Shedding light on the bimolecular interactions of Cafaminol and human serum albumin: spectroscopic characterization and in-silico investigation

Article abstract of DOI:10.1080/07391102.2020.1863262

Cafaminol, also known as methylcoffanolamine, is a vasoconstrictor and anticatarrhal of the methylxanthine family, which is used as a nasal decongestant. This study aimed to investigate the interaction mechanisms of human serum albumin (HSA) with Cafaminol, through several spectroscopic (fluorescence quenching, UV-visible absorption, and circular dichroism (CD) spectroscopies) and molecular modeling techniques. Stern-Volmer plots were employed to specify the fluorescence quenching mechanism, while the simulation methods were utilized to deduce the approximate binding position of Cafaminol on HSA. On the other hand, thermodynamic parameters, enthalpy and entropy changes, were determined to be, respectively, ?105.88 (kJ mol^?1) and ?282.34 (J mol^?1 K^?1), using the Van't Hoff equation and analyzed later to specify the main acting forces between Cafaminol and HSA. Overall results revealed the binding of Cafaminol to the site I of HSA, as a result of an enthalpy-driven process, mainly through the van der Waals and hydrogen bonding interactions. Static quenching mechanism was found to be responsible for the fluorescence quenching of HSA in the Cafaminol presence, while the number of binding sites and apparent binding constant were measured accordingly. Docking results proposed that Cafaminol and HSA interact with a binding free energy (ΔG) of ?6.5 kcal mol^?1 Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2020.1863262

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Shedding light on the bimolecular interactions
of Cafaminol and human serum albumin:
spectroscopic characterization and in-silico
investigation
Negar Parvizi , Delara Mohammad-Aghaie , Mohammad Navid Soltani Rad ,
Somayeh Behrouz & Mohamad Mehdi Alavianmehr
To cite this article: Negar Parvizi , Delara Mohammad-Aghaie , Mohammad Navid
Soltani Rad , Somayeh Behrouz & Mohamad Mehdi Alavianmehr (2021): Shedding light
on the bimolecular interactions of Cafaminol and human serum albumin: spectroscopic
characterization and in-silico investigation, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2020.1863262
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2020.1863262
Shedding light on the bimolecular interactions of Cafaminol and human serum
albumin: spectroscopic characterization and in-silico investigation
Negar Parvizi, Delara Mohammad-Aghaie, Mohammad Navid Soltani Rad, Somayeh Behrouz and
Mohamad Mehdi Alavianmehr
Department of Chemistry, Shiraz University of Technology, Shiraz, Iran
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 11 August 2020
Accepted 8 December 2020
Cafaminol, also known as methylcoffanolamine, is a vasoconstrictor and anticatarrhal of the methylxan-
thine family, which is used as a nasal decongestant. This study aimed to investigate the interaction
mechanisms of human serum albumin (HSA) with Cafaminol, through several spectroscopic (fluores-
cence quenching, UV-visible absorption, and circular dichroism (CD) spectroscopies) and molecular
modeling techniques. Stern-Volmer plots were employed to specify the fluorescence quenching mech-
anism, while the simulation methods were utilized to deduce the approximate binding position of
Cafaminol on HSA. On the other hand, thermodynamic parameters, enthalpy and entropy changes,
KEYWORDS
Human serumal bumin;
fluorescence spectroscopy;
molecular dynamics
simulation; Cafaminol
were determined to be, respectively, ꢀ105.88 (kJ molꢀ1) and ꢀ282.34 (J molꢀ1
K
ꢀ1), using the Van’t
Hoff equation and analyzed later to specify the main acting forces between Cafaminol and HSA.
Overall results revealed the binding of Cafaminol to the site I of HSA, as a result of an enthalpy-driven
process, mainly through the van der Waals and hydrogen bonding interactions. Static quenching
mechanism was found to be responsible for the fluorescence quenching of HSA in the Cafaminol pres-
ence, while the number of binding sites and apparent binding constant were measured accordingly.
Docking results proposed that Cafaminol and HSA interact with a binding free energy (DG)
of ꢀ6.5 kcal molꢀ1
1. Introduction
binding affects on absorption, distribution and excretion of
drugs (Seedher & Bhatia, 2006).
Human Serum Albumin (HSA, shown in Figure 1) has a fun-
damental role in the transport and deposition of many
endogenous and exogenous substances in blood. It is a sin-
gle-chain protein of 585 aminoacids and a molecular mass of
66.5 kDa, with a usual concentration of around 42 mg/ml
(Peters, 1995). This heart shaped molecule with approximate
dimensions of 80 ꢁ 80 ꢁ 30 Å, has three specific homolo-
gous a-helical domains, I, II and III, while each of them are
divided into sub-domains, A and B (Peters, 1995; Sugio
et al., 1999).
Various small molecules can chemically bound to HSA in
sites I (the Warfarine binding site), II (the Benzodiazepine
binding site) and III, respectively, located in domains IIA, IIIA
and IB, while the last site is relatively novel (Bos et al., 1988,
1988). When encountering with HSA, different drugs may
compete for the same binding site.
A wide variety of experimental and computational meth-
ods have been used for analysis of the interactions between
proteins and ligands. Some widely used experimental techni-
ques are X-ray scattering, nuclear magnetic resonance (NMR),
Laue X-ray diffraction, single-molecule fluorescence spectros-
copy, UV–vis absorption and circular dichroism spectroscopy
(Du et al., 2016). On the other hand, molecular dynamics and
docking simulations are able to provide microscopic insight
into the drug protein interactions.
