Molecular docking and spectroscopic study on the interaction of serum albumin with iron(III) diamine sarcophagine

Article abstract of DOI:10.1071/CH14485

Iron(III) diamine sarcophagine (DiAmsar) has attracted great attention in biological and medical applications. In particular, for any potential in vivo application, knowledge about the interaction of iron(III) DiAmsar with serum albumin is crucial. As a step towards the elucidation of the fate of iron(III) DiAmsar introduced into an organism, first, iron(III) DiAmsar was synthesised and characterised. In the next step, interactions of iron(III) DiAmsar with human serum albumin (HSA) and bovine serum albumin (BSA) were systematically investigated by various spectroscopic methods (Fourier-transform infrared, UV-visible, fluorescence) and cyclic voltammetry and molecular docking techniques under simulated physiological conditions. The fluorescence intensities of HSA and BSA decreased remarkably with increasing concentration of iron(III) DiAmsar. The Stern-Volmer quenching constant K_SV at different temperatures and corresponding thermodynamic parameters such as ΔH^o, ΔG^o, and ΔS^o were calculated. The binding distance of iron(III) DiAmsar with HSA and BSA was also determined using the theory of fluorescence energy transfer. Further, the conformational changes of HSA and BSA induced by iron(III) DiAmsar were analysed by means of Fourier-transform (FT)-IR. In addition, molecular docking was performed to explore the possible binding sites and the microenvironment conditions around the bound iron(III) DiAmsar.

Full text of DOI:10.1071/CH14485

CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
http://dx.doi.org/10.1071/CH14485
Molecular Docking and Spectroscopic Study on the
Interaction of Serum Albumin with Iron(III) Diamine
Sarcophagine
A
,
C
,
B C
Bahman Vasheghani Farahani,
Ghasem Rezanejade Bardajee,
A
A
Farzaneh Hosseinpour Rajabi, and Zari Hooshyar
A
Department of Chemistry, Faculty of Science, Imam Khomeini International University,
PO Box 288, Qazvin 34149, Iran.
B
Department of Chemistry, Payame Noor University, PO Box 19395-3697, Tehran, Iran.
C
Corresponding authors. Email: bvasheghanif@yahoo.com; rezanejad@pnu.ac.ir
Iron(III) diamine sarcophagine (DiAmsar) has attracted great attention in biological and medical applications. In particular,
for any potential in vivo application, knowledge about the interaction of iron(III) DiAmsar with serum albumin is crucial.
As a step towards the elucidation of the fate of iron(III) DiAmsar introduced into an organism, first, iron(III) DiAmsar was
synthesised and characterised. In the next step, interactions of iron(III) DiAmsar with human serum albumin (HSA) and
bovine serum albumin (BSA) were systematically investigated by various spectroscopic methods (Fourier-transform
infrared, UV-visible, fluorescence) and cyclic voltammetry and molecular docking techniques under simulated
physiological conditions. The fluorescence intensities of HSA and BSA decreased remarkably with increasing
concentration of iron(III) DiAmsar. The Stern–Volmer quenching constant K at different temperatures and correspond-
SV
o
o
o
ing thermodynamic parameters such as DH , DG , and DS were calculated. The binding distance of iron(III) DiAmsar with
HSA and BSA was also determined using the theory of fluorescence energy transfer. Further, the conformational changes
of HSA and BSA induced by iron(III) DiAmsar were analysed by means of Fourier-transform (FT)-IR. In addition,
molecular docking was performed to explore the possible binding sites and the microenvironment conditions around the
bound iron(III) DiAmsar.
Manuscript received: 2 August 2014.
Manuscript accepted: 22 October 2014.
Published online: 7 January 2015.
Introduction
the stage of human diseases around the world and in obtaining
[
15,16]
Diamine sarcophagine (DiAmsar, Fig. 1a) with an encapsulated
metal ion (clathrochelates) is representative of a relatively new
class of compounds with unusual chemical, physical, and
body images in a non-invasive manner.
One important way
to improve the image quality in MRT is to introduce imaging
agents. The imaging agents are a unique class of pharmaceu-
ticals that enhance various portions of the MRT by changing,
[
1–3]
physicochemical properties.
The keen interest of researchers
[17]
in these compounds is affected by the unique properties of a
metal ion completely encapsulated in the three-dimensional
cavity of a macropolycyclic ligand and largely isolated from
usually decreasing, the relaxation time of tissue water.
According to the literature, iron(III) DiAmsar is a moderate
relaxation agent for water protons; however, on binding of its
functionalised derivatives with proteins, the relaxation efficien-
cy markedly increases because of an increase in rotational and
[
4,5]
external factors.
Moreover, they belong to a special type of
extraordinarily thermodynamically stable and kinetically inert
polynuclear metallochelates that result from self-organisa-
[
14]
translational correlation times.
The common administration
[6]
tion. Their thermodynamic and kinetic stability, as well as
their availability, intense colouring, low toxicity, unique redox
properties, and ability to form ordered molecular structures have
attracted the attention of researchers working in the fields of
pathway is intravenous injection; hence, the interaction between
the imaging agents and serum albumin (SA) has been extensively
[
18–20]
studied.
SA is the most abundant protein in blood plasma, accounting
for ,60 % of total protein, corresponding to a concentration of
[
7–9]
chemistry and biochemistry.
intensively studied and applied as prospective radiodiagnostic
and radiotherapeutic compounds
For instance, they have been
ꢀ
3
42 g dm
[21]
blood.
and providing 80 % of the osmotic pressure of
It has many physiological functions, such as main-
[
10]
and as molecular scaffolds
[
11]
for the design of modern photoelectronic devices
and sen-
as well as mild electron carriers for biochemical
taining the osmotic pressure and pH of blood and as carrier for
transporting a great number of endogenous and exogenous
compounds such as fatty acids, amino acids, drugs, and phar-
[
12]
sors
applications.
Recently, iron(III) DiAmsar (Fig. 1b) was proposed as an
[
13]
[
22–25]
maceuticals.
The most important property of this group of
[
14]
imaging agent for magnetic resonance tomography (MRT).
MRT has become widely used in diagnosing and determining
proteins is that they serve as transporters for numerous exoge-
nous and endogenous compounds in blood. It has been reported
Journal compilation Ó CSIRO 2015
www.publish.csiro.au/journals/ajc

