A spectroscopic and molecular docking study of interactions of tetracarboxyphenyl porphyrin and chlorin e6 with bovine serum albumin

Article abstract of DOI:10.1007/s11696-021-01670-3

Abstract: In order to use tetrapyrrolic macrocyclic species in medical applications, the interactions of bovine serum albumin (BSA) with tetrakis-para-carboxyphenyl-porphyrin (H₂T(p-COOH) PP or TCPP) and Chlorin e6 (Ce6), under different conditions, were investigated by fluorescence spectroscopy and UV–VIS absorption spectroscopy and contrasted with molecular docking study. The binding constant (K_b) values at three different temperatures were calculated using the modified Stern–Volmer equation. The enthalpy change (ΔH) and entropy change (ΔS) were determined based on the van’t Hoff equation. The results of fluorescence spectroscopy indicate that static quenching is the dominant process resulting from the BSA-TCPP complex formation. In the case of the BSA-Ce6 complex, static quenching was confirmed too. Thermodynamic analysis indicated that hydrogen bonds and van der Waals interactions were the predominant intermolecular forces in the binding process to stabilize the BSA-TCPP and BSA-Ce6 complexes. Molecular docking suggests that the probable binding site of the two macrocyclic species at BSA occurs in the vicinity of the Trp-134 residue. Molecular modeling study further confirmed interactions in the binding mode obtained experimentally. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11696-021-01670-3

Chemical Papers
https://doi.org/10.1007/s11696-021-01670-3
ORIGINAL PAPER
A spectroscopic and molecular docking study of interactions
of tetracarboxyphenyl porphyrin and chlorin e6 with bovine serum
albumin
Jonathan Osiris Vicente‑Escobar1 · Miguel A. García‑Sánchez1 · F. González2 · S. Cipagauta‑Díaz3 ·
A. Estrella González1
Received: 6 December 2020 / Accepted: 20 April 2021
© Institute of Chemistry, Slovak Academy of Sciences 2021
Abstract
In order to use tetrapyrrolic macrocyclic species in medical applications, the interactions of bovine serum albumin (BSA)
with tetrakis-para-carboxyphenyl-porphyrin (H2T(p-COOH) PP or TCPP) and Chlorin e6 (Ce6), under diferent conditions,
were investigated by fuorescence spectroscopy and UV–VIS absorption spectroscopy and contrasted with molecular dock-
ing study. The binding constant (Kb) values at three diferent temperatures were calculated using the modifed Stern–Volmer
equation. The enthalpy change (ΔH) and entropy change (ΔS) were determined based on the van’t Hof equation. The results
of fuorescence spectroscopy indicate that static quenching is the dominant process resulting from the BSA-TCPP complex
formation. In the case of the BSA-Ce6 complex, static quenching was confrmed too. Thermodynamic analysis indicated that
hydrogen bonds and van der Waals interactions were the predominant intermolecular forces in the binding process to stabilize
the BSA-TCPP and BSA-Ce6 complexes. Molecular docking suggests that the probable binding site of the two macrocyclic
species at BSA occurs in the vicinity of the Trp-134 residue. Molecular modeling study further confrmed interactions in
the binding mode obtained experimentally.
* Miguel A. García-Sánchez
mags@xanum.uam.mx
1
Departmento de Química, Universidad Autónoma
Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186,
C.P. 09340 Ciudad de México, México
2
Departamento de Ingeniería de Procesos E Hidráulica,
Universidad Autónoma Metropolitana-Iztapalapa, Av. San
Rafael Atlixco No. 186, C.P. 09340 Ciudad de México,
México
3
Departamento de Química, Catedra CONACyT, Universidad
Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco
No. 186, C.P. 09340 Ciudad de México, México
Vol.:(0123456789)
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Graphic abstract
Keywords Chlorin e6 · Tetracarboxyphenyl porphyrin · Bovine serum albumin · Molecular docking
binding site for compounds such as bilirubin or hemin (Ghu-
man et al. 2011). Generally, weak interactions cause a short
lifetime or low distribution of the complexes formed, while
a higher binding force decreases the free drug concentra-
tion in plasma (Samari et al. 2012). In this regard, several
studies in this area can provide information on the structural
characteristics that determine the therapeutic efectiveness of
drugs, standardize drug scanning processes, and limit dose
setting (González 2017; Busch 2009).
Introduction
Quantitative as well as qualitative descriptions of biological
processes are some of the main areas of research in the life
sciences, such as physiology, biochemistry and cell biology.
For example, molecular recognition between proteins and
ligands through non-covalent associations plays a fundamen-
tal role in virtually all biological processes (Williams 2013).
An application category where it acquires great relevance
is the search and development of new pharmaceutical com-
pounds, which in their later stages are focused on the study
of pharmacokinetic parameters such as absorption or endog-
enous distribution. The latter is mediated by plasma proteins,
thus infuencing the concentration of free drug molecules
that reach target cells (Dmitry et al. 2020). Most drugs are
transported to the target site (target), either by human serum
albumins (HSA) or low-density lipoproteins (Karsima and
Mochizuki 1999; Chakraborty and Basu 2010).
In particular, bovine serum albumin (BSA) is a probe pro-
tein for interaction studies of both exogenous and endoge-
nous molecules. BSA consists of a chain of 583 amino acids
with an α-helix structure and a weight of 66 kDa. Struc-
turally, there are nine loops that connect three homologous
domains, consisting of domains I (1–195), II (196–383), and
III (384–585) (Peters 1995; Jamie 2005). The BSA has six
ligand binding sites (Jacobson et al. 2019). Specifcally, in
the subdomain IA (site III), there is tryptophan 134 (Trp-
134), frequently used to monitor drug binding through fuo-
rescence spectroscopy techniques because in the presence
of molecules interacting with BSA, signifcant fuorescence
emission modifcations can occur (Peters 1999).
