was financially supported by the University of Shahrekord, Iran. The authors also
thank the University of Tehran for helping in CD experiments.
References
1.
2.
3.
4.
5.
Kumar, A.; Attri, P.; Venkatesu, P. Effect of polyols on the native structure of αꢀ
chymotrypsin: A comparable study. Thermochim. Acta 2012, 536, 55ꢀ62.
Chowhan, R. K.; Singh, L. R. Polyamines in modulating protein aggregation. J. Proteins
Proteomics 2013, 3, 141ꢀ150.
Casero, R.; Pegg, A. Spermidine/spermine N1ꢀacetyltransferaseꢀꢀthe turning point in
polyamine metabolism. FASEB J. 1993, 7, 653ꢀ661.
Chattopadhyay, M. K.; Tabor, C. W.; Tabor, H. Polyamines protect Escherichia coli cells
from the toxic effect of oxygen. Proc. Natl. Acad. Sci. 2003, 100, 2261ꢀ2265.
Geall, A. J.; Eaton, M. A.; Baker, T.; Catterall, C.; and Blagbrough, I. S. The regiochemical
distribution of positive charges along cholesterol polyamine carbamates plays significant
roles in modulating DNA binding affinity and lipofection. FEBS Lett. 1999, 459, 337ꢀ342.
Hoet, P. H.; Nemery, B. Polyamines in the lung: polyamine uptake and polyamineꢀlinked
pathological or toxicological conditions. Am. J. Physiol.ꢀLung Cell. Mol. Physiol. 2000, 278,
L417ꢀL433.
Til, H.; Falke, H.; Prinsen, M.; Willems, M. Acute and subacute toxicity of tyramine,
spermidine, spermine, putrescine and cadaverine in rats. Food Chem. Toxicol. 1997, 35, 337ꢀ
348.
Kimberly, M. M.; Goldstein, J. Determination of pKa values and total proton distribution
pattern of spermidine by carbonꢀ13 nuclear magnetic resonance titrations. Anal. Chem. 1981,
53, 789ꢀ793.
RezaeiꢀGhaleh, N.; EbrahimꢀHabibi, A.; MoosaviꢀMovahedi, A. A.; NematꢀGorgani, M.
Effect of polyamines on the structure, thermal stability and 2, 2, 2ꢀtrifluoroethanolꢀinduced
aggregation of αꢀchymotrypsin. Int. J. Biol. Macromol. 2007, 41, 597ꢀ604.
Moelbert, S.; Normand, B.; De Los Rios, P. Kosmotropes and chaotropes: modelling
preferential exclusion, binding and aggregate stability. Biophys. Chem. 2004, 112, 45ꢀ57.
Hu, X.; Yu, Z.; Liu, R. Spectroscopic investigations on the interactions between isopropanol
and trypsin at molecular level. Spectrochim. Acta Part A 2013, 108, 50ꢀ54.
6.
7.
8.
9.
10.
11.
ACS Paragon Plus Environment

Page 11 of 20
The Journal of Physical Chemistry
1
2
12.
13.
Liu, W.; Liu, J.ꢀP.; Zou, L.ꢀQ.; Zhang, Z.ꢀQ.; Liu, C.ꢀM.; Liang, R.ꢀH.; Xie, M.ꢀY.; Wan, J.
Stability and conformational change of methoxypolyethylene glycol modification for native
and unfolded trypsin. Food Chem. 2014, 146, 278ꢀ283.
Kudou, M.; Shiraki, K.; Fujiwara, S.; Imanaka, T.; Takagi, M. Prevention of thermal
inactivation and aggregation of lysozyme by polyamines. Eur. J. Biochem. 2003, 270, 4547ꢀ
4554.
3
4
5
6
7
8
9
14.
15.
Zhang, Z.; He, Z.; Guan, G. Thermal stability and thermodynamic analysis of native and
methoxypolyethylene glycol modified trypsin, Biotechnol. Tech. 1999, 13, 781ꢀ786.
