Spectroscopic investigations of the binding interaction of a new indanedione derivative with human and bovine serum albumins

Article abstract of DOI:10.3390/molecules14041614

Binding of a newly synthesized indanedione derivative, 2-(2-hydroxy-3- ethoxybenzylidene)-1,3-indanedione (HEBID), to human and bovine serum albumins (HSA and BSA), under simulated physiological conditions was monitored by fluorescence spectroscopy. The b

Full text of DOI:10.3390/molecules14041614

Molecules 2009, 14, 1614-1626; doi:10.3390/molecules14041614
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Spectroscopic Investigations of the Binding Interaction of a New
Indanedione Derivative with Human and Bovine Serum
Albumins
Dana Stan 1,*, Iulia Matei 2, Carmen Mihailescu 1, Mihaela Savin 3, Mihaela Matache 1, Mihaela
Hillebrand 2 and Ion Baciu 1
1
Department of Organic Chemistry, Bucharest University, 00133, Sos. Panduri No. 90-92,
Romania;E-mails: carmen_mihail28@yahoo.com(C.M.),mihaela.matache@gmail.com(M.M.)
ion_baciu2004@yahoo.com (I.B.)
2
Department of Physical Chemistry, Faculty of Chemistry, University of Bucharest, Bd. Regina
Elisabeta, No. 4-12, Bucharest, Romania; E-mails: iulia.matei@yahoo.com (I.M.),
mihaela.hillebrand@gmail.com (M.H.)
3
Telemedica, Str. Ion Calin, No.13, Bucharest, Romania; E-mail: mihaelasvn@yahoo.com (M.S.)
* Author to whom correspondence should be addressed; dana_stan2005@yahoo.com; Tel.: +40 21
410 4009, Fax: +40 21 410 4009.
Received: 17 March 2009; in revised form: 15 April 2009 / Accepted: 20 April 2009 /
Published: 24 April 2009
Abstract: Binding of a newly synthesized indanedione derivative, 2-(2-hydroxy-3-
ethoxybenzylidene)-1,3-indanedione (HEBID), to human and bovine serum albumins
(HSA and BSA), under simulated physiological conditions was monitored by fluorescence
spectroscopy. The binding parameters (binding constants and number of binding sites) and
quenching constants were determined according to literature models. The quenching
mechanism was assigned to a Förster non-radiative energy transfer due to the HEBID-SA
complex formation. A slightly increased affinity of HEBID for HSA was found, while the
number of binding sites is approximately one for both albumins. The molecular distance
between donor (albumin) and acceptor (HEBID) and the energy transfer efficiency were
estimated, in the view of Förster’s theory. The effect of HEBID on the protein
conformation was investigated using circular dichroism and synchronous fluorescence
spectroscopies. The results revealed partial unfolding in the albumins upon interaction, as
well as changes in the local polarity around the tryptophan residues.

Molecules 2009, 14
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Keywords: Indanedione; Serum albumin; Fluorescence; Circular dichroism; Fluorescence
resonance energy transfer.
1. Introduction
Serum albumins (SAs) are the major soluble protein constituents of the circulatory system and have
many physiological functions, including acting as transporters for numerous endogenous and
exogenous ligands (e.g. drugs, fatty acids, etc.). Drug binding to SAs is an important determinant of
drug pharmacokinetics, restricting the unbound concentration and affecting drug distribution and
elimination [1].
Human serum albumin (HSA) is the major protein component of blood plasma (approx. 60% of the
total protein) [2]. It is a single, non-glycosylated polypeptide that organizes to form a heart-shaped
protein with approximately 67% α-helix [3]. HSA is capable of binding reversibly a wide variety of
drugs, resulting in an increased solubility in plasma, decreased toxicity, and/or protection against
oxidation of the bound ligand. The protein’s capability of binding aromatic and heterocyclic
compounds largely depends on the existence of two major binding regions, namely Sudlow’s sites I
and II [4]. Site I, also known as the warfarin binding site, is formed by a pocket in subdomain IIA and
contains the only tryptophan of HSA (Trp 214) [5]. Site II is located in subdomain IIIA and is known
as the benzodiazepine binding site [5]. Of these sites, site I seems to be the more versatile, because it
can bind, with a high affinity, to ligands that are very different from a chemical point of view [2].
Bovine serum albumin (BSA) is constituted by 582 amino acid residues and, on the basis of the
distribution of the disulfide bridges and of the amino acid sequence, it can be regarded as composed of
three homologous domains (I, II and III) linked together. Each domain can be subdivided into two
subdomains, A and B. BSA has two tryptophans, Trp-134 and Trp-212, embedded in subdomains IB
and IIA, respectively [6].
During the last three decades, substituted indane-1,3-dione derivatives have shown wide
applicability in the fields of medicine [7,8] and biology [9]. Many studies on the biological activity of
these compounds report their anticoagulant [10], anti-inflammatory [11], analgesic [12], antibacterial
[13] or bronchial dilating [14] action. However, literature data on their interaction with proteins is
relatively scarce [15-17]. Because they are compounds of important biological activity, we considered
that such studies could elucidate important aspects of drug pharmacokinetics.
In the present report we focus on a newly synthesized indanedione derivative, 2-(2-hydroxy-3-
ethoxybenzylidene)-1,3-indanedione (HEBID), studying its interaction with human and bovine serum
albumins (HSA and BSA) by means of fluorescence and circular dichroism spectroscopies.
Fluorescence is a practical method for studying protein interactions with various ligands [18-24], as it
yields a vast amount of information on the magnitude of binding and on the microenvironment
surrounding the protein residues. In our work, we monitored the changes in the intrinsic fluorescence
of the SAs upon binding to HEBID. A circular dichroism study was also undertaken in order to
complement the findings by fluorescence spectroscopy, allowing us to ascertain the influence of
HEBID binding upon the secondary structures of SAs. Therefore, the aim of this study was to