Albumins are multifunctional proteins that have an
extraordinary capacity for establishing reversible binding
with a great number of ligands (Majorek et al. 2012). The
binding capacity of albumin varies widely for different
ligands. Although there are up to seven long-chain fatty acid
binding sites in this molecule, there is only one principal
In this sense, the study of the non-covalent interactions
between protein-ligands is of paramount importance in
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selecting and formulating new therapeutic agents, such as
photosensitizing agents for use in photodynamic therapy
(PDT) (Kenneth 1999; Klebe 2015). Porphyrins, whether
synthetic or natural derivatives, and phthalocyanines are two
of the most efcient and common photosensitizers used in
PDT studies. It is because these tetrapyrrolic macrocycles
have very important intrinsic characteristics such as strong
absorption in the visible region of the electromagnetic spec-
trum, long halftime in the triplet-excited state, and efcient
phototoxicity to cancer cells (Sunaina et al. 2015). Studies
of the interactions between porphyrin derivatives with car-
rier proteins such as BSA have focused mainly on the dis-
covering or developing new photosensitizing agents. Thus,
the possible associations of various structural derivatives of
porphyrins with BSA have been studied, such as glycocon-
jugates (Obata Makoto et al. 2009), tetraphenylporphyrins
(Lebedeva et al. 2019; Ping et al. 2012), metalloporphyrins
(Klein et al. 2020), and lanthanoid porphyrins (Lu et al.
2009). These studies have provided very interesting pro-
posals for the search of possible photosensitizing agents for
use in PDT.
Experimental
Materials
All chemicals were of analytical grade and used as received
without further purifcation. All solvents and all solid rea-
gents employed in this work were obtained from Sigma-
Aldrich (St. Louis, USA). Phosphate bufer solution (PBS)
was made from K2HPO4, KH2PO4. All tests were carried
out using ultrapure water.
Synthesis or extraction of macrocycles
The TCPP was synthesized by the Adler method (Adler and
Longo 1964), using pyrrole and terephthaladehydic acid as
a starting reagent. Both reagents are refuxed with an excess
of propionic acid. The synthetical procedure is shown in Fig.
S1. Ce6 extraction and purifcation proceeded according to
the protocol shown in Fig. S2. The extraction of chlorophyll
a was carried out by the Keiji Iriyama method (Iriyama and
Takamiya 1974), followed by saponifcation of chlorophyll
a to produce chlorophyllin a (Geraldo 1964; Iman 2017),
whereas Ce6 was obtained by acid hydrolysis of chlorophyl-
lin. TCPP and Ce6 were dissolved in ethanol and diluted
with phosphate bufer pH 7.
The use of experimental techniques along with compu-
tational tools, such as molecular docking, provides a guide
for a better explanation of all the processes inherent to inter-
molecular interaction in protein–ligand systems (Douglas
and Decornez 2004). Due to the facts mentioned above,
we present herein some results on the BSA interaction
with two tetrapyrrolic macrocyclic species with peripheral
carboxyl groups: tetrakis-para-carboxyphenyl-porphyrin
(H2T(p-COOH)PP or TCPP) and the Chlorin e6 (Ce6), a
derivative of chlorophyll a (Fig. 1). The characterization of
systems was carried out by UV–VIS and fuorescence spec-
troscopy. We also performed molecular coupling studies for
both analyzed systems to contrast them with experimental
results. It was aimed to determine the nature of the principal
interactions to be able to propose a model for the complexes
formed.
Characterization
The UV–VIS spectroscopy measurements were performed
on a Varian Cary 500E spectrophotometer using the conven-
tional quartz cuvettes of 10 mm width. UV–VIS spectra of
BSA solutions were recorded in the absence and presence
of TCPP or Ce6, from 200 to 800 nm at room tempera-
ture. The BSA concentration was kept at 3.2 × 10–6 M. In
each measurement, the blank was the dissolution medium:
ethanol-phosphates 20:80 v/v.
Fig. 1 Structure of TCPP a
and Ce6 b. In schemes red is
oxygen, blue nitrogen and white
is carbon
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Fluorescence quenching study
Molecular docking
The fuorescence spectroscopy measurements were conducted
on an Edinburgh Instruments FLS920 spectrometer. Because
of the nature of the compounds and experimental conditions
used, the inner flter fuorescence efect was considered insig-
nifcant, so no method to avoid or eliminate it was applied
and fuorescence data were used as taken from the instrument.
The concentration of BSA was fxed at 3.2×10–6 M, while
TCPP or Ce6 solutions were modifed from 4.4×10–6 M to
6.7×10–5 M. Fluorescence studies were performed in dupli-
cate at three temperatures (298, 304, and 315 K). The inter-
action monitoring was performed by excitation at 295 nm
wavelength and recording the corresponding spectra in a
range of 300–550 nm. Data analysis was carried out using the
Stern–Volmer graphical method (Mocz 2013). This model was
used to determine, in the frst instance, whether only one type
of quenching is present in Eq. (1): (Lakowicz 2008)
Rigid molecular and fexible molecular docking In order to
understand the possible intermolecular interactions in the
BSA-tetrapyrrolic macrocycles systems, molecular rigid
docking studies were carried out. The binding interac-
tions of TCPP or Ce6 with BSA were analyzed by the rigid
docking method implemented in Autodock Vina software
(Olen and Olsen 2010). The crystallographic structure of
BSA (PDB ID: 3V03) was downloaded from the Protein
Data Bank (http://www.pdb.org/pdb/home/home.do). As
required, all water molecules and co-crystallized ligands
were removed, and hydrogen atoms were added, followed by
the calculation of Gasteiger charges. The optimized three-
dimensional structures of TCPP and Ce6 were obtained
with the Gaussian 9.0 (Frisch et al. 2016) software using the
density functional theory (DFT) and the Becke-3-Lee Yang
Parr (B3LYP) method at level 6-31G(d). All torsion bonds
of both macrocycles were considered free.