Ajloo, D.; Shabanpanah, S.; Shafaatian, B.; Ghadamgahi, M.; Alopour, Y.; Lashgarbolouki,
T.; Saboury, A. A. Interaction of three new tetradentates Schiff bases containing N2O2 donor
atoms with calf thymus DNA. Int. J. Biol. Macromol. 2015, 77, 193ꢀ202.
Divsalar, A.; Razmi, M.; Saboury, A. A.; MansouriꢀTorshizi, H.; Ahmad, F. Biological
Evaluation of a New Synthesized Pt (II) Complex by Cytotoxic and Spectroscopic Studies.
Cell Biochem. Biophys. 2014, 71, 1415ꢀ1424.
Vignesh, G.; Manojkumar, Y.; Sugumar, K.; Arunachalam, S. Spectroscopic investigation on
the interaction of some polymer–cobalt (III) complexes with serum albumins. J. Lumin. 2015,
157, 297ꢀ302.
Bernt, E.; Bergmeyer, H.; Bergmeyer, H. Methods of enzymatic analysis; by HU Bergmeyer,
Academic Press, New York, 1974.
Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson,
A. J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor
flexibility. J. Comput. Chem. 2009, 30, 2785ꢀ2791.
Laxmi, D.; Priyadarshy, S. HyperChem 6.03. Biotech Software Internet Rep.: Comput.
Software J. Sci. 2002, 3, 5ꢀ9.
Adinarayana, K.; Devi, R. K. ProteinꢀLigand interaction studies on 2, 4, 6ꢀtrisubstituted
triazine derivatives as antiꢀmalarial DHFR agents using AutoDock. Bioinformation 2011, 6,
74ꢀ77.
Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson,
A. J. Automated docking using a Lamarckian genetic algorithm and an empirical binding free
energy function, J. Comput. Chem. 1998, 19, 1639ꢀ1662.
Sudha, N.; Chandrasekaran, S.; Sameena, Y.; Israel, V. Mode of encapsulation of linezolid by
βꢀcyclodextrin and its role in bovine serum albumin binding. Carbohydr. Polym. 2015, 115,
589ꢀ597.
Rahban, M.; Divsalar, A.; Saboury, A. A.; Golestani, A. Nanotoxicity and spectroscopy
studies of silver nanoparticle: Calf thymus DNA and K562 as targets. J. Phys. Chem. C 2010,
114, 5798ꢀ5803.
Farhadian, S.; Shareghi, B.; Saboury, A. A. Exploring the thermal stability and activity of αꢀ
chymotrypsin in the presence of spermine. J. Biomol. Struct. Dyn. 2016, In press, DOI
10.1080/07391102.2016.1147984.
Saboury, A. A.; Moosavi‐Movahedi, A. A. Derivation of the thermodynamic parameters
involved in the elucidation of protein thermal profiles. Biochem. Educ. 1995, 23, 164ꢀ167.
Pace, C. N. Measuring and increasing protein stability. Trends biotechnol. 1990, 8, 93ꢀ98.
Moradi, M.; Divsalar, A.; Saboury, A.; Ghalandari, B.; Harifi, A. Inhibitory effects of
deferasirox on the structure and function of bovine liver catalase: a spectroscopic and
theoretical study. J. Biomol. Struct. Dyn. 2015, 33, 2255ꢀ2266.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
Van de Weert, M.; Stella, L. Fluorescence quenching and ligand binding: a critical discussion
of a popular methodology. J. Mol. Struct. 2011, 998, 144ꢀ150.
Zong, W.; Liu, R.; Sun, F.; Teng, Y.; Fang, X.; Chai, J. A new strategy to identify and
eliminate the inner filter effects by outer filter technique. J. fluoresc. 2011, 21, 1249ꢀ1254.
Koutsopoulos, S.;, Patzsch, K.; Bosker, W. T.; Norde, W. Adsorption of trypsin on
hydrophilic and hydrophobic surfaces. Langmuir 2007, 23, 2000ꢀ2006.