B
B. V. Farahani et al.
(a)
(b)
NH
NH
HN
HN
HN
HN
NH
NH
H N
NH2
H N
3+
2
2
Fe
NH2
NH
HN
NH
HN
Fig. 1. Molecular structure of (a) diamine sarcophagine (DiAmsar); and (b) iron(III) DiAmsar.
that the distribution and metabolism of a great number of
biologically active compounds, such as metabolites, drugs,
and even some toxins, in blood are dependent on their affinities
sodium cyanide, iron(III) nitrate, and fatty acid- and globulin-
free BSA and HSA were purchased from Sigma–Aldrich Che-
micalsandused without further purification. Tris(hydroxymethyl)
aminomethane hydrochloride (Tris-HCl) was obtained from
Acros (Geel, Belgium). Double-distilled water (DDW) was used
in all experiments.
[
26,27]
towards SA to a certain extent.
HSA) is a globular protein composed of three structurally
similar domains (I, II, and III), each containing two subdomains
Human serum albumin
(
[
28,29]
(A and B) and stabilised by 17 disulfide bridges.
Aromatic
and heterocyclic ligands are known to bind within two hydro-
phobic pockets in subdomains IIA and IIIA, namely site I and
Measurements
The FT-IR spectra of the compounds were recorded using a
Jasco 4200 FT-IR spectrophotometer in the range 400–
[
site II. HSA has one tryptophan residue (Trp-214), located in
30]
[31]
site II.
Also, several drug-binding sites are localised in
ꢀ1
4
500 cm , by KBr pellet technique. Absorption spectra were
subdomains IB, IIIA, and IIIB, and on the subdomain interfaces.
However, the bovine serum albumin (BSA) molecule is made
up of three homologous domains (I, II, III) that are divided into
recorded at room temperature using a Shimadzu UV-vis 1650
PC spectrophotometer. Fluorescence measurements were car-
ried out on an Scinco fluorescence spectrometer FluoroMate
FS-2 in a 1-cm path-length quartz cell with the excitation and
emission wavelengths set at 295 and 310–540 nm respectively.
The CV measurements were carried out with Autolab
PGSTAT12 controlled by an external PC and utilising a three-
electrode configuration at 298 K. The working electrode was
glassy carbon (GC). AgCl/Ag was employed as the reference
electrode. Also, a platinum wire was selected as auxiliary.
[
31]
nine loops (L1–L9) by 17 disulfide bonds.
Each domain is
composed of two subdomains (A and B). Intrinsic fluorescence
of BSA is due to the presence of two tryptophan residues (Trp-
1
IB and Trp-212 located within the hydrophobic binding pocket
34 and Trp-212), Trp-134 located on the surface of subdomain
[32]
of subdomain IIA.
HSA and BSA solutions exhibit an
intense fluorescence emission with lem,max ¼ 343 and 351 nm
respectively, due to the tryptophan residues, when excited at
2
1
13
H and C NMR spectra were obtained at 400 MHz with a
[
25,31]
95 nm.
These optical characteristics of HSA and BSA
Varian Mercury 400 spectrometer and a Bruker Avance III
spectrometer respectively, using deuterium oxide as solvent.
Elemental analysis was performed on a Leco Elemental Ana-
lyser CHNS O932 instrument.
provide a sensitive spectroscopic method to study their interac-
tions with various ligand molecules.
In this paper, first we synthesised and characterised iron(III)
DiAmsar and then we studied in vitro interaction of iron(III)
DiAmsar with HSA and BSA by spectroscopic (Fourier-
transform infrared (FT-IR), UV-visible (UV-vis), fluorescence),
cyclic voltammetry (CV), and molecular docking methods. FT-
IR, a powerful technique for the study of hydrogen bonding,
has recently become very popular for structure characterisation
Synthesis of Iron(III) DiAmsar
The synthesis of iron(III) DiAmsar was carried out according to
the method of Sargeson et al.
Scheme 1):
[33]
in four steps as follows
(
[
27]
of proteins.
structural changes and to determine the complex formation.
UV-vis measurement is applicable to explore
[
3
þ
Step 1: Synthesis of [Co-(NO ) -sar] ,Cl3
2 2
26]
Fluorescence measurements can give some information on the
binding of small-molecule substances to proteins, such as
binding constants, binding mechanism, binding mode, binding
First, tri(ethylenediamine) cobalt(III) chloride dihydrate (2 g)
was dissolved in a mixture of DDW (3 mL) and aqueous
formaldehyde (37 %, 10.4 equiv., 4.5 mL). Then, nitromethane
(1.06 mL) was added to the solution. The resulting solution was
cooled to 48C in an ice-water bath. Aqueous sodium hydroxide
(4 M, 5 mL) was cooled to 48C and mixed with the solution
above. The combined solution was stirred magnetically for
90 min in an ice–water bath while maintaining the temperature
at 48C. The mixture rapidly turned deep brown from the initially
orange colour. After 90 min, the reaction mixture was allowed to
come to room temperature. At the end, the reaction was
quenched by the addition of concentrated hydrochloric acid
[
29]
sites, and intermolecular distance.
obtain electrochemical information about the interaction of
CV can be applied to
[
31]
small molecules with biomolecules.
The molecular docking
method can offer a molecular-level explanation allowing the
estimation of the participation of specific chemical groups and
[
30]
their interactions in complex stabilisation. Knowledge about
the interaction and binding of iron(III) DiAmsar to HSA and
BSA may open new avenues for future application of these kinds
of compound in pharmacology.
(37 %, 60 mmol, 5 mL). The orange precipitate was collected by
filtration after cooling on ice for 90 min, and washed with
methanol. Then, the orange powder was dried at room tempera-
ture. dH (400 MHz, D O) 2.94–2.96 (d, J 8, 6H, –NH–CH –
Experimental
Reagents and Chemicals
2
2
Ethanol, tri(ethylenediamine) cobalt(III) chloride dihydrate,
methanal, sodium hydroxide, hydrochloric acid, methanol,
tin(II) chloride dihydrate, sodium hydroxide, cobalt(II) chloride,
CH –NH–), 3.36–3.40 (d, J 11, 6H, –NH–CH –C–NO ), 3.58–
2 2 2
3.60 (d, J 8, 6H, –NH–CH –CH –NH–), 3.89–3.93 (d, J 11,
2
2
6H, –NH–CH –C–NO ). d (400 MHz, D O) 51.28, 54.62,
2
2
C
2

Interaction of Serum Albumin with Iron(III) Diamine Sarcophagine
C
NO2
Co
NH
NH
NH
HN
HN
HN
NaOH
ꢂC
[Co(en) ]3 , 3Cl ꢀ 6 HCHO ꢀ 2 CH NO2
ꢀ
ꢁ
Co3ꢀ
(
1)
2)
O N
NO2
3
3
2
4
ꢁ
NO2
Co
ꢀNH3
(CL)3
NO2
SnCl /HCl
2
(
Co
70ꢂC, 4 h
Co-DiAmsar
NO2
ꢀNH3
ꢀ
NH3
NH
NH
HN
HN
NaOH/NaCN
0ꢂC, 7 h
H N
(
3)
4)
Co
2
NH2
7
NH
HN
ꢀ
NH3
DiAmsar
NH
NH
NH
HN
HN
HN
NH
NH
HN
(
H O
2
HN
HN
H N
NH2
Fe (NO3)
H N
Fe3ꢀ
2
ꢀ
2
2
NH2
1
00ꢂC
NH
Iron(III) DiAmsar
Scheme 1. Synthesis of iron(III) diamine sarcophagine (DiAmsar).
8
7.53. m/z (high-resolution mass spectrum, electron impact
almost yellow (,7 h). The final solution was dried under air
flow, and the residue was extracted with boiling acetonitrile
(three times, 10 mL). The volume of total extract was reduced
under vacuum (10 mL), and cooled to ꢀ108C to precipitate
white crystals of the product. The product was recovered by
(
HRMS EI)) 431.2689; calc. for C H CoN O : 431.3565
14 31 8 4
[
M ꢀ 3HCl]þ.
5þ
Step 2: Synthesis of [Co-(NH ) -sar] ,Cl5
3
2
filtration and dried for further experiments. d (400 MHz, D O)
H
The tin(II) chloride dihydrate (13 equiv., 3 g) was introduced
2
2
C–NH ). d (400 MHz, D O) 48.1, 51.7, 57.4. m/z (HRMS EI)
.80 (s, 12 H, –NH–CH –CH –NH–), 2.88 (s, 12 H, –NH–CH –
2 2 2
into a round-bottom flask under a nitrogen atmosphere. Then,
concentrated hydrochloric acid (37 %, 145 equiv., 12 mL) was
added, followed by ethanol (6 mL). The resulting solution was
stirred magnetically and heated to 708C under nitrogen until the
2
C
2
þ
3
15.3858; calc. for C H N : 315.4813 [M þ H] .
14 34
8
3
þ
solution became colourless. Then, the [Co-(NO ) -sar] ,Cl3
2
2
Step 4: Synthesis of Iron(III) DiAmsar
complex (1 equiv., 600 mg) was added to the solution under
strong stirring. The resulting solution was heated at 708C for 4 h.
The colour of the solution turned green from orange–brown and
then became darker. After that, water (6 mL) was added to the
solution, and it turned orange. The solution was cooled at room
temperature for 15 min before the flask was immersed in an
ice–water bath. The orange precipitate formed was filtered and
An appropriate amount of purified DiAmsar (0.040 g,
.120 mmol) was dissolved in water (5 mL) at room tempera-
ture. Then, iron(III) nitrate (0.038 g, 0.120 mmol) in water
5 mL) was added dropwise to the above solution and the
reaction was allowed to proceed for 24 h at 1008C. The iron(III)
DiAmsar obtained was characterised by absorption and FT-IR
spectra, and elemental analysis.
0
(
5þ
dried. The [Co-(NH ) -sar] ,Cl complex was obtained with a
3
2
5
yield of ,90 %. d (400 MHz, D O) 2.93–2.97 (m, 12H, –NH–
H
2
þ
CH –CH –NH–), 3.51–3.57 (m, 12H, –NH–CH –C–NH ). d
C
Methods
2
2
2
3
(
400 MHz, D O) 51.4, 54.7, 58.5.
2
Tris-HCl buffer (0.10 M, pH 7.40) containing 0.10 M NaCl was
selected to keep the pH value constant and maintain the ionic
strength of the solution. Appropriate amounts of HSA and BSA
were directly dissolved in pH 7.40 Tris-HCl buffer to prepare
stock solutions at a final concentration of 1.0 ꢁ 10ꢀ M and were
stored in the dark at 0–48C.
Step 3: Synthesis of DiAmsar
Sodium hydroxide (2.11 equiv., 77 mg) was dissolved in
deoxygenated water (7 mL) and the solution was purged with
nitrogen to eliminate oxygen. The bubbling was continued for
5
5
0 min. Then, [Co(NH ) -sar] ,Cl complex (1 equiv., 500 mg)
þ
3
A solution in Tris-HCl (2 mL) containing the appropriate
M
3
2
5
ꢀ
6
and cobalt(II) chloride (1.01 equiv., 118 mg) were dissolved in
the basic solution. After homogenisation of the solution, sodium
cyanide (17.7 equiv., 783 mg) was added to the resulting solu-
tion under strong stirring. The mixture was heated to 708C and
vigorously stirred under nitrogen until the solution had become
concentration of iron(III) DiAmsar was added to 5.0 ꢁ 10
HSA and BSA solutions and mixed at 298 K. UV-visible spectra
of all solutions were recorded in the range of 200–800 nm. The
respective blanks of Tris-HCl buffer were used for the correc-
tion of all spectra.