F0
A 3D box with a grid spacing of 1 Å and a size of
84 Å×76 Å×81 Å in x, y, and z directions was created to
analyze the entire surface of BSA. The binding site III of the
BSA was analyzed using a grid box with a spacing of 1 Å
and a size of 35 Å×23 Å×30 Å and centered at the coordi-
nates: 37.9 Å, 36.4 Å, and 42.5 Å. Additionally, it was con-
sidered that at pH 7, the carboxyl groups of the TCPP and
Ce6 species remain in a neutral state and that all torsional
bonds were free. For each proposed system, ffty docking
runs were conducted with a population of 100 poses, and
those with the lowest afnity, released by the Autodock Vina
program, were chosen. For the TCPP and Ce6 species, the
afnity was−29.7 kJ/mol and 25.5 kJ/mol, respectively.
= 1 + KSV[L]
(1)
F
where F0 and F are the fluorescence intensities in the
absence and presence of ligand (TCPP or Ce6) at each titra-
tion point, KSV is the Stern–Volmer constant, and [L] is the
total concentration of ligand. To obtain the binding constant,
quenching data from the fuorescence titration were analyzed
through the modifed Stern–Volmer Eq. (2):
F0 − F
log
= log Kb + n log [L]
(2)
F
where F0, F, and L are the same as in Eq. (1). The graph of
log [F0−F]/F] versus log [L] allows determining the values
of n, the number of binding sites from the slope, and Kb, the
binding constant from the intercept of the line.
Flexible molecular docking
To establish the nature of the interactions present in the
analyzed systems, the values of the binding enthalpy change
(ΔHu) and entropy change (ΔSu) were determined using the
van’t Hof model Eq. (3) (James and Murphy 2002):
Flexible molecular docking was performed using the binding
site found by rigid docking studies but considering that both
species, the ligand and the protein are fexible entities during
the docking process. So, to carry out these studies, the pose
with the lowest afnity was considered for each system and
for a series of BSA residues that each species interacted with
at a maximum distance of 5 Å, except for Trp-134. The grid
spacing, volume, and size of the box were the same as in the
BSA site III analysis.
ΔHu ΔSu
+
InKb = −
(3)
RT
R
where Kb is the binding constant at temperature T, and R is
the universal constant of gases. The values of ΔHu and ΔSu
are calculated from the slope and intercept, respectively, of
the straight line for the graph representing ln Kb versus 1/T.
The free energy change (ΔG) can be estimated from Eq. (4):
Results and discussion
ΔG = ΔH − TΔS.
(4)
UV–VIS and fuorescence spectroscopy
Figure 2a shows a characteristic UV–VIS spectrum of free-
based porphyrin monomers, whose Soret band is located at
416 nm, and the four Q bands in the range of 513–652 nm.
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Fig. 2 a UV–VIS absorption spectra and b fuorescence spectra (λexc: 280 and 419 nm) of TCPP (at pH 7; 298 K; solution ethanol-phosphate)
It should be mentioned that the predominantly aqueous envi-
ronment does not infuence the aggregation of TCPP. This
molecule was also characterized by fuorescence spectros-
copy considering prior interaction studies with BSA at two
diferent excitation lengths: 280 and 419 nm, corresponding
to protein absorption and maximum absorption of tetrap-
henylporphyrin derivative, respectively. The spectrum of
Fig. 2b shows a strong fuorescence emission at 640 nm for
both excitation lengths and the presence of a lower peak at
around 550 nm when porphyrin is excited at 280 nm, i.e
., there is a large separation between the excitation and
absorption maxima. However, TCPP emission interference
is not possible because BSA displays fuorescence bands at
320–350 nm.
structural presence of three free carboxylic groups, which,
in addition to improving solubility, also promote intermo-
lecular interactions, such as hydrogen bonds that are of great
interest in protein–ligand interaction studies.
The UV–VIS spectrum (Fig. 3a) of the Ce6 ethanoic solu-
tion is very similar to that of chlorophyllin a. The Soret
band is slightly shifted to 402 nm, and the Q band exhib-
its a hypsochromic shift to 664 nm, while the absorption
spectrum is unchanged in ethanol-phosphates solution pH
7. This indicates that no aggregation phenomenon occurs in
this dissolution medium. Ce6 emission spectrum (Fig. 3b)
consists of an intense emission band located at 623 nm when
an excitation length of 402 nm is used. Similarly, as occurs
in the case of TCCP, there are large separations between
the excitation and absorption maxima for Ce6. Furthermore,
the system’s spectroscopic properties were preserved, which
suggests that chlorin does not show evidence of aggregation
Ce6 is poorly soluble in water compared to chlorophyllin
a, but highly soluble in alcohols and therefore in aqueous
solutions. The great importance of this derivative lies in the
Fig. 3 a UV–VIS absorption spectra and b fuorescence spectra (λexc: 290 and 402 nm) of Ce6 (at pH 7; 298 K; solution ethanol-phosphates)
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or protonation in the ethanol-phosphate medium, thus being
an interesting and relevant candidate for the interaction anal-
ysis. For the purpose of conducting these interaction studies
with BSA and discovering whether there is an interference
of Ce6 emissions with respect to BSA, its emission spectrum
was also recorded using excitation with light at a wavelength
of 295 nm. As a result, no signifcant emission was observed
in the range of 320–350 nm, the region in which it could
interfere with the BSA maximum emission.