Garabagiu, S. A spectroscopic study on the interaction between gold nanoparticles and
hemoglobin. MRS Bulletin 2011, 46, 2474ꢀ2477.
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 12 of 20
1
2
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
Hu, Y.ꢀJ.; Liu, Y.; Zhang, L.ꢀX.; Zhao, R.ꢀM.; Qu, S.ꢀS. Studies of interaction between
colchicine and bovine serum albumin by fluorescence quenching method. J. Mol. Struct.
2005, 750, 174ꢀ178.
Yue, Y.; Chen, X.; Qin, J.; Yao, X. Spectroscopic investigation on the binding of
antineoplastic drug oxaliplatin to human serum albumin and molecular modeling. Colloids
Surf. B 2009, 69, 51ꢀ57.
Singh, U. K.; Dohare, N.; Mishra, P.; Singh, P.; Bohidar, H. B.; Patel, R. Effect of
pyrrolidinium based ionic liquid on the channel form of gramicidin in lipid vesicles. J.
Photochem. Photobiol. B 2015, 149, 1ꢀ8.
Maurya, J. K.; Mir, M. U. H.; Singh, U. K.; Maurya, N.; Dohare, N.; Patel, S.; Ali, A.; Patel,
R. Molecular investigation of the interaction between ionic liquid type gemini surfactant and
lysozyme: A spectroscopic and computational approach. Biopolymers 2015, 103, 406ꢀ415.
Faridbod, F.; Ganjali, M. R.; Larijani, B.; Riahi, S.; Saboury, A. A.; Hosseini, M.; Norouzi,
P.; Pillip, C. Interaction study of pioglitazone with albumin by fluorescence spectroscopy and
molecular docking. Spectrochim. Acta Part A 2011, 78, 96ꢀ101.
Ghalandari, B.; Divsalar, A.; Saboury, A. A.; Parivar, K. βꢀLactoglobulin nanoparticle as a
chemotherapy agent carrier for oral drug delivery system. J. Iran. Chem. Soc. 2015, 12, 613ꢀ
619.
Divsalar, A.; Saboury, A. A.; MansooriꢀTorshizi, H.; Ahmad, F. Design, synthesis, and
biological evaluation of a new palladium (II) complex: βꢀlactoglobulin and K562 as targets. J.
Phys. Chem. B 2010, 114, 3639ꢀ3647.
Kumari, M.; Maurya, J. K.; Tasleem, M.; Singh, P.; Patel, R. Probing HSAꢀionic liquid
interactions by spectroscopic and molecular docking methods. J. Photochem. Photobiol. B
2014, 138, 27ꢀ35.
Lu, D.; Zhao, X.; Zhao, Y.; Zhang, B.; Zhang, B.; Geng, M.; Liu, R. Binding of Sudan II and
Sudan IV to bovine serum albumin: comparison studies. Food Chem. Toxicol. 2011, 49,
3158ꢀ3164.
Li, H.; Pu, J.; Wang, Y.; Liu, C.; Yu, J.; Li, T.; Wang, R. Comparative study of the binding of
Trypsin with bifendate and analogs by spectrofluorimetry. Spectrochim. Acta Part A 2013,
115, 1ꢀ11.
Ross, P. D.; Subramanian, S. Thermodynamics of protein association reactions: forces
contributing to stability. Biochem. 1981, 20, 3096ꢀ3102.
Saeidifar, M.; MansouriꢀTorshizi, H.; Saboury, A. A. Biophysical study on the interaction
between two palladium (II) complexes and human serum albumin by Multispectroscopic
methods. J. Lumin.2015, 167, 391ꢀ398.
Momeni, L.; Shareghi, B.; Saboury, A. A. Spectroscopic analysis of the interaction between
NiO nanoparticles and Bovine trypsin. J. Biomol. Struct. Dyn. 2016, In press, DOI
10.1080/07391102.2016.1185041.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
43.
44.
45.
46.
Kumar, A.; Venkatesu, P. Overview of the stability of αꢀchymotrypsin in different solvent
media. Chem. Rev. 2012, 112, 4283ꢀ4307.