Molecules 2009, 14
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characterize the HEBID-HSA and HEBID-BSA interactions, by determining the binding parameters,
the efficiency of energy transfer and the conformational changes occurring in the proteins upon
binding.
2. Results and Discussion
2.1. Fluorescence quenching of serum albumins in presence of HEBID
The fluorescence of HSA and BSA mainly resides in the emissions from the tryptophan, Trp (~340
nm) and tyrosine, Tyr (~315 nm) residues. Upon excitation at 286 nm, HSA and BSA show strong
emissions, with peaks at 340 nm and 345 nm, respectively. It was observed that the fluorescence
emission intensities of both SAs were quenched, in a concentration-dependent manner, when the
proteins interacted with HEBID (a typical example is shown in Figure 1).
Figure 1. The quenching of BSA and HSA (inset) fluorescence at increasing HEBID
concentrations. [HEBID]/[SA] = 0 (1), 0.48 (2), 0.97 (3), 1.67 (4), 2.53 (5), 3.83 (6), 5.19
(7), 7.61 (8), 10.00 (9). λex = 286 nm.
1
250
1
200
200
150
100
150
8
50
0
300
350
400
450
500
100
50
0
λ (nm)
9
300
350
400
λ (nm)
450
500
Quenching of the intrinsic protein fluorescence can be used to retrieve many ligand-protein binding
information. Fluorescence quenching is described by the well-known Stern-Volmer equation [25]:
F0
=1+ KSV [Q]
(1)
F
where F0 and F denote the steady-state fluorescence intensities in the absence and presence of the
quencher (HEBID), respectively, KSV is the Stern-Volmer quenching constant and [Q] the quencher
concentration. Hence, Equation (1) can be applied to determine KSV by linear regression of a plot of
F0/F against [Q] (Figure 2). KSV was estimated at 5.55×104 M-1 for the HEBID-HSA interaction
(correlation coefficient R = 0.9988) and at 4.80×104 M-1 for the HEBID-BSA interaction (R = 0.9976).

Molecules 2009, 14
Figure 2. Stern-Volmer plots for the quenching of HSA and BSA by HEBID in the d/p
1617
range 0-10. F0 and F denote the fluorescence intensities in the absence and presence of the
quencher (λex = 286 nm).
2.2. Estimation of the binding parameters
The estimation of the binding parameters was made using two formulae that consider the presence
of a single class of binding sites. The first formula, Equation (2), starts from the classical Scatchard
equation [26] and expresses the free ligand concentration at equilibrium as a function of the analytical,
total concentration of the ligand [27,28]:
(F − F)
1
0
log
= nlog
+ nlog K
(F0 − F)
F
(2)
[HEBID]−[SA]
F
0
K and n are the binding constant and number of binding sites, and [HEBID] and [SA] are the total
ligand concentration and the total protein concentration, respectively. Linear fits according to Equation
(2), i.e. log(F0-F)/F vs. log(1/([HEBID]-[SA](F0-F)/F0)), are presented in Figure 3. The binding
parameters, evaluated from the slope and intercept of these plots, are listed in Table 1. It can be seen
that n is about 1 for both SAs, suggesting that one molecule of albumin combines with one molecule of
HEBID in the drug to protein (d/p) molar ratio under study, ranging from 0 to 10. We can also observe
that the binding affinity of HEBID to HSA is slightly higher than that to BSA.
The second formula, equation (3), is based on the approximation that the free ligand concentration,
at equilibrium, can be replaced by the total analytical concentration of the ligand, approximation that is
only valid when the ligand is in excess, like in our working conditions [29, 30].
(F − F)
0
log
= log K + nlog[HEBID]
(3)
F
It can be observed from Table 1 that, in our case, both equations lead to similar values of the
binding constants and attest a one to one ligand-protein interaction, implying the binding of the ligand
to the Trp 214 residue.