Alavianmehr et al. (2014) determined the binding proper-
ties of N,N’,N"-triethylenethiophosphoramide (thioTEPA), with
human serum albumin in aqueous solution, at pH 7.4. They
employed the UV/vis absorption and fluorescence spectros-
copies along with molecular dynamics and docking simula-
tions. ThioTEPA was found to be located at domain I of HSA,
resulting in both static and dynamic fluorescence quenching
mechanisms, while no significant changes in the local and
overall secondary structure, polarity and hydrophobicity of
HSA were observed.
It is of great importance to determine the interaction of
different drugs with HSA, not only to comprehend their
transport and metabolism in the body, but also to shed light
on their therapeutic effectiveness. It is well known that the
binding mechanism has an important pharmacokinetic and
Recently, Poursoleiman et al. (2019) studied the inter-
action of HSA with the two Polymyxins (B & E), which are
pharmacodynamics implications, and the degree of protein members of a group of cationic charged cyclic antibiotic
CONTACT Delara Mohammad-Aghaie
d_aghaie@sutech.a.ird; delara.a@gmail.com
Department of Chemistry, Shiraz University of Technology, Shiraz,
71555-313, Iran
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2020.1863262
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
N. PARVIZI ET AL.
lipopeptides, to get quantitative and qualitative insight into hand, results of the three-dimensional fluorescence spectra
the binding characteristics. Considering the values of thermo- indicated the remarkable changes in the structures of HSA,
dynamic parameters revealed that binding interaction was HTF, and (HSA-HTF), in the presence of Nano-CUR, within the
spontaneous and entropy driven. Electrostatic interactions
were found to be responsible for binding of polymixins to a
binary and ternary systems.
In continuation of our previous works (Alavianmehr et al.,
moiety of HSA, between domains I and III. The results of 2014; Keshavarz et al., 2013; Mohammad-Aghaie et al., 2019;
both fluorescence anisotropy and Far-UV CD spectroscopies,
in agreement with the computational studies, demonstrated
minor changes in the HSA conformational structure.
Shahsavani et al., 2016), this study aims to elucidate different
aspects of Cafaminol-HSA binding interactions, through sev-
eral spectroscopic and simulation techniques. To the best of
our knowledge, there has not been any reported study on
the interaction of Cafaminol with the human serum albumin.
Cafaminol [CAS No: 30924-31-3] with the IUPAC name of
(8-[(2-ydroxyethyl) (methyl)amino]-1,3,7-trimethyl-3,7-dihydro-
1H-purine-2,6-dione), also known as methylcoffanolamine,
has been clinically applied as a nasal decongestant and anti-
catarrhal, since 1974. This drug which is derived from xan-
thine (a purine base) (Rogowski & Chodynicki, 1985), select-
ively binds to alpha-1 receptors, causing blood vessels
to constrict.
In the present study, binding of Cafaminol to HSA was
studied by UV/vis absorption and fluorescence spectroscop-
ies, the fluorescence quenching mechanism was specified
and the drug’s effect on the protein’s secondary structure
was explored using the circular dichroism (CD) method. In
order to assess the nature of dominant acting forces
between HSA and Cafaminol, thermodynamic parameters
were analyzed. Furthermore, docking and molecular dynam-
ics simulation methods were employed to get a more solid
understanding of the structural features, induced by
Cafaminol on HSA, and determine their approximate binding
position. These results will be of biological importance in
pharmacology as well as clinical medicine.
Sohrabi et al. (2018) investigated the mutual interactions
of lomefloxacin (LMF) and a calf thymus DNA-histone H1
(ctDNA-H1) complex, through several spectroscopic techni-
ques, along with the viscometry and molecular modeling
methods. Analysis of thermodynamic parameters, such as
DG0 and DS0, revealed the important role of van der Waals
forces and hydrogen bonding, in the LMF binding to ctDNA
and the ctDNA-H1 complex. On the other hand, CD spectros-
copy confirmed the interaction of LMF with ctDNA and
ctDNA-H1, through an outside binding mode, leading to the
conformational changes in ctDNA. Both in the absence and
presence of H1, the interaction mechanism was found to
be static.
In the study of the effects of Nano-Curcumin (Nano-CUR)
binding on HSA-HTF (holo transferrin) interactions, Mokaberi
et al. (2020) found that Nano-CUR is capable of quenching
both proteins with a static mechanism and obtained the
binding constants for the formation of binary and ternary
complexes. Analysis of thermodynamic parameters revealed
the outstanding roles of van der Waals forces and hydrogen
bonding interactions, in complex formations. On the other
2. Materials and methods
2.1. Materials
HSA was purchased from sigma Aldrich and used without fur-
ther purification. Cafaminol was synthesized in our laboratory
in two synthetic steps, shown in Figure 2. Primarily, the caf-
feine was brominated using NBS in DCM/Water (50:50, V/V)
solution at room temperature for 5 days, then the obtained 8-
bromocaffeine was reacted with N-methylethanol amine, in a
neat condition at 170 ꢂC, to obtain the pure Cafaminol as a
white solid (mp: 162–164 ꢂC). The physical and spectral data
assignments of Cafaminol, comprising 1HNMR, 13CNMR and IR,
have been provided as supplementary data.
The stock solution of HSA 30 micro molar (mM) was pre-
pared by dissolving protein in phosphate buffer solution
0.1 M (Na2HPO4 and NaH2PO4 in pure aqueous medium, with
Figure 1. The ribbon model of albumin, derived from X-ray crystallography,
where the subdivision of albumin into domains (I, II, III) and subdomains (A and
B) is indicated. C and N show the C-terminal and N-terminal ends, respectively.
PH
¼
7.4). Before sample preparation, the protein
Figure 2. The synthetic procedure of Cafaminol from Caffeine.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
concentration was determined spectrophotometrically using conformational and structural information about proteins. In
an extinction coefficient of 42,000 Mꢀ1 cmꢀ1, at 280 nm
this study, excitation and emission wavelengths were set
ꢀ
(Spiewak et al., 2015). The Cafaminol working solutions were
prepared by dissolving appropriate amounts of Cafaminol in
the same buffer. All solutions were stored at 0–4 ꢂC in the
dark for a week. Doubly distilled water was used throughout
all over experiments.
between 200 and 500 nm, while other scanning parameters
were the same as those for the fluorescence quench-
ing spectra.