Fig. 1: Structure of spermidine
ACS Paragon Plus Environment

Page 13 of 20
The Journal of Physical Chemistry
1
2
3
4
5
6
7
8
9
A)
1
0.8
0.6
0.4
0.2
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0 mM
1 mM
2 mM
3 mM
4 mM
303
308
313
318
323
328
333
338
343
T (K)
8000
6000
4000
2000
0
B)
0 mM
1 mM
2 mM
3 mM
4 mM
310
312
314
316
318
320
322
324
326
328
-2000
-4000
-6000
T (K)
Fig. 2A): The fraction unfolded of trypsin in various concentrations of spermidine, 2B): The
effect of spermidine on ∆G° of trypsin in different concentrations of spermidine at pH 8.0
700
(A) 298 K
600
0 mM spermidine
1 mM spermidine
500
2 mM spermidine
400
300
200
100
0
3 mM spermidine
4 mM spermidine
5 mM spermidine
6 mM spermidine
7 mM spermidine
290
310
330
350
370
390
410
430
450
Wavelenght (nm)
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 14 of 20
1
2
3
4
5
6
7
8
9
700
600
500
400
300
200
100
0
0 mM spermidine
1 mM spermidine
2 mM spermidine
3 mM spermidine
4 mM spermidine
5 mM spermidine
6 mM spermidine
7 mM spermidine
8 mM spermidine
9 mM spermidine
(B) 308 K
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
290
310
330
350
370
390
410
430
450
Wavelenght (nm)
Fig. 3A): Fluorescence emission of trypsin in the absence and presence of different
concentrations of spermidine (0ꢀ9 mM) at 298 K, and 3B): at 308 K in the 50 mM Trisꢀ HCl
buffer.
1.4
1.3
1.2
1.1
308 K
298 K
1
0.9
0
1
2
3
4
5
6
7
8
1010 [Q] (mM)
Fig. 4: Stern–Volmer plots for the quenching of trypsin by spermidine at two temperatures of
298 K (●) and 308 K (■), λexi= 280 nm, λemi= 290ꢀ450 nm, and pH 8.0.
0
-0.2
-0.4
-0.6
-0.8
-1
308 K
-1.2
-1.4
298 K
-1.6
-1.8
-9.2
-9
-8.8
-8.6
-8.4
-8.2
-8
log [Q]
ACS Paragon Plus Environment

Page 15 of 20
The Journal of Physical Chemistry
1
2
3
4
5
Fig. 5: The plot of log [(F0 ꢀ F)/F
(●) for trypsin with spermidine, λexi= 280 nm, λemi= 290ꢀ450 nm, and pH 8.0.
] vs. log [Q] at two temperatures of 298 K (■) and 308 K
6
7
8
9
0 mM
A)
0.25
0.2
0.15
0.1
0.05
0
1 mM
2 mM
3 mM
4 mM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
260
270
280
290
300
310
Wavelength (nm)
0.26
0.25
0.24
0.23
0.22
0.21
0.2
B)
0.19
0
1
2
3
4
5
6
Spermidine concentration (mM)
Fig. 6: A) UV–Vis spectra of Trypsin in the presence and absence of spermidine (0ꢀ4 mM) at
pH 8.0 and 310 K and B) Absorbance at 280 nm in the presence and absence of spermidine
(0ꢀ4 mM).
10
A)
5
0
-5
-10
0 mM
-15
2 mM
-20
4 mM
-25
-30
-35
200
210
220
230
240
250
260
Wavelength (nm)
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 16 of 20
1
2
3
4
5
2
0
B)
6
-2
7
8
-4
0 mM
4 mM
2 mM
9
-6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-8
-10
-12
260
270
280
290
300
310
320
wavelength (nm)
Fig.7A): FarꢀUV CD and 7B): NearꢀUV CD spectra of trypsin in the absence and presence of
spermidine (dotted line: 2 mM and dash line: 4 mM) in the 50 mM Trisꢀ HCl buffer and at
pH 8. The Yꢀaxis is the meanꢀresidue ellipticity with the unit of degree cm2/mol.