Spectrofuorometric interaction studies
The quenching fuorescence technique was used to inves-
tigate the possible binding mechanism of TCPP or Ce6 to
BSA. To determine this binding constant, studies were per-
formed at pH 7 and three diferent temperatures (298, 304,
and 315 K). The intrinsic fuorescence of BSA is mainly
related to tryptophan (Trp), tyrosine (Tyr), and phenylala-
nine (Phe) residues, providing a good comprehension of the
binding interaction of BSA with proteins. The Trp residue is
responsible for the strongest fuorescence emission, whose
position and intensity are more sensitive to changes in the
microenvironment, indicating conformational changes after
binding with proteins. BSA contains two tryptophan residues
(Trp-134 and Trp-213); Trp-134 is located on the surface
and Trp-213 inside the molecule, but both mainly contribute
to the intrinsic fuorescence of BSA (Wu and Cheng 2017).
Tryptophan emission changes are common in response to
conformational transitions, binding to a substrate, or protein
denaturation. Excitation at 295 nm allows detecting only
the emission of Trp residues, while at the wavelength of
280 nm, the excitation of both Trp and Tyr can be observed
in proteins (Wang 2013). Since the BSA fuorescence emis-
sion is attributed almost entirely to the Trp-134 residue and
to a minimal extent to Trp-213 (Guha and Mitra 1999), the
monitoring of BSA quenching was performed at the excita-
tion length of 295 nm (λexc).
Interaction studies
To investigate the interactions of the BSA-TCPP and BSA-
Ce6 systems, studies were performed at pH 7. Due to the
low solubility of both TCPP and Ce6 in water, they were
dissolved in an ethanol-phosphate mixture 20:80 v/v, as the
maximum presence of 2% v/v ethanol in titration solutions
does not modify the structure and stability of BSA (Hiroki
and Tsutomu 2012). Spectrophotometric studies (Fig. 4a,
b) show a general trend of increased absorption at 280 nm,
related to increased ligand concentrations. It indicates a pos-
sible change in the microenvironment in the proximity of
BSA aromatic residues (since these are responsible for the
characteristic absorption in the UV–visible region of pro-
teins). First, an approximation to the Trp-134 residue can
be assumed to take place since it is exposed on the surface
of BSA. Figure 4a, b shows an increase and concomitant
hypsochromic shift of the absorption bands from 422 to
416 nm and 418 to 406 nm for the BSA-TCPP and BSA-
Ce6 systems, respectively. This phenomenon may be due to
the free concentration of ligands, which could be associated
with the quantity of TCPP or Ce6 molecules not interacting
with BSA. Therefore, the absorption of the Soret band is
observed at shorter wavelengths.
Figure 5a, b exhibits the fuorescence spectra of BSA-
TCPP and BSA-Ce6 systems, respectively. A progressive
decrease in the BSA fuorescence emission at 329 nm is due
to the increase in the concentration of both TCPP and Ce6,
suggesting the proximity of these species to the Trp residue
of BSA near the binding site. These changes indicated that
TCPP or Ce6 could quench the inner fuorescence of BSA
Fig. 4 UV–VIS absorbance spectra of titration of a BSA-TCPP and b BSA-Ce6 in (at pH 7; T=298 K)
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Fig. 5 Fluorescence spectra of titration of a BSA-TCPP and b BSA-Ce6 (at pH 7; T=298 K)
and clearly suggest the binding of BSA to TCPP or Ce6 and
the interactions between TCPP or Ce6 and BSA. Moreover,
an isosbestic point, close to 450 nm, is observed for the
TCPP titration case, indicating the presence of two species
in equilibrium, which could be the complex and the free pro-
tein, or the binding state and the free state. Additionally, no
displacement of the maximum BSA emission was observed
during the titration process, which suggests that there is no
change in the three-dimensional structure of BSA during the
process of interaction with both ligands.
Stern–Volmer analysis
BSA‑TCPP
Fluorescence quenching is associated with a decrease in the
Fig. 6 Stern–Volmer plot of BSA spectrofuorometric titration with
quantum performance (fuorescence intensity) of the fuoro-
phore (BSA), but this can be induced by diverse molecular
interactions with deactivated species (TCPP). In order to
confrm the nature of the quenching phenomenon occurring
in the BSA-TCPP system, either dynamic (due to intermo-
lecular contact between the protein and the ligand) or static
(formation of a complex) fuorometric titration experiments
were performed at 298, 304, and 315 K. Certain fuorescence
quenching reactions generate Stern–Volmer graphs that
diverge from linearity with either positive or negative cur-
vatures (Roy and Mukherje 1987). As observed in Fig. 6, the
BSA-TCPP titration at 298 K shows a slight deviation with
negative curvature of the Stern–Volmer graph (Miguel and
Manuel 1988). The tendency of F0/F versus [TCPP] graph
TCPP (at pH 7; solution in ethanol-phosphate)
of fuorophores (Trp-134 and Trp-213), one of them being
inaccessible to deactivation.