2.2.4. UV-visible absorption spectroscopy
Absorption spectra of HSA were recorded both in the
absence and presence of Cafaminol, in the range of
250–400 nm, employing the carry spectrophotometer
equipped with 1-cm quartz cell. HSA concentration was fixed
at 20 lmol Lꢀ1, while the Cafaminol concentration was varied
2.2. Methods
2.2.1. Fluorescence spectroscopy
Fluorescence quenching occurs due to the decrease in the
quantum yield of fluorophore, induced by the quencher mol-
ecule. The inherent fluorescence of HSA comes mainly from
tryptophan-214 and is very sensitive to the environment,
around this residue (Lakowicz, 2013).
from 0 to 40 lmol Lꢀ1
.
2.2.5. Circular dichroism (CD) measurement
Here, Cafaminol acts as a quencher for HSA molecule.
Fluorescence quenching is classified into dynamic and static
quenching. These two mechanisms are distinguished, based
on their response to the temperature. To examine the mech-
anism of Cafaminol induced quenching, the Stern-Volmer
equation was employed.
Fluorescence spectra and intensities were measured with
carry-Eclipse spectrophotometer (Varian model-Australia)
equipped with a thermostat bath and 1-cm quartz cell.
Excitation and emission bandwidths were set at 5 and
10 nm, respectively.
CD is an optical technique to monitor the protein’s conform-
ational changes. In this study, CD spectra were recorded
using Aviv Circular Dichroism Spectrometer Model-215, in
the range of 190–260 nm, at room temperature under con-
stant nitrogen flush rate of 30 l/min. Measurements were
taken at constant 10 lmol Lꢀ1 HSA concentration, while
Cafaminol was added gradually to cover the concentration
range of 0 to 74 lmol Lꢀ1
.
2.2.6. Molecular dynamics simulation
MD simulation is the most comprehensive computational
method that allows one to predict the time evolution of a
molecular system of interacting particles. In this study, two
separate MD simulations were performed on pure HSA and
HSA-Cafaminol complex, respectively, in order to find the
equilibrium structure of HSA at semi-physiological conditions
and to analyze the conformational changes of HSA upon
binding to Cafaminol.
In this respect, initial structure of HSA (PDB ID: 4k2c) was
downloaded from the RCSB website and required topology
and interaction parameters were created using the standard
GROMOS9643a1 force field. The PRODRG 2.5 automated ser-
The steady-state fluorescence emission spectra of HSA
were measured in the 300–450 nm range, at 289, 296, 303
and 310 K, with the wavelength of 295 nm. In each tempera-
ture, a 1 ml portion of 10 lmol Lꢀ1 HSA solution was titrated
manually by successive additions of stock solution of
Cafaminol, to span the drug concentration range, from 0 to
40 lmol Lꢀ1. After each drug addition, the complex solution
was left for 3 min, to reach equilibrium at the desired tem-
perature; then fluorescence intensities were measured.
2.2.2. Synchronous spectroscopy
Synchronous fluorescence (SF) spectrometry is able to pro-
vide information on the molecular environment in the vicin-
ity of the chromophore molecules. This technique has
several advantages such as spectral bandwidth reduction,
simplification of the emission spectra, contraction of the
spectral range and avoiding different perturbing effects
(Lloyd & Evett, 1977).
€
ver (Schuttelkopf & Van Aalten, 2004), a tool for quickly gen-
erating topologies was employed to produce topology
parameters for Cafaminol, within the framework of the
GROMOS96-43a1 forcefield.
After insertion of protein in a cubic box of simple point
charge (SPC) water molecules, the steepest descent algo-
rithm was employed to run energy minimization, followed
by the two short position restrained MD simulations, in NVT
and NPT ensembles, each for 100 ps. The system was sub-
jected to these equilibration steps, in order to reach to the
specified temperature (310 K) and pressure (1 bar).
It should be mentioned that velocity verlet algorithm [40]
was used for allocating initial velocities to atoms, based on
the Maxwell distribution. Cutoff value for short range van
der Waals interactions was set to 1.4 nm, while the Particle
Mesh Ewald was employed to treat the long-range electro-
static interactions.
SF spectra were recorded by simultaneously scanning the
emission and excitation monochromators, when Dk between
excitation and emission wavelengths was set to 15 or 60 nm,
giving characteristic information about the Tyr and Trp resi-
dues, respectively. Measurement of synchronous spectra was
done by titrating 1 ml portion of 10 lmol Lꢀ1 HSA, with suc-
cessive additions of Cafaminol to reach the maximum con-
centration of 40 lmol Lꢀ1
.
2.2.3. 3D fluorescence spectroscopy
After NVT and NPT equilibration steps, the system was
Three-dimensional fluorescence spectroscopy is an effective
and powerful method to provide comprehensive subjected to
a 20 ns MD simulation (using leap-frog

4
N. PARVIZI ET AL.
Figure 4. Stern-Volmer plots for the fluorescence quenching of HSA, at differ-
ent temperatures. HSA: 10 mmol Lꢀ1, Cafaminol: 0–40 mmol Lꢀ1, pH ¼ 7.4.
Figure 3. Conventional fluorescence spectra of HSA, at 289 K. HSA: 10 lmol
ꢀ1; Cafaminol (a–i): 0–40 lmol Lꢀ1
L
.