D
B. V. Farahani et al.
A known concentration of iron(III) DiAmsar in Tris-HCl
iron(III) DiAmsar formation. The FT-IR spectra of DiAmsar and
iron(III) DiAmsar were obtained and are presented in Fig. 2b. For
the DiAmsar spectrum shown in Fig. 2b, the bands characteristic
buffer solution was added to 1.0 ꢁ 10ꢀ M HSA and BSA
solutions and mixed at 298, 303, 308, and 313 K. The fluorescent
intensity of the solution was recorded at an excitation wave-
length of 295 nm with slit widths for excitation and emission
of 5 nm.
5
of the stretching vibration absorption of ꢀNH and ꢀNH are
2
ꢀ
1
ꢀ1
located at 3000–3600 cm . The bands at 2800–2900 cm are
attributed to the asymmetric and symmetric stretching vibra-
ꢀ
1
tions of –CH –. The strong bands at 1700 and 1550 cm are
For the voltammetric assay, first a blank CV was run with the
Tris-HCl buffer, HSA, and BSA at 298 K separately, which
showed no electroactivity in the potential range of interest to us
2
assignable to the stretching vibration absorption of ꢀNH
ꢀ
1
groups. The vibration bands between 1400 and 1500 cm are
three groups’ –N–C– bond absorption peaks. The bands at 950
(
ꢀ1.3 to 0 V). Cyclic voltammograms of iron(III) DiAmsar were
ꢀ
1
recorded from ꢀ1.3 to 0 V before and after the addition of
different volumes of the stock HSA and BSA solutions corre-
sponding to final concentrations of HSA, and BSA ranging from
and 1150 cm were assigned to the C–N asymmetric and
symmetric stretching vibrations respectively. The FT-IR spec-
trum of iron(III) DiAmsar is also shown in Fig. 2b. As can be
ꢀ
ꢀ
8
ꢀ8
1
1
.0 ꢁ 10 to 5.0 ꢁ 10 M within the cell. A scan rate of
seen from this figure, the –CH – vibration bands of DiAmsar
2
1
00 mV s was used throughout the experiments. Prior to each
are almost unchanged, but the bands of C–N at 1122.37 and
1045.23 cm
ꢀ
1
experiment, the GC electrode was polished with alumina pow-
der and rinsed thoroughly with double-distilled water for 30 s.
Each spectrum was the average of three successive scans.
shift to lower wavenumbers (1121.40 and
ꢀ1
1042.34 cm respectively). This can be interpreted in terms
of strong interaction between iron(III) inside DiAmsar cavities
and nitrogens as strongly electronegative atoms. Furthermore, a
new medium to weak band assigned to iron(III) appeared at a
Molecular Docking
ꢀ1
lower frequency (572.72 cm ), which confirms the presence of
iron(III) within the DiAmsar cavities.
The 3D structures of HSA and BSA were taken from Protein
Data Bank (PDB). AutoDock 4.2 and AutoDock Tools software
was applied to study the interaction of iron(III) DiAmsar with
Elemental analysis was also done to determine the composi-
tion of iron(III) DiAmsar. The percentage of chemical elements
was calculated: carbon (6.22 %), hydrogen (6.79 %), and
nitrogen (58.06 %). These results basically demonstrated the
expected composition of iron(III) DiAmsar.
˚
HSA and BSA. Grid maps were generated with 0.575-A spacing
and set to encompass the residues perturbed on fragment addi-
tion. The Lamarckian genetic algorithm (LGA) implemented in
AutoDock was used to estimate the possible conformations of
the aforementioned systems. In this approach, the number of
conformers was considered to be 200. The conformer with the
lowest binding energy was selected for further analyses. Visu-
alisation of the docked pose was carried out using PyMol and
visual molecular dynamics (VMD) molecular graphic programs.
Interaction of Iron(III) DiAmsar with HSA
and BSA by UV-Vis Spectroscopy
The interaction of iron(III) DiAmsar with HSA and BSA was
studied by UV-vis spectroscopy. As we know, the quenching
mechanism (which distinguishes between dynamic quenching
(a collisional process) and static quenching (ground-state
complex formation)) and the binding constant (K ) of the reac-
Results and Discussions
a
Characterisation of Iron(III) DiAmsar
[33]
tion can be obtained from the absorption spectra.
The
1
13
H and C NMR spectroscopic methods were used to charac-
absorption spectra of solutions containing proteins (HSA and
BSA) and iron(III) DiAmsar were investigated by keeping the
3
þ
terise the structures of [Co-(NO ) -sar] ,Cl , [Co-(NH ) -
3 2
2
2
3
5þ
sar] ,Cl , and DiAmsar. Fig. S1 (Supplementary Material)
5
ꢀ6
concentration of proteins constant at 5.0 ꢁ 10 M and varying
1
shows the H NMR spectrum of [Co-(NO ) -sar] ,Cl , which
3þ
ꢀ5
2
2
3
the iron(III) DiAmsar concentrations from 0 to 5.4 ꢁ 10 M in
contains four doublet peaks (in fact, two of them are doublets of
doublets) of methylene diastereotopic hydrogens in the target
1
molecule. Fig. S2 (Supplementary Material) shows the H NMR
Tris-HCl buffer at pH 7.4 at 298 K. The absorption spectra of
HSA and BSA in the absence and presence of iron(III) DiAmsar
are shown in Fig. 3. The addition of iron(III) DiAmsar led to a
gradual increase in the absorption intensity of HSA and BSA. As
static, but not dynamic, quenching (which only affects the
5þ
spectrum of [Co-(NH ) -sar] ,Cl , which includes two doub-
3
2
5
lets for each diastereotopic –CH (ꢀCH between the secondary
2
2
amines and the ꢀCH close to the ammonium groups), at
2
excited state of the fluorophore) often leads to a change in
[32]
2
.83–2.91 and 3.40–3.54 ppm. Fig. S3 (Supplementary Material)
absorption spectra via ground-state complex formation,
the
1
shows the H NMR spectrum of the DiAmsar target molecule.
This spectrum contains one singlet at 2.76 ppm resulting from
above results suggest ground-state complex formation between
proteins and iron(III) DiAmsar. The equilibrium between the
protein and iron(III) DiAmsar can be shown by Eqns 1 and 2:
the ꢀCH between the secondary amines and one singlet at
2
2
.85 ppm resulting from the ꢀCH close to the primary amines.
2
Because of the symmetry of the molecule, each singlet inte-
grates for 12 protons. An NMR spectrum of iron(III) DiAmsar
could not be obtained, as the iron(III) is paramagnetic and it does
not show clear signals.
Protein þ ironðiiiÞ DiAmsar 2 Protein2ironðiiiÞ DiAmsar
ð1Þ
Ka ¼ ½Protein2ironðiiiÞ DiAmsarꢂ=
ð2Þ
Fig. 2a shows the absorption spectral change of DiAmsar
solution before and after addition of iron(III) nitrate. As can be
seen in this figure, in the presence of iron(III), a new absorption
band showed up between 450 and 700 nm, which was ascribed to
iron(III) ions. At the same time, the characteristic absorption of
DiAmsar was shifted to higher wavelength. These results indicate
ð½Proteinꢂ½ironðiiiÞ DiAmsarꢂÞ
where K
HSA and BSA. K
intensity of absorption and expressed as Eqn 3:
is the association constant of iron(III) DiAmsar with
a
can be obtained from the changes of the
a
[
32,33]
[
32]
that a stable complex formed between DiAmsar and iron(III).
At the same time, the FT-IR spectra also provided evidence for
ð1=ðA ꢀ A0ÞÞ ¼ fð1=KaDeÞð1=½QꢂÞg þ 1=De
ð3Þ