The Trp-213 residue (Fig. 7) is located in the hydrophobic
cavity of BSA (domain II), which is not accessible to the sol-
vent. Additionally, the cavity aperture is around 10 Å in size
(Ghuman et al. 2005), which would severely restrict access
to porphyrin, and induces steric impediment that would
not allow an efective contact between TCPP and the Trp-
213 residue. This phenomenon is also frequently observed
in the fuorescence quenching of proteinic tryptophan by
polar deactivator species or those with dipolar, partial, or
formal charge regions or groups, such as carboxylic groups
(–COOH) of TCPP. Because these and other molecules do
not easily penetrate the protein’s interior, only the fuores-
cence of tryptophan residues on the protein surface under-
goes quenching (Halina et al. 2007).
is not linear in the concentration range from 0.65×10–6
M
to 6.98×10–6 M. These events cannot be attributed to con-
ventional static quenching, that is, the one that establishes
the formation of a non-fuorescent complex. However, they
can be attributed to the presence of two diferent species
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process, attributable to the formation of a non-fuorescent
BSA-TCCP complex.
BSA‑Ce6
In the respective Stern–Volmer graph of the BSA-Ce6 sys-
tem, a major degree of linearity over the entire concentra-
tion range was observed when titration data were adjusted
to diferent temperatures (Fig. 8b). This result suggests that
only one type of quenching is present. However, the KSV
constants (Table 1) are one order of magnitude smaller com-
pared to TCPP, suggesting lower quenching efectiveness,
that is, a higher concentration of Ce6 is required to observe
similar efects.
At pH less than 5, the Ce6 form aggregates (Halina et al.
2007); however, in the pH range studied, several ionized spe-
cies may exist in equilibrium, as the carboxylic acid groups
on the macrocycle periphery and pyrrolic nitrogen (N–H)
may become mainly depleted. For practical purposes, it is
assumed that, at pH 7, there must be a similar concentration
Fig. 7 BSA structure and location of the two Trp residues
However, a linear relationship can be observed when the
total TCPP concentration is less than 2.98× 10–6 M; con-
sequently, the data can be adjusted to the Stern–Volmer
equation in this concentration range (Fig. 8a). At this point,
we clarify that no method of elimination was applied to
eliminate the inner flter efect because the wavelength of
absorption and excitation and the concentration and width
of the sample cell allowed us to consider it insignifcant at
the experimental conditions used.
Table 1 Stern–Volmer quenching constants KSV for the binding of
BSA to TCPP and Ce6 at diferent temperatures
System
pH
7
T (K)
KSV (×10–5 mol L−1
)
R2
BSA-TCPP
298
304
315
298
304
315
1.35 0.28
0.48 0.03
0.41 0.02
4.38 0.18
3.71 0.21
3.12 0.40
0.9845
0.9878
0.9818
0.9893
0.9895
0.9907
In all cases, fuorescence data show that KSV decreases
with rising temperature, indicating that the fuorescence
quenching between BSA and TCPP is a static quenching
BSA-Ce6
7
Fig. 8 Stern–Volmer plots for the fuorescence quenching of a BSA-TCPP and b BSA-Ce6 at diferent temperatures
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Table 2 Binding parameters for the BSA-TCPP and BSA-Ce6 system
at diferent temperatures
of dissociated species, such as those with the –COO– ani-
ons. The progressive reduction of KSV is indicative of static
quenching. Compared with TCPP species, the efective-
ness of Ce6 quenching on BSA is lower. As was previously
mentioned, the nature of BSA quenching observed is static,
making it possible to determine the binding constant and the
number of binding sites through the modifed Stern–Volmer
equation.
System
pH T (K)
n
Kb (×106 mol L−1
)
R2
BSA-TCPP
7
7
298
304
315
298
304
315
1.26 1.53 1.23
1.00 0.49 0.01
0.39 0.26 0.01
0.94 2.89 0.02
0.81 2.81 0.02
0.77 0.42 0.03
0.9862
0.9946
0.9887
0.9917
0.9803
0.9831
BSA-Ce6
Determination of binding constant Kb and number
of binding sites n for interaction of BSA with TCPP
and Ce6
Bearing in mind a non-fuorescent BSA-Ce6 complex,
the Kb and n binding parameters were obtained from the
Stern–Volmer double logarithm graph (Fig. 9b). The bind-
ing constant for Ce6 is two orders of magnitude lower
than for TCPP (Table 2). In the same way that the value
of binding constants as a function of temperature was
found to diminish, the value of binding sites n was found
to decrease as well. However, based on the dissolution
values of 0.77–0.90 at pH 7 and temperatures of 298 and
304 K, respectively, a 1:1 binding stoichiometry between
BSA and Ce6 was established. These results are consistent
with the number of sites permissible for BSA in biologi-
cal structures and porphyrinic molecules. Because of the
reduction in the binding constant value at a temperature
of 315 K, it was assumed that a signifcant dissociation of
the BSA-complex takes place. The Kb values around 104
at temperatures of 298 and 304 K are within the moderate
range of interactions.
For static quenching interactions (assuming that all bind-
ing sites are equal and independent in BSA), the modifed
Stern–Volmer equation was used to achieve information on
the binding constant between BSA and TCPP or Ce6. Plots
of log [(F0 −F)/F] versus log [L] at 298, 304, and 315 K for
the TCPP and Ce6 systems are given in Fig. 9a, b.