These two mechanisms are distinguished based on their
responses to the temperature. Taking into account that
higher temperatures give rise to larger diffusion coefficients,
and the dependence of dynamic quenching on the diffusion,
it is expected that bimolecular quenching constants increase
with the increasing temperature. Contrarily, in static quench-
ing, temperature increase leads to the decrease in complex
stability, so lowers the quenching constant values (Hu
et al., 2005).
integrator), at 310 K, while the trajectory was analyzed by the
Gromacs package tools. Above mentioned steps were
repeated for the HSA-Cafaminol mixture, to assess the struc-
tural changes in HSA, induced by Cafaminol. All calculations
and analyzes were performed using the Gromacs 5.1.4 simu-
lation package.
2.2.7. Docking
Cafaminol’s structure was generated by the Chemdraw ultra
8, and energy minimized in a two-step process. Energy mini-
mization was first implemented by AM1, a semi-empirical
method to ensure that the main structure is good enough
for initiating the main energy minimization step, by the
Guassian 09 software. In the next step, the Auto Dock Tools
version 1.5.6 software (http://mgltools.scripps.edu) was used
to prepare the docking input files, by Reading drug coordi-
nates, adding necessary charges, merging non-polar hydro-
gens, and defining the rotatable bonds. Auto Dock Vina was
In this study, HSA, acting as a fluorophore, has 1 trypto-
phan, 18 tyrosine and 32 phenylalanine residues. The intrin-
sic fluorescence of HSA comes mainly from tryptophan
alone, because phenylalanine has a very low quantum yield,
and the fluorescence of tyrosine is totally quenched if it is
ionized or is near an amino group, a carboxyl group or a
tryptophan residue (Sułkowska, 2002).
When studying HSA fluorescence, in order to prevent
tyrosine from getting excited, normally an excitation of
295 nm is used. Figure 3 depicts the fluorescence quenching
then employed to dock drug molecule, into the equilibrated spectra of HAS, in the presence of different concentrations of
receptor (HSA) structure (obtained from the MD simulation). Cafaminol (acting as a quencher). It is clear that the fluores-
During the docking process, HSA structure was set rigid, cence intensity is decreased gradually with the increasing
while considering flexibility for the ligand, via gen- concentrations of Cafaminol, which in turn confirms the
binding of Cafaminol to HSA.
etic algorithm.
The Stern-Volmer equation is often employed to deter-
mine the fluorescence quenching mechanism, induced by a
ligand, here the Cafaminol.
3. Results and discussion
3.1. Fluorescence quenching
Fluorescence quenching is
involved in molecular interaction between fluorophore and
quencher, which lowers the intensity of emitted light from
the fluorescent molecules. This phenomenon occurs due to a
variety of events, such as excited state reactions, energy
transfer, collisional quenching and ground state complex for-
mation (Lakowicz, 2013).
There are two distinct mechanisms for the fluorescence
quenching, namely dynamic and static. Dynamic (or colli-
sional) quenching occurs due to the diffusive encounter
between fluorophore (F) and quencher (Q); while on the
other hand, static (or contact) quenching is mainly based on
F0
F
½ ꢃ
KSV ¼ kqs0
¼ 1 þ KSV
Q
(1)
(2)
a
physicochemical process
where F0 and F are the fluorescence intensities, respectively,
in the absence and presence of the quencher. kq is the
bimolecular quenching rate constant, [Q] is the concentration
of quencher, and Ksv is the Stern-Volmer quenching constant,
providing a direct measure of the quenching efficiency.s0 is
the average lifetime of the fluorophore (without quencher),
where the fluorescence lifetime of biopolymers is about
10ꢀ8 s (Feng et al., 1998), so the values of ksv are similar to
kq values, except for the difference in power.
Figure 4 represents the Stern-Volmer plots at different
the formation of non-fluorescent (FQ) complex (Chen temperatures, which are linear, with the decreasing slopes
et al., 1990). against temperature, providing an evidence for the static

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
Table 1. Stern-Volmer quenching constants (KSV), binding constants (Kb), binding sites (n) and Thermodynamic parameters.
Quenching mechanism
Binding parameters
T (K)
KSVꢁ103 (Mꢀ1
10.46 0.02
8.92 0.02
7.70 0.02
6.70 0.02
)
DG (kJ molꢀ1
)
DH (kJ molꢀ1
ꢀ105.88
)
DS (J molꢀ1 Kꢀ1
ꢀ282.34
)
R2
N
Kb (ꢁ103 Mꢀ1
28.44 0.04
7.71 0.04
2.59 0.04
1.47 0.04
)
R2
289
296
303
310
ꢀ24.64
ꢀ22.03
ꢀ19.76
ꢀ18.762
0.99
0.99
0.99
0.99
1.10
0.98
0.89
0.85
0.98
0.99
0.98
0.99
Figure 5. Double log plots (log ðF ꢀFÞ vs. log [Q]) for HSA fluorescence quenching at different temperatures. HSA: 10 mmol Lꢀ1, Cafaminol: 0–40 mmol Lꢀ1, pH
0
F
¼ 7.4
quenching mechanism. Table 1 reports the Ksv values (Stern- where a plot of log (F0ꢀF)/F versus log [Q] is employed to
determine Kb. Figure 5 depicts these double log plots, for
the fluorescence quenching of HSA, at different tempera-
tures. The correlation coefficients which are greater than 0.99
(reported in Table 1) show acceptable agreement between
the HSA-Cafaminol interaction, and the site binding, underly-
ing Eq. (3). Kb and n values, obtained from Eq. (3), are also
reported in Table 1. n values were found to be approxi-
mately 1, meaning that HSA has only a single binding site
for Cafaminol. In other words, regardless of the present num-
ber of Cafaminol molecules in solution, HSA is able to carry
just one Cafaminol molecule.
Volmer quenching constants), obtained from the slopes of
Stern-Volmer plots, at different temperatures.