Interaction of Serum Albumin with Iron(III) Diamine Sarcophagine
E
(
a) 3.0
2
2
1
1
0
.5
.0
.5
.0
.5
0
Iron(III) DiAmsar
DiAmsar
350
300
400
450
500
550
600
650
700
750
800
Wavelength [nm]
(
b)
DiAmsar
045.23 cm 1
ꢁ
1
92.196 cm 1
ꢁ
9
ꢁ
1
ꢁ
301.72 cm 1
92.6 cmꢁ1
7
1
666.2 cm 1
469.49 cm 1
122.37 cm 1
ꢁ
1
ꢁ
1
ꢁ1
573.719 cm
Iron(III) DiAmsar
893.66 cm 1
ꢁ
2
042.34 cm 1
ꢁ
1
294.79 cm 1
ꢁ
3
ꢁ1
9
97.982 cm
ꢁ
1
789.707 cmꢁ1
11
301.72 cm
ꢁ
1657.52 cm
472.38 cm 1
ꢁ
1
121.4 cmꢁ1
1
892.7 cm 1
ꢁ
2
294.79 cm 1
ꢁ
3
4000
3000
2000
1000
400
Wavenumber [cmꢁ1]
Fig. 2. (a) Absorption, and (b) Fourier-transform (FT)-IR spectra of diamine sarcophagine (DiAmsar) (3.50ꢁ 10ꢀ M) and iron(III) DiAmsar (3.50 ꢁ 10ꢀ3 M)
3
solutions.
where (A – A ) is the change in the absorbance at 280 nm, De is
the differential extinction coefficient at 280 nm, and [Q]
represents the concentration of iron(III) DiAmsar. The values
Changes in the emission spectra of tryptophan are usually
in response to protein conformational transitions, subunit
0
[
22,26]
association, substrate binding, or denaturation.
The
of 1/(A – A ) were calculated and were plotted against 1/[Q]
0
conformation changes in HSA and BSA were evaluated by
measuring the intrinsic fluorescence intensity of protein
tryptophan residues in the absence and presence of iron(II)
DiAmsar. Fig. 4 shows the fluorescence emission spectra (at
lex ¼ 295 nm, 298 K) obtained from HSA and BSA at pH 7.4
titrated with iron(III) DiAmsar. The maximum emission
wavelengths were near 351 and 343 nm for HSA and BSA
respectively. The same trends were also observed at other
temperatures. It can be seen that a greater excess of iron(III)
DiAmsar led to more effective quenching of the chromophore
molecular fluorescence.
according to Eqn 3 as shown in Fig. 3. After linear fitting, the
slope equals the value of 1/K De with an intercept of 1/De on the
a
ordinate. In this way, the association constant was estimated to
5
5
ꢀ1
be K ¼ 0.79 ꢁ 10 and 0.87 ꢁ 10 M for HSA and BSA
a
respectively.
Interaction of Iron(III) DiAmsar with HSA and BSA
by Fluorescence Quenching Spectra
A valuable feature of the intrinsic fluorescence of proteins is
23]
the high sensitivity of tryptophan to its local environment.
[

F
B. V. Farahani et al.
(
a)
0
0
0
0
0
.5
.4
.3
.2
.1
0
.4 ꢄ 10ꢁ5
14
5
4
4
3
M
M
M
M
.8 ꢄ 10ꢁ
.2 ꢄ 10ꢁ
5
y ꢃ 10.85x ꢀ 8.576
5
2
1
1
2
0
R
ꢃ 0.986
.6 ꢄ 10ꢁ
5
5
3
ꢄ 10ꢁ
M
5
2
.4 ꢄ 10ꢁ
M
M
0.2
0.3
0.4
0.5
0.6
1/([Q] ꢄ 10ꢁ
5)
.8 ꢄ 10ꢁ5
1
0
250
300
350
400
450
500
550
600
650
700
Wavelength [nm]
(
b)
0
0
0
0
0
.5
.4
.3
.2
.1
0
28
2
2
6
4
5
5
.4 ꢄ 10ꢁ
M
M
M
M
22
20
4
4
3
.8 ꢄ 10ꢁ5
18
y ꢃ 26.35x ꢀ 3.163
R2 ꢃ 0.997
1
1
1
1
6
4
2
0
8
6
.2 ꢄ 10ꢁ5
.6 ꢄ 10ꢁ
5
ꢄ 10ꢁ5
3
M
0
.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
2
.4 ꢄ 10ꢁ
5
M
M
1
/([Q] ꢄ 10ꢁ5)
.8 ꢄ 10ꢁ5
1
0
250
300
350
400
450
500
550
600
650
700
Wavelength [nm]
ꢀ6
Fig. 3. Absorption spectra of aqueous solutions of 5.0 ꢁ 10 M (a) human serum albumin (HSA), and (b) bovine
serum albumin (BSA) with varying concentrations of iron(III) diamine sarcophagine (DiAmsar). Insert shows the
0 0
straight line dependence of 1/(A – A ) on the reciprocal concentration of iron(III) DiAmsar, where (A – A ) is the
change in the absorbance at 280 nm.
Fluorescence quenching can be induced by different
mechanisms, which are usually classified into dynamic and
static quenching. Dynamic and static quenching can be distin-
The fluorescence quenching data at 295, 305, 308, and 313 K
were analysed with the Stern–Volmer equation:
[
25]
guished by their different dependence on temperature.
F0=F ¼ 1 þ Kqt0½Qꢂ ¼ 1 þ KSV½Qꢂ
and F are the steady-state fluorescence intensities in
0
ð4Þ
Higher temperature results in faster diffusion and larger
amounts of dynamic quenching and will typically lead to the
dissociation of weakly bound complexes and smaller amounts of
static quenching. Therefore, the quenching constant increases
with a temperature increase for dynamic quenching. However, it
decreases with a temperature increase for static quenching.
where F
the absence and presence of iron(III) DiAmsar respectively; K is
q
the quenching rate constant of the biomolecule; KSV is the
Stern–Volmer quenching constant; [Q] is the concentration of
iron(III) DiAmsar; and t is the average fluorescence lifetime of
0