Results of n and Kb values, calculated from the slope and
the intercept of Eq. (2), are shown in Table 2. It was found
that the BSA-TCPP binding stoichiometry was 1:1 since
the number of binding sites fuctuated slightly above 1.0
at both mentioned temperatures, which is indicative of the
interaction occurring between one molecule of BSA and one
molecule of TCPP at these temperatures. For the titration at
315 K, it was observed that both the number of binding sites
and the Kb were signifcantly reduced, indicating a signif-
cant complex dissociation at this temperature (Table 2). At
298 K, the complex formed is thermodynamically stable,
and the order of magnitude at pH 7 is in the range of strong
interactions.
Fig. 9 Modifed Stern–Volmer plots for a BSA-TCPP and bBSA-Ce6 b systems at three diferent temperatures. Inset: van’t Hof plot for a BSA-
TCPP and b BSA-Ce6
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Waals forces play major roles in the BSA-TCPP and BSA-
Ce6 binding reactions and contribute to the stability of the
complexes.
Thermodynamic analysis of interaction
between BSA‑TCPP and BSA‑Ce6
In order to obtain more details of the acting forces between
the BSA and both ligands, the temperature dependence on
thermodynamic variables was examined. Generally, small
molecules are bound to proteins through four binding
modes: hydrogen bonding, van der Waals, electrostatic, and
hydrophobic interactions. The sign and magnitude of ther-
modynamic parameters, such as ΔH (enthalpy change) and
ΔS (entropy change) for protein–ligand interactions, reveal
details on the formation and stability of a complex (Yan
et al. 2010). Ross and Subramanian (Ross and Subrama-
nian 1981) have characterized the sign and magnitude of
thermodynamic parameters associated with various kinds of
interactions that may take place in protein binding processes:
(1) ΔH > 0 and ΔS > 0 were associated with hydrophobic
forces; (2) ΔH<0 and ΔS<0 were associated with van der
Waals interactions and hydrogen bonds, and (3) ΔH<0 and
ΔS>0 with electrostatic interactions.
The computational approach, molecular docking
studies
Experimental analyses combined with molecular docking
simulations of protein–ligand interaction studies provide a
better understanding of the type of intermolecular interac-
tions that govern such processes. The in silico studies of the
BSA-TCPP and BSA-Ce6 systems were carried out consid-
ering the entire protein surface. However, exhaustive moni-
toring was carried out at the BSA site III (Shen et al. 2015),
because this is the site of binding to endogenous ligands
structurally related to the porphyrin nucleus, such as hemin,
and bilirubin. Based on the BSA fuorescence quenching
observed in experimental studies, monitoring focused on
the vicinity of the Trp-134 residue was also carried out.
The energy range obtained for the BSA-TCPP system was
from−29.7 to−21.3 kJ/mol.
In the present study, ΔH and ΔS values were calculated
according to van’t Hof Eq. (3). The inset in Fig. 9a, b,
depicts the van’t Hof plots used for analysis of the interac-
tion modes between BSA with TCPP and Ce6.
BSA‑TCPP and BSA‑Ce6
In order to analyze the nature of the coupling and interac-
tions between the species, we chose a pair of representative
poses for the macrocyclic species TCPP or Ce6 (Figs. 10a
and 11a). The chosen conformations were lower energy con-
formations, determined with the Autodock Vina program in
the proximity of the Trp-134 residue. Intermolecular interac-
tions in BSA are mainly allowed in a hydrophobic environ-
ment, mostly the hydrogen bond interactions and, to a lesser
extent, electrostatic interactions (Rola and Helene 2017).
However, due to the existence of the resonant π-bond system
in the porphyrinic macrocycle, this species is predominantly
hydrophobic and can establish nonpolar interactions, which
may be the forces that most contribute to the stabilization
of the formed complex. In addition, this species has at least
three carboxyl groups (–COOH), which are able to establish
hydrogen bonds, dipole–dipole and dipole–dipole induced
interactions. Then, the coupling can result from all these
interactions, especially those of hydrophobic nature and
hydrogen bonding. The area surrounding the Trp- 134 resi-
due is nonpolar; however, there are a few polar amino acids
such as lysine (Lys), glutamine (Glu), and glycine (Gly),
which can increase the energy of the interactions, inducing
a stabilizing polar environment for the complex formation.