It should be mentioned that the maximum scatter colli-
sion quenching constant (kq) for various quenchers of biopol-
ymers, is about 2.00 ꢁ 1010 Mꢀ1 Sꢀ1 (Yuan et al., 2011). Here,
the quenching constants of HSA induced by Cafaminol
(reported in Table 1) are greater than 2.00 ꢁ 1010 Mꢀ1 Sꢀ1
,
which is another proof that the quenching process occurs by
the complex formation between HSA and drug.
For static quenching interaction, the binding constant and
stoichiometry can be calculated using the following equa-
tion, if it is assumed that there are comparable and inde-
pendent binding sites in the biomolecule (Lakowicz, 2013):
According to the Table 1, Kb values for binding of
Cafaminol to HSA are on the order of 103 M, showing the
moderate binding affinity between these two molecules. This
justification is based on the fact that high affinity binding
ðF0ꢀFÞ
log
¼ logKb þ nlog½Qꢃ
(3)
constants of ligand-protein are in the range of 106–108
M
F
Again F and F0 are the fluorescence intensities with and
without quencher, respectively. Kb is the binding constant
(53). Such a moderate binding affinity is benefited for the
efficient transport of drug, and its subsequent release, at the
and n is the number of binding sites per albumin molecule, target site.

6
N. PARVIZI ET AL.
DG ¼ ꢀRTlnK
Here T is the temperature in Kelvin and R is the universal
gas constant (8.31 J molꢀ1 ꢀ1). Examination of the
(6)
K
Thermodynamic parameters (reported in Table 1) showed
that Cafaminol-HSA complex formation is an exothermic and
spontaneous process. On the other hand, negative DS and
DH values indicated that van der Waals forces and hydrogen
bonding play the dominant roles in the binding of Cafaminol
to HSA. The binding process was found to be enthalpy
driven, because the main contribution to the DG value was
from DH, while the DS factor had smaller portion. It is known
that enthalpy driven interactions favor hydrogen bonding in
the low dielectric moieties like water, and Van der Waals
interactions get into the game, directly duo to hydrophobic
interactions (Kang et al., 2004; Ross & Subramanian, 1981).
Figure 6. Overlap spectra of Cafaminol UV absorption spectrum and the fluor-
escence emission spectrum of HAS. [HSA] ¼ 30 ꢁ 10ꢀ6 mol Lꢀ1
,
[Cafaminol] ¼ 30 ꢁ 10ꢀ6mol Lꢀ1
.
3.3. Fluorescence resonance energy transfer (FRET) from
HSA to Cafaminol
FRET is a non-destructive spectroscopic mechanism, describ-
ing energy transfer between two light-sensitive molecules.
Energy may transfer from the donor, initially in its electronic
excited state, to an acceptor through the non-radiative long
range dipole-diploe interactions (Helms, 2018).
In order to reduce the inner filter effect, all fluorescence
intensities were corrected for absorption of the exciting light
and reabsorption of emitted light, using the following rela-
tionship (Lakowicz, 2013; Rashidipour et al., 2016)
According to the FRET theory, the energy transfer will
occur under the following three conditions: (I) Donor can
produce fluorescence light, (II) Fluorescence emission spec-
trum of donor and UV-vis absorbance spectrum of acceptor
have overlap and, (III) Distance between donor and acceptor
is less than 8 nm (Szabo & Ostlund, 2012). Spectral overlap of
the donor (HSA) and acceptor (Cafaminol) is shown in
Figure 6.
The efficiency of energy transfer (E) is inversely propor-
tional to the sixth power of the distance (r0) between donor
and acceptor, and the critical energy transfer distance (R0). E
is calculated through the following equation:
ðA þA
ex em
Þ
=
2
Fcor ¼ F
e
(4)
obse
where Fcor and Fobs are, respectively, the corrected and
observed fluorescence intensities. In addition, Aex and Aem
are the system absorptions at excitation and emission wave-
lengths, respectively.
3.2. Thermodynamic parameters and the
binding constant
When a small molecule binds to a macromolecule, several
types of non-covalent interactions such as Van der Waals
forces, hydrogen bonding, electrostatic and hydrophobic inter-
actions may occur. It is evident from Table 1 that temperature
is an important parameter affecting the binding process, while
the lower temperatures favor the stability of the HSA-
Cafaminol complex. Therefore the temperature dependent
thermodynamic parameters (free energy change, and the
entropy and enthalpy changes) were analyzed in order to char-
acterize the nature of main contributing forces to the HSA-
drug complex formation (Ross & Subramanian, 1981).
Simultaneous DH > 0 and DS > 0 imply a hydrophobic
association; while DH < 0 and DS < 0 confirm the existence
of the van der Waals forces, or the formation of hydrogen
bonding, and electrostatic forces are specified by DH < 0 and
DS > 0 (Ross & Subramanian, 1981).
F
F0
R06
R60 þ r06
E ¼ 1 ꢀ
¼
(7)
where F and F0, respectively, are the fluorescence intensities
of HSA after the ligand addition, and free HSA.
R0, the critical distance at 50% efficiency, is obtained from
Eq. (8).
R60 ¼ 8:79ꢄ10ꢀ25K2Nꢀ4/J
(8)
Here, K2 is the spatial orientation factor, related to the
geometry of dipoles; N is the average refractive index of
medium (in the wavelength range, where spectral overlap is
significant); / is the fluorescence quantum yield of the
donor; and J is the overlap integral between the emission
spectrum of donor and the absorbance spectrum of
acceptor, given by:
The enthalpy and entropy changes were determined,
respectively, from the slope and the y-intercept of the van’t
Hoff equation.
RFkekk4Dk
J ¼
(9)
RFkDk
where F(k) is the fluorescence intensity of donor and e(k) is
The free energy change was later calculated using the fol- the molar absorption coefficient of the acceptor, both at
lowing relationship: wavelength k.