Interaction of Serum Albumin with Iron(III) Diamine Sarcophagine
G
(
a)
6
5
4
3
2
1
00
00
00
00
00
00
0
2
3
3
4
5ꢂC
0ꢂC
5ꢂC
0ꢂC
1
1
.5
.0
0
0.5
0
1.2 ꢄ 10 4
ꢁ
M
M
M
.8 ꢄ 10 4
ꢁ
1
2
1
2
3
4
5
4
6
7
8
.4 ꢄ 10 4
ꢁ
[Q] ꢄ 10 M
ꢄ 10 4
ꢁ
M
3
.6 ꢄ 10 4
ꢁ
M
M
M
M
3
4.2 ꢄ 10 4
ꢁ
.8 ꢄ 10 4
ꢁ
4
5
.4 ꢄ 10 4
ꢁ
ꢄ 10 4
ꢁ
M
6
350
400
450
500
550
600
650
700
Wavelength [nm]
(
b)
1
1
0
.5
.2
.9
2
30ꢂC
5ꢂC
2
1
1
000
500
000
3
4
5ꢂC
0ꢂC
0
1
.2 ꢄ 10 4
ꢁ
M
M
M
0.6
1.8 ꢄ 10 4
ꢁ
0.3
.4 ꢄ 10 4
ꢁ
2
0
ꢄ 10 4
ꢁ
M
3
1
2
3
4
5
4
6
7
8
.6 ꢄ 10 4
ꢁ
M
M
M
M
3
4
4
5
[Q] ꢄ 10 M
.2 ꢄ 10 4
ꢁ
.8 ꢄ 10 4
ꢁ
.4 ꢄ 10 4
ꢁ
6 ꢄ 10 4
ꢁ
M
500
0
350
400
450
500
550
600
650
700
Wavelength [nm]
Fig. 4. Fluorescence spectra of (a) human serum albumin (HSA), and (b) bovine serum albumin (BSA) in
different concentrations of iron(III) diamine sarcophagine (DiAmsar) at 298 K. Inset: Stern–Volmer curves for the
binding of HSA and BSA with iron(III) DiAmsar at different temperatures (lex 295 nm).
the biomolecule without iron(III) DiAmsar. The results are
shown in Fig. 4. The KSV and correlation coefficient values, R
maximum scatter collision quenching constant K of various
q
1
0
ꢀ1 ꢀ1 [25]
quenchers with biopolymers is 2.0 ꢁ 10 L mol
s . Obvi-
(the linear relationship between (F /F) – 1 and [Q]), obtained at
0
ously, the rate constants of the HSA and BSA quenching
procedure initiated by iron(III) DiAmsar were greater than the
K of the scatter procedure. This proves that the quenching was
different temperatures are listed in Table 1. The KSV values
decreased with increasing temperature, which is consistent with
the static type of quenching mechanism. As a result, possible
quenching mechanisms of HSA and BSA with iron(III) DiAmsar
are suggested as static quenching but not dynamic. That is, HSA
and BSA bound to iron(III) DiAmsar, which resulted in the
formation of iron(III) DiAmsar–HSA and iron(III) DiAmsar–
BSA complexes. Because the fluorescence lifetime of the
q
not initiated by dynamic collision but by the formation of a
complex.
The binding constant (K ) and binding site number (n) can be
a
obtained from the site binding model, which assumes the
existence of n independent binding sites for a quencher (Q) in
a biomolecule as expressed in Eqn 5:
ꢀ8
[24]
protein is 10 s,
equation in Fig. 4. According to Eqn 4, the quenching constant
K is the slope of the linear regression
q
logððF0 ꢀ FÞ=FÞ ¼ log Ka þ n log½Qꢂ
ð5Þ
Kq was calculated and is listed in Table 1. However, the

H
B. V. Farahani et al.
q a
Table 1. The Stern–Volmer constants (KSV), quenching constants (K ), binding constants (K ), and number of binding
sites (n) of the iron(III) diamine sarcophagine (DiAmsar)–human serum albumin (HSA) or bovine serum albumin (BSA)
systems at different temperatures
4
ꢀ1
12 ꢀ1 ꢀ1
A
5
[10 M
ꢀ1
B
A
Serum albumin
HSA
T [K]
K
SV [10 M
]
K
q
[10
M
s
]
R
K
a
]
n
R
double-logarithm equation
298
0.34
0.32
0.29
0.28
0.28
0.27
0.25
0.24
0.34
0.32
0.29
0.28
0.28
0.27
0.25
0.24
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.79
0.57
0.17
0.072
0.87
0.63
0.16
0.12
1.44
1.39
1.24
1.13
1.41
1.33
1.26
1.22
0.99
0.99
0.98
0.99
0.99
0.98
0.98
0.98
3
3
3
03
08
13
BSA
298
3
3
3
03
08
13
A
R is the correlation coefficient.
B
The binding site (n) approximated to 1.
(
a)
0
0
.4
.2
0
Table 2. Thermodynamic parameters of the iron(III) diamine sarco-
phagine (DiAmsar) interactions with human serum albumin (HSA) and
bovine serum albumin (BSA) at different temperatures
Serum
Temperature
[K]
DS
DH
[kJ mol
DG
[kJ mol ]
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
albumin
(J mol
K
)
]
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
0.2
0.4
0.6
0.8
HSA
298
ꢀ340.45
ꢀ340.45
ꢀ340.45
ꢀ340.45
ꢀ377.70
ꢀ377.70
ꢀ377.70
ꢀ377.70
ꢀ129.92
ꢀ129.92
ꢀ129.92
ꢀ129.92
ꢀ142.80
ꢀ142.80
ꢀ142.80
ꢀ142.80
ꢀ28.466
ꢀ28.76
3
3
3
03
08
13
ꢀ25.06
ꢀ23.36
40ꢂC
5ꢂC
30ꢂC
5ꢂC
BSA
298
ꢀ29.521
ꢀ27.633
ꢀ25.744
ꢀ23.856
3
3
3
3
03
08
13
2
1.0
ꢁ
4.2 ꢁ4.1 ꢁ4.0 ꢁ3.9 ꢁ3.8 ꢁ3.7 ꢁ3.6 ꢁ3.5 ꢁ3.4 ꢁ3.3 ꢁ3.2 ꢁ3.1
log [Q]
the binding site number (n), and the intercept on the y axis was
equal to logK . Table 1 gives the results of K and n for
interactions of HSA and BSA with iron(III) DiAmsar. It was
(
b)
0
0
.4
.2
0
a
a
2
3
3
4
5ꢂC
0ꢂC
5ꢂC
0ꢂC
found that the values of K for the aforementioned interactions
a
decreased with a rise in temperature, which implies that the
stability of iron(III) DiAmsar–HSA and iron(III) DiAmsar–
BSA complexes is weakened at higher temperatures. Further-
more, it was found that the binding site numbers were close to
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
0.2
0.4
0.6
0.8
1
, which suggests that there is only one binding site for iron
24]
[
(
III) DiAmsar interactions with HSA and BSA.
These
results are in agreement with the above Stern–Volmer equa-
tion, and further reveal that the quenching mechanisms of iron
(III) DiAmsar interactions with HSA and BSA are static
quenching procedures.
1.0
The intermolecular forces between compounds and bio-
molecules may involve hydrophobic forces, electrostatic inter-
actions, van der Waals interactions, and hydrogen bonds.
From enthalpy change (DH) and entropy change (DS) data, the
type of interaction between the compound and biomolecules can
ꢁ
4.0 ꢁ3.9 ꢁ3.8 ꢁ3.7 ꢁ3.6 ꢁ3.5 ꢁ3.4 ꢁ3.3 ꢁ3.2 ꢁ3.1 ꢁ3.0
log [Q]
Fig. 5. Plot of log((F
constants of iron(III) diamine sarcophagine (DiAmsar) interactions with
a) human serum albumin (HSA), and (b) bovine serum albumin (BSA) at
98 K. F and F are the steady state fluorescence intensities in the absence
0
– F)/F) vs log[Q] for the calculation of binding
[27]
be deduced: (1) DH . 0 and DS . 0, hydrophobic forces;
(2) DH , 0 and DS , 0, van der Waals interactions and hydro-
(
2
0
gen bonds; (3) DH , 0 and DS . 0, electrostatic interactions. In
order to elucidate the type of interaction of iron(III) DiAmsar
with HSA and BSA, we calculated the thermodynamic para-
meters. The plot of ln K versus 1/T (Eqn 6), where K is the
and presence of iron(III) DiAmsar, respectively, and [Q] is the concentration
of iron(III) DiAmsar.
a
a
where F and F indicate the fluorescence intensities before
0
binding constant at the corresponding temperatures, T is tem-
perature, and R is the gas constant, allows the determination
of DH and DS. If the temperature does not vary significantly, DH
can be regarded as a constant. Based on the binding constants at
and after the addition of the quencher, and [Q] is the total
quencher concentration. The plots of log((F – F)/F) versus
log[Q] were straight lines (Fig. 5), whose slope was equal to
0