Figure 10b reveals the formation of two hydrogen bonds
of carboxyl groups with the Asn-42 and Glu-14 residues
for the BSA-TCPP system. In addition, polar residues such
as lysine and glutamine can promote interactions with the
carboxyl groups of porphyrins (Fig. 10c). Although there is
no direct interaction with Trp-134, the distances to observe
Thermodynamic parameters of BSA-ligand complexes
are listed in Table 3. The negative values obtained for both
ΔH and ΔS indicate that the forces involved in complexes
BSA-Ce6 and BSA-TCPP result from a combination of van
der Waals and hydrogen bonds’ interactions. The negative
sign for ΔG° values determined in the case of both ligands
used (TCPP and Ce6) demonstrates the existence of spon-
taneous interactions. Based on the obtained data, it can be
suggested that tetraphenyl porphyrin substituents establish a
larger number of interactions or provide a greater contribu-
tion to the association with BSA if compared to Ce6. The
negative values of both ΔH and ΔS parameters show that the
interaction between BSA-TCPP is enthalpically driven and
not entropically favorable. For BSA-Ce6 complex, similar
results are found. Finally, the thermodynamic results sum-
marized above indicate that hydrogen-bonding and van der
Table 3 Thermodynamic parameters for binding of BSA to TCPP
and Ce6
System
pH Tem-
ΔH (kJ/mol) ΔS (J/mol
K)
*ΔG (kJ/
mol)
perature
(K)
BSA- TCPP
BSA-Ce6
7
7
298
304
315
298
304
315
−789.9
−98.84
−2505.9
−242.7
−41.57
−33.11
−23.50
−25.45
−25.89
−23.51
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Chemical Papers
Fig. 10 a Lower energy poses
close to the Trp-134 residue,
obtained with Autodock Vina
program, b interaction map
between BSA and TCPP, the
hydrogen bond was added by
Maestro program (Schrödinger
2020-2), c determination of dis-
tances and types of interaction
by BSA- TCPP system, using
Ligplot program (Amber and
David 2014)
Fig. 11 a Lower energy poses
close to the Trp-134 residue,
obtained with Autodock Vina
program, b interaction map
between BSA and Ce6, the
hydrogen bond was added by
Maestro program, (Schrödinger
2020-2) c determination of
distances and types of interac-
tion by BSA- Ce6 system, using
Ligplot program (Amber and
David 2014)
quenching of tryptophan are larger than 15 Å (ligand-fuo-
rophore) (Amber and David 2014). Trp-134 appears in the
proximity of macrocyclic species in Fig. 9b but cannot be
observed in the diagram of Fig. 10c; then, the quenching
phenomenon is feasible to occur. Similarly, a correlation
between experimental evidence and the models coming from
the molecular docking could exist. The Trp -134 residue
appears to maintain a weak interaction between the phenyl
ring of tryptophan and the aromatic central region of the
porphyrin macrocycle. The above-mentioned facts clearly
show that there is a correlation between the experimental
data and the models simulated through molecular docking.
For the BSA-Ce6 system, the size of peripheral sub-
stituents with carboxyl groups of Ce6 species seems to be
more accessible to the cavity where the Trp-134 residue
is found (Fig. 11b). The porphyrinic nucleus of the Ce6
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Chemical Papers
Table 4 Distance, binding mode between ligands and amino acids
involved in the interaction, obtained from molecular docking simula-
tion
Flexible molecular docking
In the case of the BSA-TCPP systems, the fexible dock-
ing analysis reveals that the carboxyl group (−COOH) of
Glu-15 points to the center of TCPP, whereas the hydroxyl
group (−OH) of Tyr-153 and the amino or amido (−N−H
or−CON=) groups of Lys-157, Phe-124 and Glu-128 resi-
dues point to two of the closer carboxyl (-COOH) groups
of the macrocycle. However, the free bond rotation of the
amino acid residues allows a conformation closer to the
macrocycle, principally Trp-134, which appears at 3.2 Å of
separation from the phenyls of the TCPP species, contrast-
ing strongly with 9.1 Å determined with the rigid docking
(Fig. 12). This distance is sufcient to consider that energy
transfer between aromatic systems is possible. As the rigid
molecular docking suggests, this residue remains at a dis-
tance of about 9.1 Å, possibly as a result of the steric efect
with nonpolar regions of His-16 and Tyr-153 residues. How-
ever, fexible molecular coupling results suggest that the
species evolved adopts a conformation that allows a closer
approach to Trp-134. All the information mentioned above
suggests that, because of the inherent size and structure of
the TCPP macrocycle, its approach to the active site could
occur pointing the two carboxylic groups to nearer amine,
amide (N–H or -CON =) or polar regions of the protein.
Subsequently, the central nitrogen atoms of the macrocy-
cle interacting with the polar regions of residues, such as
Glu-15 or His-16, reinforce the coupling. Finally, the weak
Ligand Residue atom Donor Angle Distance (Å) Binding mode
TCPP Glu-14
Asn-42
114.4
103.0
3.2
3.3
Hydrogen
bonding
Hydrogen
bonding
Lys-18
Lys-18
Asn-159
–
–
–
3.8
5.3
2.9
π-Cation*
Salt bridge*
Ce6
Hydrogen
bonding
Pro-279
Lys-129
–
3.7
5.3
Hydrophobic*
Salt bridge*
* Classifcation of interactions based on the protein-ligand interaction
profler (PLIP) (Salentin et al. 2015)
species proves able to establish hydrophobic interactions
with Tyr-153, Pro-279, and His-16 residues (Fig. 11c).
However, there are other residues that could induce charge
repulsions, such as Lys-157, localized near the pyrrolic
nitrogen of porphyrin and Gln-128, which can establish
hydrogen bond interactions with the oxygen atoms of car-
boxyl groups (Fig. 11c).
The distance obtained from docking simulation between
BSA and Ce6 or TCPP and the main amino acid residues are
shown in Table 4. These results provide a base to explain
the efcient fuorescence quenching of BSA emission in the
presence of Ce6 or TCPP. Furthermore, the same results
suggest that the main interactions between BSA with Ce6
and TCPP are dominated by hydrogen-bonding forces and
imply that the formation of hydrogen bonds increases the
stability of BSA-TCPP and BSA-Ce6.
The higher-score poses determined by the Autodock Vina
program give an interesting and useful idea of the confor-
mations adopted by coupling species; that is, the tetrapyr-
rolic macrocycles in the region near the Trp-134 residue, as
well as the other residues with that compound, can establish
stabilizing or unfavorable interactions. Therefore, a deeper
analysis of the electrostatic and hydrophobic contributions
was needed to further explore their infuence. Also, the
fuorescence quenching observed experimentally could be
related to the approach to the Trp-134 residue, which ensures
the establishment of mainly polar interaction of macrocycles
with the surrounding residues for both systems analyzed.