DΗ DS
lnK ¼ ꢀ
þ
(5)
RΤ
R

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Figure 7. UV–vis spectra of HSA in the absence and presence of Cafaminol at
room temperature and pH 7.4. [HSA] ¼ 20 ꢁ 10ꢀ6mol Lꢀ1; [Cafaminol] ¼ (0–40)
ꢁ10ꢀ6 mol Lꢀ1
.
In the present study for HSA-Cafaminol complex, n ¼ 1.36
and /¼ 0.07 (Hu et al., 2005), so based on Eqs. (7)–(9),
J ¼ 2.60 ꢁ 10ꢀ14 cm3 L molꢀ1; R0 ¼ 2.63 nm, and the binding
distance r ¼ 3.28 nm. The value of binding distance (r), falling
in the (0.5 R0 < r < 1.5 R0) range, confirms the occurrence of
energy transfer from HSA to Cafaminol. On the other hand, r
value is greater than R0, which is another evidence for the
static quenching mechanism (Shi et al., 2020).
Figure 8. CD spectra of HSA in the presence of various concentrations of
Cafaminol
(T ¼ 298 K).
C
[HSA] ¼ 10 ꢁ 10ꢀ6mol
Lꢀ1
,
and
C
[Cafaminol] ¼ 0–74.07 ꢁ 10ꢀ6 mol Lꢀ1
.
intensities at all wavelengths of the far-UV, without any sig-
nificant shifts of the peak positions. This observation reveals
the peptide strand unfolding and the decrease of a-helical
content of HSA. It should be mentioned that the main spec-
tra shape of HSA remains similar in the presence and
absence of Cafaminol, showing that the structure of HSA is
predominantly a-helical.
The CD result can be expressed as MRE (mean residue
ellipticity) in deg cm2 dmolꢀ1, according to the following
equation (Chen et al., 1972):
3.4. UV-vis absorption spectra
UV–vis measurement is a simple and effective spectroscopic
method to explore the structural changes and specify the
complex formation. Native HSA spectra depicts two absorp-
tion peaks at 233 and 278 nm, respectively, due to the char-
ꢄ
acteristic p–p transition of polypeptide backbone structure
of C ¼ O, and n–p transition of aromatic amino acids, i.e.
ꢄ
Trp, Tyr and Phe (Shu et al., 2011).
ObservedCDðmdegreeÞ
It is well known that the dynamic quenching only affects
the excited state of fluorophore, without changing the
absorption spectra. In contrast, the complex formation in the
ground state is able to change the absorption spectrum of
the fluorophore (Lakowicz, 2013).
As shown in Figure 7, the UV absorption intensity of HSA
was increased with the increasing amount of Cafaminol,
while the maximum absorption slightly shifted towards the
longer wavelengths. It should be mentioned that the UV-vis
absorption spectra of the protein were obtained through
subtracting the absorption contribution of free Cafaminol
(Figure S4), from that of HSA-Cafaminol mixture.
MRE ¼
(10)
Cpnl ꢁ 10
where Cp, n and l are the molar concentration of the protein
(HSA), number of amino acid residues (585 for HSA) and the
path-length of the cell (1 cm), respectively. The alpha-helix
contents of free and combined HSA were calculated from
the MRE values at 208 nm, using Eq. (11) (Khan et al., 2008).
MRE208ꢀ4000
ð Þ
/ ꢀHelix % ¼
ꢁ 100
(11)
33000 ꢀ 4000
Calculated results showed the decrease of alpha-helicity
in the secondary structure of HSA from 75% to 60.91% (with
the increasing concentrations of Cafaminol), which is in
acceptable agreement with the CD spectra.
These observations support the idea of complex formation
between HSA and Cafaminol, accompanied by the induced
changes in HSA structure, which provide another proof for
the static quenching mechanism.
3.6. Synchronous fluorescence
Complementary to the CD measurements, synchronous fluor-
escence spectra of HSA, with various amounts of Cafaminol,
were carried out to assess the micro environmental changes
in a vicinity of the chromophore molecules. When the excita-
tion and emission monochromators of a fluorimeter are
scanned simultaneously, the synchronous fluorescence spec-
tra are obtained. According to Miller (1979), the fluorescence
spectra of HSA with Dk of 60 and 15 nm, are characteristics
of tryptophan and tyrosine residues, respectively.
3.5. Circular dichroism (CD) spectra analysis
Due to the remarkable sensitivity of CD spectra in UV region
to the backbone conformation of proteins, this technique is
employed to assess alterations in the secondary and tertiary
structures of HSA protein. In the CD diagram of free HSA,
two negative bands at 208–209 and 222–223 nm (in the far
ꢄ
ꢄ
ultra-violet region) are contributed to the p–p and n–p
transfers, characterizing the albumin’s alpha-helical structure.
As seen in Figure 8, with the increasing concentrations of
Figure 9 shows the effect of Cafaminol on the synchron-
Cafaminol, the CD spectra of HSA show decreasing band ous fluorescence spectra of HSA. It is clear that the intensity

8
N. PARVIZI ET AL.
Figure 9. The synchronous fluorescence spectra of HSA-Cafaminol. (A)
Dk ¼ 60 nm; (B) Dk ¼ 15 nm. C [HSA] ¼ 10 ꢁ 10ꢀ6 mol Lꢀ1 while concentra-
tions of Cafaminol were 0.0–40.0 ꢁ 10ꢀ6 mol Lꢀ1 (a–i).
Figure 10. Three-dimensional fluorescence contour diagrams of (A) HSA, (B)
HSA-Cafaminol, at T ¼ 298 K and pH
¼
7.4. [HSA] ¼ 30 mM;
[Cafaminol] ¼ 30 mM.
of tryptophan residue is greater than that of tyrosine resi-
dues, indicating that tryptophan contributes strongly to the
quenching of intrinsic fluorescence of HSA.