Interaction of Serum Albumin with Iron(III) Diamine Sarcophagine
I
(
a)
1
0
0
0
0
.0
.8
.6
.4
.2
0
1.0
0.8
0.6
0.4
0.2
0
400
Fluorescence spectra of HSA or BSA
Absorption spectrum of iron(III) DiAmsar
0
ꢁ400
800
ꢁ
ꢁ
ꢁ
1200
1600
J
400
500
600
700
800
ꢁ2000
Wavelength [nm]
ꢁ1.2
ꢁ1.0
ꢁ0.8
ꢁ0.6
ꢁ0.4
ꢁ0.2
0
E [V]
Fig. 6. The fluorescence spectra of human serum albumin (HSA) and
bovine serum albumin (BSA) and the absorption spectrum of iron(III)
diamine sarcophagine (DiAmsar). J represents the overlap integral of the
fluorescence emission spectrum of the donor and the absorption spectrum of
the acceptor.
(
b)
5
00
0
0
different temperatures, the free energy change (DG) can be
estimated from Eqn 7 (Table 2).
ꢁ5
ꢁ5
ꢁ
500
1.8 ꢄ 10
M
M
2.4 ꢄ 10
ꢁ
5
3
ꢄ 10
M
ꢁ1000
ꢁ5
3
4
.6 ꢄ 10
M
M
ln Ka ¼ ꢀðDH=RTÞ þ ðDS=RÞ
DG ¼ DH ꢀ TDS ¼ ꢀRT ln Ka
ð6Þ
ð7Þ
ꢁ5
.2 ꢄ 10
ꢁ1500
ꢁ2000
As shown in Table 2, for iron(III) DiAmsar interactions with
HSA and BSA, the main source of the DG value was a large
contribution of the DH term with a little contribution from DS.
So the main interaction among iron(III) DiAmsar–HSA and iron
ꢁ1.2
ꢁ1.0
ꢁ0.8
ꢁ0.6
ꢁ0.4
ꢁ0.2
0
E [V]
(
c)
(III) DiAmsar–BSA complexes is believed to be van der Waals
interactions and hydrogen bonds. The negative values of DG
reveal that the interaction processes are spontaneous.
5
00
[
27]
0
Fluorescence resonance energy transfer (FRET) is a reliable
method for studying protein–ligand interactions and evaluating
the distance between the ligand and tryptophan residues of the
0
ꢁ500
ꢁ
5
[
22]
1.8 ꢄ 10
M
protein.
The fluorescence spectra of HSA and BSA and the
ꢁ5
2
.4 ꢄ 10
M
absorption spectrum of iron(III) DiAmsar are shown in Fig. 6.
The energy transfer efficiency (E) is defined by the following
equation:
ꢁ5
ꢁ
1000
1500
2000
3 ꢄ 10
M
ꢁ5
3.6 ꢄ 10
M
M
ꢁ
5
4.2 ꢄ 10
ꢁ
ꢁ
6
6
6
E ¼ 1 ꢀ ðF=F0Þ ¼ R =R þ r
ð8Þ
0
0
ꢁ1.2
ꢁ1.0
ꢁ0.8
ꢁ0.6
ꢁ0.4
ꢁ0.2
0
where r is the distance between the acceptor and the donor, and
R0 is the F o¨ rster critical distance, at which 50 % of the excitation
energy is transferred to the acceptor. F and F are the fluores-
E [V]
0
Fig. 7. Cyclic voltammetry (CV) curves of iron(III) diamine sarcophagine
DiAmsar) in the absence (a), and presence of (b) human serum albumin
cence intensities of HSA and BSA in the presence and absence
of iron(III) DiAmsar (3.6 ꢁ 10ꢀ M). R0 can be calculated
from donor (HSA and BSA) emission and acceptor (iron(III)
(
4
(HSA) and (c) bovine serum albumin (BSA) with different concentrations
at 298 K.
[
28]
DiAmsar) absorption spectra using the F o¨ rster formula,
which is the following equation:
the fluorescence emission spectrum of the donor and the
absorption spectrum of the acceptor. J(l) is given by:
R ¼ 8:8 ꢁ 10 k Nꢀ4FDJðlÞ
6
0
ꢀ25
2
ð9Þ
X
X
4
JðlÞ ¼
FðlÞeAðlÞðl Dl=
FðlÞDlÞ
ð10Þ
2
2
where k is the spatial orientation factor of the dipole (k ¼ 2/3);
N is the refractive index of the medium (N ¼ 1.36); ^ is the
D
fluorescence quantum yield of the donor (^ ¼ 0.15 and 0.1 for
where F(l) is the fluorescence intensity of the fluorescent donor
D
HSA and BSA, respectively); and J(l) is the overlap integral of
at wavelength l; and e (l) is the molar absorption coefficient of
A