Then, to have a more conclusive idea of the conformation
adopted by the residues in the binding site, in silico stud-
ies of fexible molecular docking were carried out. For this
purpose, in the proximity of the binding site of the BSA-
TCPP complex, only the Glu-15, His-16, Phe-124, Glu-128,
Trp-134, Tyr-153, and Lys-157 residues were considered as
fexible.
Fig. 12 Flexible molecular docking of the BSA-TCPP system. Black
or gray residues correspond to positions obtained from the rigid or
fexible docking in silico analysis, respectively. The distance between
the phenyl rings of the TCPP and Trp-134 appears strongly reduced
in the fexible docking studies, compared with those determined with
the rigid docking analysis (deep red and blue color)
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Chemical Papers
interactions between the peripheral phenyl of the macrocy-
cle and the non-polar or aromatic regions in Tyr-153, Phe-
125, or Trp-134 complete the set of interactions that allow
the complex formation. So, in this complex, the macrocycle
penetrates and remains relatively parallel to the depth of the
active site as a coin inside a groove.
size allows interactions of the closer amino acid residues
with its pyrrole groups or central regions. While all this evi-
dence suggests that the Ce6 species penetrates the active site
less than TCPP, because of its smaller size, this macrocyclic
species remains perpendicular to the Trp-134 ring and with
its molecular plane approximately parallel to the aperture
plane. So, it looks like a small coin deposited in the bottom
of the groove.
For Ce6, eight amino acids of site III were considered
fexible residues: Glu-15, His-116, Phe-124, Glu-128, Trp-
134, Glu-163, Tyr-153, and Lys-157. In this analysis, Glu-
163 points to the center of Ce6 and Tyr-153 and Lys-157 res-
idues point their respective hydroxyl (–OH) or amino (N–H)
group to the carboxyl (–COOH) groups of the macrocycle.
As occurs with the TCPP species, the free bond rotation of
the closer residues leads to a closer confguration of spe-
cies. Consequently, in this simulation, the indole aromatic
rings of Trp-134 and a pyrrole ring of the macrocycle of Ce6
remains at 4.6 Å (Fig. 13), which suggests the possibility
of its coupling through π-π interactions, but the approach
between these species occurs at a greater distance than with
TCPP. Probably, this diference of interaction distance with
the Trp-134 residue is the principal reason for the lower
quenching observed for the BSA-Ce6 system. At the same
time, Glu-128 and Phe-124 appear to point to the interaction
region of Trp-134 and the macrocycle. Furthermore, this
simulation reveals that the approximation of N–H groups
in both species is possible and could be a crucial event for
stabilizing the formed complex. However, the fexible dock-
ing analysis suggests that the approximation of Ce6 points
its two adjacent carboxyl groups to the active site, and its
Finally, it is possible to state that the number and locali-
zation of the carboxylic groups in the periphery of TCPP
or Ce6 are mainly responsible for the approach to the BSA.
However, the interactions with the central nitrogen atoms
of the macrocycle are also important in the formation of the
fnal complexes. Additionally, the interactions of aromatic
regions of the macrocycles and the non-polar regions of the
surrounding amino acids have an important role as well. In
this regard, the interactions with the central nitrogen atoms
prove to be more signifcant for the Ce6 species, due to its
smaller size, compared to the TCPP species. In contrast, as
a result of the steric efect of the TCPP peripheral phenyl
groups, the protein residues are prevented from approaching
the central regions of that macrocycle. However, the macro-
cycle interactions with slightly polar or aromatic regions of
protein residues, such as Phe- 124, Tyr-153, or Trp-134, are
possible. In other words, the size and structure of the mac-
rocyclic species used have an important efect on how the
fnal complex is formed and, on its structure, and stability.
Conclusions
The interaction of BSA and tetrapyrrole macrocycles, such
as TCPP or Ce6, was analyzed by spectroscopic techniques
and molecular docking modeling.
The results of fuorescence experiments clearly reveal that
the BSA quenching mechanism is of static nature, induced
by TCPP or Ce6 proximity and due to the formation of the
respective BSA- TCPP and BSA-Ce6 complexes.
A single binding site on BSA is found for both tetrapyr-
role species. The binding constants (Kb) at diferent tem-
peratures reveal that there is a strong binding interaction of
TCPP and Ce6 with BSA.
Based on the calculated thermodynamic parameters, such
as ΔG, ΔH, and ΔS of the binding process, it was estab-
lished that the main binding forces between TCPP and Ce6
with BSA were the van der Waals force and hydrogen bond-
ing. The negative free energy change (ΔG) shows that their
interaction allows the coupling of the species through an
energetically favored process. The negative enthalpy change
(ΔH) exposes the occurrence of an enthalpy-driven process,
and the negative entropy change (ΔS) suggests an entropi-
cally unfavorable process. In other words, the evidence
Fig. 13 Flexible molecular docking of the BSA-Ce6 system. Black or
gray residues correspond to the positions obtained from the rigid or
fexible docking in silico analysis, respectively. The distance between
the center of the Ce6 pyrrole rings and the Trp-134 group appears
reduced in the fexible docking studies if compared with those deter-
mined with the rigid docking analysis (deep red and blue color)
1 3

Chemical Papers
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Considering the residues of the binding site fexible, a
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Supplementary Information The online version contains supplemen-
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Acknowledgements J. Osiris Vicente thanks the National Science
and Technology Council of Mexico (CONACYT) for the scholarship
number 130442.
Declarations
Conflict of interest On behalf of all authors, the corresponding author
states that there is no confict of interest.
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