Observing a small blue shift (1.07 nm and 1.1 nm, respect-
ively, for Dk ¼ 15 nm and Dk ¼ 60 nm) in both A and B spec-
tra implies an increase in the hydrophobicity and decrease in
the polarity around the tryptophan and tyrosine residues (Hu
et al., 2005).
of higher excited electronic states of the aromatic residues,
present in the protein (Bortolotti et al., 2016).
It is clear that both fluorescence peaks (1 and 2) of HSA
have been quenched in response to the Cafaminol addition,
which is another proof for the binding of Cafaminol to HSA
that leads to the change in protein’s conformation. Close
look at the 3D fluorescence spectra of pure HSA, and HSA-
Cafaminol mixture (Figure 10) shows that changes in the
fluorescence intensity of peaks 1 & 2 are, respectively, 84.55
and 43.38 a.u. This conformational change is along with
unfolding some hydrophobic regions of HSA, which were
buried before.
3.7. 3D fluorescence spectra
The three dimensional fluorescence spectra depict the micro-
environmental changes around the protein fluorophores (Tyr
& Trp). Successively using the excitation and emission mono-
chromators makes it possible to measure emission spectra
for different excitation wavelengths, with a constant step.
The emission-excitation matrices have two dimensions, exci-
tation wavelengths (kex) and emission wavelengths (kem),
and provide a type of fluorescence map of all the fluoro-
phores (Locquet et al., 2006).
3.8. Molecular dynamics simulation
At the end of 20 ns MD simulation, some analyzes were per-
formed on the trajectory file of pure HSA, to ensure that it
has reached to the equilibrium state in a semi-physiological
condition. Simultaneous analyzes were also implemented on
HSA-Cafaminol simulated complex, to assess the induced
structural and conformational changes on HSA molecule, due
to the complex formation.
In the three following subsections, analysis of root mean
square deviation (RMSD), radius of gyration (Rg) and root
mean square fluctuations (RMSF) for both pure HSA and
The contour map provides bird’s eye view of 3D fluores-
cence spectra. Shown in Figure 10, peaks a and b are,
respectively, the Rayleigh scattering peak (kex ¼ kem) and the
ꢄ
second-order scattering peak (kex ¼ 2kem), due to the p–p
transition of the characteristic polypeptide backbone struc-
ture C ¼ O of HSA (Pan et al., 2011).
Two other peaks are also observed in 3D fluorescence
spectra of protein, Peak 1 indicates the spectral characteris-
tics of Trp and Tyr residues, while peak 2 is due to excitation HSA-Cafaminol complex will be discussed.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
contributes the most to a molecular motion. In this study,
RMSF was calculated using Eq. (15), to get an estimation of
the ith particle’s position from its reference position
(Moeinpour et al., 2016).
3.8.1. Root mean square deviation (RMSD)
Root mean square deviation is a common method to deter-
mine the stability of a system (e.g. backbone of a protein)
and provides quantitative measure of the similarity between
two superimposed atomic coordinates. The value of RMSD
between two sets of coordinates, from two time points of
simulation trajectory, is a measure of how much the protein
conformation has been changed.
This property is calculated through the following equa-
tion, where N is the number of atoms, and ui and ꢀi are the
initial and final atomic points, respectively.
2
3
1=2
T
X
1
T
2
4
5
RMSFi ¼
jjriðtjÞꢀriref jj
(15)
tj¼1
where T is the time over which one wants to take average
and riref is the reference position of particle i. Generally this
reference position will be the time-averaged position of the
same particle i, i.e. riref ¼ ri:
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u
N
u X
1
Inspection of Figure 11(C) reveals that most regions of
protein are stabilized after binding to Cafaminol, showing
lower RMSF values for protein atoms. Greater fluctuations of
the protein’s terminal residues in both free and complex
form are due to their ability to move more freely.
t
ð Þ
RMSD t ¼
ðui ꢀ ꢀiÞ
(12)
N
i¼1
As depicted in Figure 11(A), the RMSD value of C-a atoms
of free HSA, first rises from 0.2 to about 0.4 nm, and then
oscillates around the average value of 0.43 nm, mainly after
9 ns of the simulation time. This evidence confirms the estab-
lishment of quasi-equilibrium state for HSA.
3.8.4. Molecular docking
In general, spectroscopic studies cannot provide information
on binding at the atomic level, so molecular modeling stud-
ies are employed as complementary approach to obtain
microscopic information on the interactions between small
molecules and receptor. Molecular docking is able to predict
the preferred orientation and binding position of one mol-
ecule to the second, in order to form a stable complex.
In the present study, docking of Cafaminol into the equili-
In the case of HSA-Cafaminol complex, backbone RMSD
shows lower oscillations and increases from 0.13 to about
0.28 nm, where it remains almost constant afterwards. This is
an indication of higher protein’s backbone stability, in a
complex form.
3.8.2. Radius of gyration (Rg)
Radius of gyration which is another parameter for describing brated structure of HSA (obtained from MD simulation at
310 K) was implemented with the Auto Dock vina (Trott &
Olson, 2010). This program needs both the receptor and lig-
and files in the PDBQT format, so, the 3D coordinate file of
receptor (HSA) was saved into this format. In order to obtain
the energy minimized structure of Cafaminol, Gaussian 09
was employed at the B3LYP level of theory, along with the
6–31 þþ g(2d) basis set (Frisch et al., 2003). Center of the
docking grid was set to x ¼ 49.34, y ¼ 53.25 and z ¼ 66.46,
with the maximum spacing of 1 Å, where the grid-size was
kept at 90 ꢁ 76 ꢁ 90 Å, to cover the entire receptor molecule.