J
B. V. Farahani et al.
ꢀ
16
the acceptor at wavelength l. We obtained J ¼ 2.76 ꢁ 10
BSA to iron(III) DiAmsar with high probability and the distance
[28]
obtained by FRET has high accuracy.
6
ꢀ1
cm L mol , R ¼ 1.39 nm, E ¼ 0.49, and r ¼ 1.40 nm for HSA
0
ꢀ16
6
ꢀ1
and J ¼ 2.75 ꢁ 10 cm L mol , R ¼ 1.39 nm, E ¼ 0.40, and
0
r ¼ 1.39 nm for BSA. The donor-to-acceptor distance is less than
Interaction of Iron(III) DiAmsar with HSA
and BSA by CV Spectra
7
nm, which indicates that the energy can transfer from HSA and
The electrochemical behaviour of iron(III) DiAmsar was studied
by CV. The CV of iron(III) DiAmsar in Tris-HCl buffer with pH
7.4 was commenced at 0.00 V and its direction was reversed at
ꢀ1.2 V. The results (Fig. 7a) showed well-defined anodic and
cathodic peaks at ꢀ0.58 and ꢀ0.95 mV respectively. Further,
the CV of iron(III) DiAmsar in the presence of increasing HSA
and BSA was investigated in Tris-HCl buffer solutions at vari-
ꢁ
ꢁ4
ꢁ
4
1
1
2
.2 ꢄ 10
.8 ꢄ 10
.4 ꢄ 10
M
M
M
M
M
M
(
a)
4
ꢁ
4
3
3
.0 ꢄ 10
.6 ꢄ 10
1
1
637.06 cmꢁ
ꢁ4
ꢁ4
ꢁ
1
1
1
1
1
637.19 cm
637.27 cm
637.35 cm
4.2 ꢄ 10
ꢁ
ꢁ
1
ꢁ
1
1
ꢀ5
1
1
637.43 cm
637.57 cm
ous concentrations (0–4.2 ꢁ 10 M) and the results are sum-
ꢁ
marised in Fig. 7b, c. As can be seen, the only anodic peak was
found to be slightly shifted towards a negative potential on the
addition of HSA and BSA. This shift in peak potential could be
related to the presence of HSA and BSA at the electrode surface
and the interaction between iron(III) DiAmsar with these. The
observed shifts in peak potentials in this study indicate that the
interaction of iron(III) DiAmsar with HSA and BSA occurred
ꢁ
ꢁ
4
4
(
b)
1.2 ꢄ 10
M
M
M
M
M
M
1
2
.8 ꢄ 10
ꢁ
4
4
.4 ꢄ 10ꢁ
ꢁ
1
3.0 ꢄ 10
3.6 ꢄ 10
4.2 ꢄ 10
1
1
1
637.27 cm
637.41 cm
637.55 cm
ꢁ
4
ꢁ
ꢁ
1
1
ꢁ4
ꢁ
1
1
637.63 cm
ꢁ
ꢁ
1
1
1
1
637.87 cm
638.23 cm
[33]
through hydrophobic interaction.
1700
1600
Wavenumber [cmꢁ1]
Interaction of Iron(III) DiAmsar with HSA and BSA
from FT-IR Spectra
Fig. 8. Fourier-transform (FT)-IR spectrograms of (a) human serum
albumin (HSA), and (b) bovine serum albumin (BSA) in the presence of
different concentrations of iron(III) diamine sarcophagine (DiAmsar).
FT-IR spectroscopy is an appropriate method for exploring the
[
secondary structures of proteins and their dynamics.
29]
In the
(
a)
(b)
(
c)
(d)
Fig. 9. Molecular docking of human serum albumin (HSA) and bovine serum albumin (BSA) with iron(III) diamine sarcophagine (DiAmsar).
(
a) Visual molecular dynamics (VMD), and (b) line-drawing method of HSA with CPK drawing method of iron(III) DiAmsar, (c) VMD and
d) line-drawing method BSA and CPK drawing method of iron(III) DiAmsar.
(

Interaction of Serum Albumin with Iron(III) Diamine Sarcophagine
aforementioned spectroscopy, the spectra of almost any bio-
K
HSA and BSA were not changed after binding of iron(III) DiA-
msar. In addition, the results of molecular docking indicate that
there are hydrogen bonds between iron(III) DiAmsar and Tyr-411,
Arg-410, Asn-391, Gln-390, Leu-387, Leu-430, and Ser-427
residues of HSA and Tyr-149, Cys-245, Glu-100, and Gln-203
residues of BSA. This study may provide important insight into
the interactions of the physiologically important HSA and BSA
with iron(III) DiAmsar. Also, useful information may be obtained
about the effect of environmental conditions on the structural
features of HSA and BSA, which may be correlated with their
physiologically activity.
[
30,31]
logical system can be achieved in different media.
In this
work, the FT-IR spectra of free HSA and BSA were acquired by
subtracting the absorption of the Tris-HCl buffer from the
spectra of the HSA and BSA solutions, which exhibited the
ꢀ
1
characteristic amide I bands between 1700 and 1600 cm , as
shown in Fig. 8. It was clear that, when adding iron(III) DiAmsar
to the HSA and BSA, the amide I bands only slightly shifted
ꢀ1
from 1637.96 to 1637.17 cm
ꢀ
for HSA and 1637.27 to
638.23 cm for BSA. Although other reasons are possible,
1
1
this indicates that the secondary structures of HSA and BSA are
perturbed, though not dramatically so.
Supplementary Material
Interaction of Iron(III) DiAmsar with HSA
and BSA by Molecular Docking
1
3þ
H NMR spectra of the [Co-(NO ) -sar] ,Cl complex, [Co-
2 2
3
5
þ
NH ) -sar] ,Cl complex, and DiAmsar are available on the
3 2 5
(
Computational molecular docking was employed to improve
understanding of the interaction of iron(III) DiAmsar with HSA
and BSA. The aim of the present study was to evaluate the exact
conformation of iron(III) DiAmsar at the binding site and to
identify the precise binding sites on HSA and BSA, which are
crucial and important for understanding the proper functioning
of iron(III) DiAmsar as a drug. The best results of the iron(III)
DiAmsar interactions with HSA and BSA in all runs of docking
procedures are shown in Fig. 9. The location of iron(III) DiAmsar
in HSA was characterised by a favourable binding energy of
Journal’s website.
Acknowledgements
We are grateful to the PNU and INSF for funding this work.
References
[
1] S. Liu, D. Li, C. Huang, L. Yap, R. Park, H. Shan, Z. Li, P. S. Conti,
Theranostics 2012, 2, 589. doi:10.7150/THNO.4295
[
2] G. A. Bottomley, I. J. Clark, I. I. Creaser, L. M. Engelhardt, R. J. Geue,
K. S. Hagen, J. M. Harrowfield, G. A. Lawrance, P. A. Lay, A. M.
Sargeson, A. J. See, B. W. Skelton, A. H. White, F. R. Wilner, Aust. J.
Chem. 1994, 47, 143. doi:10.1071/CH9940143
ꢀ
1
ꢀ
30.73 kcal mol . As seen in Fig. 9a, b, the iron(III) DiAmsar
molecule in HSA is mainly surrounded by Tyr-411, Arg-410,
Asn-391, Leu-387, Leu-430, Gln-390, Ser-427, and Phe-403. In
addition, hydrogen atoms of amine groups of iron(III) DiAmsar
formed hydrogen bonds with Tyr-411, Arg-410, Asn-391, Gln-
[3] H. Cai, J. Fissekis, P. S. Conti, Dalton Trans. 2009, 5395. doi:10.1039/
B902210D
3
(
90, Leu-387, Leu-430, and Ser-427. The docking model of iron
III) DiAmsar in BSA (Fig. 9c, d) shows that iron(III) DiAmsar
was surrounded by Tyr-149, Ser-192, Cys-245, Glu-100, Ile-
[4] P. V. Bernhardt, J. M. Harrowfield, Y. Kim, Y. H. Lee, Y. C. Park,
A. Sujandi, Bull. Korean Chem. Soc. 2007, 28, 589. doi:10.5012/
BKCS.2007.28.4.589
[
5] K. A. Lears, R. Ferdani, K. Liang, A. Zheleznyak, R. Andrews, C. D.
2
02, Lys-242, Hls-246, and Gln-203, with a binding energy of
ꢀ
1
33.46 kcal mol . Hydrogen atoms of amine groups of iron(III)
Sherman, S. Achilefu, C. J. Anderson, B. E. Rogers, J. Nucl. Med.
ꢀ
2
011, 52, 470. doi:10.2967/JNUMED.110.082826
DiAmsar formed a hydrogen bond with Tyr-149, Cys-245,
Glu-100, and Gln-203. These results are in accordance with the
binding mode results. The fluorescence quenching occurred due
to Trp-214 in the IIA domain. Furthermore, any perturbation in
Trp-214 in subdomain IIA may induce changes in IIIA domain
as well. The presence of hydrogen bonds indicates that the
formation of hydrogen bonds decreases the hydrophilicity and
increases the hydrophobicity and stability of the iron(III)
[
[
6] H. Cai, Z. Li, C. W. Huang, R. Park, A. H. Shahinian, P. S. Conti, Nucl.
Med. Biol. 2010, 37, 57. doi:10.1016/J.NUCMEDBIO.2009.09.001
7] S. D. Voss, S. V. Smith, N. DiBartolo, L. J. McIntosh, E. M. Cyr,
A. A. Bonab, J. L. J. Dearling, E. A. Carter, A. J. Fischman, S. T.
Treves, S. D. Gillies, A. M. Sargeson, J. S. Huston, A. B. Packard,
Proc. Natl. Acad. Sci. USA 2007, 104, 17489. doi:10.1073/PNAS.
0
708436104
[
[
8] T. J. Wadas, E. H. Wong, G. R. Weisman, C. J. Anderson, Curr.
Pharm. Des. 2007, 13, 3. doi:10.2174/138161207779313768
9] L. Wei, Y. Ye, T. J. Wadas, J. S. Lewis, M. J. Welch, S. Achilefu, C. J.
Anderson, Nucl. Med. Biol. 2009, 36, 277. doi:10.1016/J.NUCMED
BIO.2008.12.008
[
29]
DiAmsar–HSA and iron(III) DiAmsar–BSA systems.
Conclusions
[
[
[
10] H. Cai, Z. Li, C. Huang, R. Park, A. H. Shahinian, P. S. Conti, Nucl.
Med. Biol. 2010, 37, 57. doi:10.1016/J.NUCMEDBIO.2009.09.001
11] G. J. Christian, A. Arbuse, X. Fontrodona, M. A. Martinez, A. Lobet,
F. Maseras, Dalton Trans. 2009, 6013. doi:10.1039/B902947H
12] M. D. Hall, T. W. Failes, N. Yamamoto, T. W. Hambley, Dalton Trans.
2007, 3983. doi:10.1039/B707121C
In the present work, the synthesis and characterisation of iron(III)
DiAmsar complexes are reported. Further, the interaction of the
title complex with HSA and BSA is investigated via different
spectroscopic techniques (fluorescence, UV-vis, FT-IR) and
cyclic voltammetry and molecular docking methods under sim-
ulated physiological conditions. This study showed that iron(III)
DiAmsar binds to HSA and BSA with high affinity and quenches
the intrinsic fluorescence of HSA and BSA efficiently by a
static mechanism. The binding parameters were calculated using
the Stern–Volmer equation. The thermodynamic parameters
[13] L. Grøndahl, A. Hammershøi, A. M. Sargeson, V. J. Thom, Inorg.
Chem. 1997, 36, 5396. doi:10.1021/IC9705148
[
14] Y. Z. Voloshin, O. A. Varzatskii, Y. N. Bubnov, Russ. Chem. Bull.
007, 56, 577. doi:10.1007/S11172-007-0100-6
2
[
15] D. Martinez, M. Slifstein, N. Nabulsi, A. Grassetti, N. B. L. Urban,
A. Perez, F. Liu, S. Lin, J. Ropchan, X. Mao, L. S. Kegeles, D. C.
Shungu, R. E. Carson, Y. Huang, Biol. Psychiatry 2014, 75, 165.
doi:10.1016/J.BIOPSYCH.2013.06.026
(negative value of DH, negative value of DS, and negative value
of DG) indicate that van der Waals interactions and hydrogen
bonding interactions play a major role in the binding process. The
distances (r) between HSA and BSA (as donors) and iron(III)
DiAmsar (as acceptor) were obtained according to FRET theory.
Further, FT-IR evidence shows that the secondary structure of
[
16] E. Stein, G. C. Mueller, B. Sundaram, Radiol. Clin. N. Am. 2014, 52,
1
95. doi:10.1016/J.RCL.2013.08.002
[17] M. Østergaard, S. J. Pedersen, U. M. Døhn, Best Pract. Res., Clin.
Rheumatol. 2008, 22, 1019. doi:10.1016/J.BERH.2008.09.014