2.5
2
1.5
0 mM
1
1 mM
3 mM
5 mM
0.5
0
-2
-1
0
1
2
3
4
5
6
7
8
1/[S] (mM-1)
Fig.8: The effect of different concentrations of spermidine (0, 1, 3, 5 mM) on the activity of
trypsin in the 50 mM Trisꢀ HCl buffer and at pH 8, with 25 ꢁg/ml enzyme concentration
ACS Paragon Plus Environment

Page 17 of 20
The Journal of Physical Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 9: Hꢀbond (black) and hydrophobic (red) analysis of docking poses by Ligplot plus tool
for Spermidine ligands in Trypsin
Fig. 10: The superimposition of the docked Spermidine into Trypsin
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 18 of 20
1
2
Table 1 Thermodynamic data of trypsin at T
m
in the presence of different concentrations of
3
spermidine.
4
5
6
7
[spermidine] (mM)
Tm (K)
∆Smº (J molꢀ1Kꢀ1)
∆Hmº (kJ molꢀ1)
R2a
8
9
0
1
318.0
322.2
323.2
323.5
333.8
441.4
459.7
466.7
499.0
516.1
140.4
148.1
150.8
161.4
172.3
0.9994
0.9992
0.9985
0.9992
0.9975
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
3
4
a
.
The correlation coefficient.
Table 2 Stern–Volmer quenching constants of the trypsinꢀspermidine complexes at pH 8.0.
T (K)
298
308
Ksv (×108 Mꢀ1)
4.74
Kq (×1016 Mꢀ1sꢀ1)
R2
0.9956
0.9951
4.74
3.53
3.53
Table 3 Binding and Thermodynamic parameters of trypsinꢀspermidine interaction at pH 8.0 and two
different temperatures.
T (K)
298
308
KA (×107 Mꢀ1)
10.6
n
1.04
1.05
R2
0.996
0.994
ꢂHº (kJ molꢀ1)
ꢂGº (kJ molꢀ1)
-45.8
ꢂSº (J molꢀ1 Kꢀ1)
-18160.5
-18.3
8.4
-46.7
-18162.5
Table 4 Changes in the secondary structures of trypsin in the absence and presence of spermidine.
[spermidine] (mM)
% αꢀHelix
5.40
% β Sheet
40.80
39.68
ΒꢀTurn
19.70
19.94
20.00
% Random coil
34.10
0
2
4
5.96
6.28
34.42
34.50
39.20
Table 5 The kineticvalues of trypsin at various concentrations of spermidine.
[spermidine]
Km (mM)
0.9
kcat ×106 (sꢀ1)
Vmax (mM.sꢀ1)
3.3
kcat/Km ×106
(mM)
0
(mMꢀ1.sꢀ1)
3.4
3.1
3.3
3.9
4.7
1
3
5
3.6
3.9
4.0
0.9
1.0
1.2
3.6
4.2
5.0
ACS Paragon Plus Environment

Page 19 of 20
The Journal of Physical Chemistry
1
2
3
4
5
6
7
Table 6 Docking results for the spermidine ꢀ Trypsin system
Lowest
Binding
Energy
Estimated
Inhibition
Constant,
Ki
Final
Intermolecular
Energy
Interaction bonds
Electrostatic
Energy
(Kcal/mol)
Hꢀbond
(Kcal/mol)
8
9
complex
(Kcal/mol)
(Kcal/mol)
Hydrogenꢀ
Hydrophobicꢀ
Bonding
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bonding
Trypsinꢀ
Spermidine
Glu77
Asp71
ꢀ4.65
391.93
ꢀ5.84
ꢀ2.05
ꢀ3.79
Val 154
ACS Paragon Plus Environment

The Journal of Physical Chemistry
Page 20 of 20
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
62x44mm (300 x 300 DPI)
ACS Paragon Plus Environment