Auto Dock vina yielded nine most stable conformations,
arranged in the order of increasing binding energies, while
the conformation with the lowest binding energy was
chosen for further analysis. The best energy-ranked result of
the interaction between Cafaminol and HSA in all runs of
docking procedure is shown in Figure 12.
In addition, docking results indicated that Cafaminol inter-
acts with HSA with a binding energy (DG) of ꢀ6.5 kcal
molꢀ1, while the fluorescence data revealed that Cafaminol
prefers to fit in the area between sub-domains IIA and IIIB,
with DG of ꢀ4.48 kcal molꢀ1. In fact, our in silico obtained
results are in acceptable accordance with our experimen-
tal findings.
the equilibrium conformation of protein is an indicator of its
structure compactness. A stably folded protein will maintain
a relatively steady value of Rg, but if a protein unfolds, its Rg
will change over time (Lobanov et al., 2008b)
Rg is calculated in two steps: First, the center of mass (RC)
coordinates are determined using Eq. (13), while the hydro-
gen atoms are neglected:
X
miðri ꢀ RCÞ ¼ 0
(13)
mi and ri are the mass and position vector of the ith atom,
respectively.
Then, considering the atoms as points in a 3D space, Rg
is obtained from:
X
2
R2g ¼
miðriꢀRCÞ =M
(14)
where M is the total mass of protein atoms (Lobanov
et al., 2008a).
As indicated in Figure 11(B), Rg values for both HSA and
HSA-Cafaminol complex reach a plateau after about 9 ns,
which is another clue for achieving the quasi equilibrium
state. Lower Rg values for HSA in a complex form is due to
the conformational changes induced by the Cafaminol, lead-
ing to a little more compact structure.
Later, the LIGPLOT (A program for automatically plotting
protein ligand interactions) (Wallace et al., 1995) was
employed to analyze the HSA and Cafaminol interactions.
This program was used to specify the interactions mediated
by hydrogen bonds and hydrophobic contacts (Figure 12B).
3.8.3. Root mean square fluctuations (RMSF)
Root mean square fluctuations (RMSF) is a numerical meas-
urement of individual atom or residue flexibility (dynamism),
or how much it moves (fluctuates) during a simulation. Hydrogen bonds are indicated by dashed lines between the
Therefore one can determine which aminoacid in a protein atoms involved, while hydrophobic contacts are represented

10
N. PARVIZI ET AL.
Figure 11. (A) RMSD of the HSA backbone in the presence and absence of Cafaminol, at 310 K. (B) Time evolution of the radius of gyration (Rg) during 20 ns of
MD simulation for HSA and HSA-Cafaminol complex. (C) Root mean square fluctuations over atoms for HSA, in both free and complex form.
by an arc with spokes, radiating towards the ligand atoms spectroscopy methods, and molecular dynamics and docking
simulations.
In total, it was determined that Cafaminol binds to the
they contact. Contacted atoms are shown with spokes radiat-
ing back.
site I (Between sub-domains IIA and IIIB) of HSA, in an exo-
thermic and enthalpy driven process.
Docking results showed that Cafaminol mainly interacts
with HSA, through hydrogen bonding, which agrees well
with the thermodynamic analysis outcome.
As shown in Figure 12, interaction of Cafaminol and HSA
occurs in the area between sub-domains IIA and IIIB.
Hydrogen bonds decrease the hydrophilicity, increase the
Analysis of the Stern-Volmer plots revealed that Cafaminol
quenches the fluorescence of HSA through the static
quenching mechanism, due to the drug-protein complex for-
mation. UV-Visible absorption spectra of HSA showed an
obvious intensity increase, along with a slight red shift, due
to the Cafaminol addition. These findings support the idea of
Cafaminol-HSA complex formation, accompanied by the
induced microenvironmental perturbations around the pro-
tein’s chromophore.
Analysis of the circular dichroism (CD) spectra of HSA in
both free and complex forms demonstrated the decrease of
protein’s a-helical content, due to the complex formation.
Both negative DH and DS values confirmed that the van
der Waals and hydrogen bonding interactions stabilize the
hydrophobicity,
and
thus
stabilize
the
HSA-
Cafaminol complex.
4. Conclusion
Since albumin serves as a drug carrier, studying its inter-
action with different drugs is of importance in pharmacy,
pharmacology and biochemistry. In the present study, the
interaction of Cafaminol with the human serum albumin
(HSA) was investigated through the various molecular Cafaminol in the HSA binding domain. Moderate binding

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
Figure 12. (A) Molecular docking pose of Cafaminol in HSA. (B) Cafaminol docked in the binding pocket of HSA. (C) Two-dimensional schematic representation of
hydrogen bonding and hydrophobic interactions. Figure was plotted using the program LIGPLOT.
Chen, G. Z., Huang, X. Z., Xu, J. G., Zheng, Z. Z., & Wang, Z. B. (1990). The
affinity between Cafaminol and HSA is benefited for the effi-
cient transport of drug, and its subsequent release, at the
target site. Overall results of molecular dynamics and dock-
ing simulations indicated the binding site of Cafaminol and
methods of fluorescence analysis (2nd ed., pp. 112-145). Beijing:
Science Press.
Chen, Y.-H., Yang, J. T., & Martinez, H. M. (1972). Determination of the second-
ary structures of proteins by circular dichroism and optical rotatory disper-
sion. Biochemistry, 11(22), 4120–4131. https://doi.org/10.1021/bi00772a015
made it possible to designate the main aminoacids involved
in the drug-protein interactions.
Du, X., Li, Y., Xia, Y.-L., Ai, S.-M., Liang, J., Sang, P., Ji, X.-L., & Liu, S.-Q.
(2016). Insights into protein–ligand interactions: Mechanisms, models,
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