L
B. V. Farahani et al.
[
18] C. Tilcock, Adv. Drug Deliv. Rev. 1999, 37, 33. doi:10.1016/S0169-
09X(98)00109-4
[26] H. M. Ueno, H. Urazono, T. Kobayashi, Food Chem. 2014, 145, 90.
doi:10.1016/J.FOODCHEM.2013.07.143
4
[
19] D. R. Anderson, M. J. Duryee, R. P. Garvin, M. D. Boska, G. M. Thiele,
L. W. Klassen, Magn. Reson. Imaging 2012, 30, 96. doi:10.1016/
J.MRI.2011.07.023
[27] Y. Huang, Y. Shuai, H. Li, Z. Gao, Biochim. Biophys. Acta 2014, 1840,
970. doi:10.1016/J.BBAGEN.2013.11.011
[28] C. Hebia, L. Bekale, P. Chanphai, J. Agbebavi, H. A. Tajmir-Riahi,
J. Photochem. Photobiol. B 2014, 130, 254. doi:10.1016/J.JPHOTO
BIOL.2013.11.025
[
20] H. Ai, Adv. Drug Deliv. Rev. 2011, 63, 772. doi:10.1016/J.ADDR.
2011.03.013
[
21] M. Chruszcz, K. Mikolajczak, N. Mank, K. A. Majorek, P. J. Porebski,
W. Minor, Biochim. Biophys. Acta 2013, 1830, 5375. doi:10.1016/
J.BBAGEN.2013.06.016
[29] X. Li, D. Chen, G. Wang, Y. Lu, Eur. J. Med. Chem. 2013, 70, 22.
doi:10.1016/J.EJMECH.2013.09.033
[30] Z. Chen, Y. He, B. Shi, D. Yang, Biochim. Biophys. Acta 2013, 1830,
[
[
[
[
22] S. Fujiwara, T. Amisaki, Biochim. Biophys. Acta 2013, 1830, 5427.
5515. doi:10.1016/J.BBAGEN.2013.04.037
[31] J. Lin, Y. Liu, M. Chen, H. Huang, L. Song, J. Lumin. 2014, 146, 114.
doi:10.1016/J.JLUMIN.2013.09.054
[32] Q. Ma, H. Bai, C. Wang, G. Xi, Q. Zhang, X. Meng, Y. Chen, J. Li,
H. Ma, L. Guo, Int. J. Mass Spectrom. 2014, 357, 34. doi:10.1016/
J.IJMS.2013.10.007
[33] A. M. Sargeson, Pure Appl. Chem. 1984, 56, 1603. doi:10.1351/
PAC198456111603
doi:10.1016/J.BBAGEN.2013.03.032
23] C. Ha, N. V. Bhagavan, Biochim. Biophys. Acta 2013, 1830, 5486.
doi:10.1016/J.BBAGEN.2013.04.012
24] J. Reichenwallner, D. Hinderberger, Biochim. Biophys. Acta 2013,
1830, 5382. doi:10.1016/J.BBAGEN.2013.04.031
25] M. Anraku, V. T. G. Chuang, T. Maruyama, M. Otagiri, Biochim.
Biophys. Acta 2013, 1830, 5465. doi:10.1016/J.BBAGEN.2013.04.036