Molecules 2009, 14
Figure 3. Plots for the determination of the binding parameters, according to: (A) Equation
1618
(2) and (B) Equation (3). [SA] = 3×10-6 M, λex = 286 nm, λem = 335nm.
(A)
(B)
0.4
0.4
0.0
0.0
-0.4
-0.4
-0.8
-1.2
-1.6
-0.8
-1.2
-1.6
-2.0
HSA
BSA
HSA
BSA
4.5
5.0
5.5
6.0
6.5
-6.5
-6.0
-5.5
-5.0
-4.5
log(1/([HEBID]-(F0-F)*[SA]/F0))
log[HEBID]
Table 1. Binding parameters (binding constants, K, and number of binding sites, n)
characterizing the HEBID-HSA and HEBID-BSA interactions, estimated in the d/p range
0-10.
K (M-1) ×10-4
6.54
n
R
SD
Equation HSA
(2)
Equation HSA
(3)
0.96
0.81
0.99
0.86
0.9985 0.0279
0.9989 0.0209
0.9987 0.0262
0.9992 0.0180
BSA
5.65
5.47
BSA
1.11
2.3. Effect of HEBID on the conformations of HSA and BSA
2.3.1 Synchronous fluorescence measurements
Synchronous fluorescence is a method that provides information on the molecular environment in
the vicinity of the fluorophores of SAs. While the sensitivity of the fluorescence method is preserved,
this technique offers several advantages, such as spectral simplification, bandwidth reduction and
therefore a minimization of the effects that interfere in the steady-state technique [31, 32]. Using Δλ =
60 nm and 15 nm we obtain the characteristic synchronous fluorescence spectra of the Trp and Tyr
residues, respectively. The shifts in the position of the synchronous maxima of these residues, which
usually occur upon binding, give indications on the changes in polarity around these particular
fluorophores and thus on the proximity of the ligand. Figure 4 shows the effect of HEBID addition on
the position of the synchronous emission maxima of the Trp and Tyr residues of HSA.

Molecules 2009, 14
Figure 4. The HEBID effect on the synchronous fluorescence emission of HSA (Δλ = 60
1619
nm). Inset: Δλ = 15 nm.
250
200
150
100
d/p=0
λem= 339 nm
λem= 298 nm
100
80
60
40
20
0
d/p=0
d/p=10
300
350
λ (nm)
400
d/p=10
300
λem= 343 nm
50
0
350
400
450
λ (nm)
As evidenced in Figure 4, the emission maximum of Tyr does not show a significant shift upon
binding, which indicates that the polarity around the Tyr residues of HSA remains unchanged.
Differently, the red shift of approximate 4 nm in the emission maximum of the Trp residue suggests
that the HEBID-HSA interaction results in a more polar environment for the Trp residue. A similar
trend was observed for BSA.
2.3.2. Circular dichroism results
When a ligand binds to a globular protein, the intramolecular forces responsible for maintaining the
secondary and tertiary structures can be altered, resulting in a conformational change of the protein
[33]. The observed fluorescence quenching of the Trp and Tyr residues suggests that the ligand-
albumin interaction has indeed changed the microenvironment of these residues. Moreover, the red
shift in the synchronous fluorescence of Trp shows an increased polarity of the microenvironment after
binding.
Conformational changes upon binding are also reflected in the circular dichroism (CD) spectrum of
the protein. The CD spectra of SAs exhibit two negative bands in the UV region at 209 and 222 nm,
characteristic for the α-helical structure [34]. The spectra recorded in presence of low HEBID
concentrations did not exhibit appreciable changes in the conformation of SAs in terms of α-helicity.
However, significant changes in α-helicity values were noticed at higher concentrations of HEBID.
The CD spectra of HSA and BSA in the absence (d/p = 0) and presence (d/p = 18) of HEBID are
shown in Figure 5. The alterations of protein secondary structure after HEBID complexation were
estimated as follows. The results of CD measurements can be expressed as MRE (mean residue
ellipticity) in deg·cm2·dmol-1, defined as:

Molecules 2009, 14
1620
θ
MRE =
(4)
10rl[SA]
where θ is the observed CD (in milli-degrees), r is the number of amino acid residues (585 for HSA
and 582 for BSA), and l is the path length of the cell (in cm). The helical content of free and bound SA
was calculated from the MRE values at 209 nm using the following equation, as described by Lu et al.
[35]:
−MRE209nm − 4000
%α − helix =
×100
(5)
33000 − 4000
It was observed that the percent of α-helix was reduced from 59.98% in free BSA to 57.58 % upon
binding to HEBID (d/p =18). The fact that the CD spectra of BSA in the presence and absence of
HEBID are similar in shape indicates that the structure of BSA remains predominantly α-helical after
binding to HEBID. Similarly, for HSA, the protein α-helix structure decreased gradually and the
reduction reached about 5% for protein binding with HEBID at a d/p = 18, indicating partial unfolding
of HSA.
Figure 5. The CD spectra of HEBID-HSA and HEBID-BSA (inset) systems. [HSA] and
[BSA] = 0.75×10-6 M. [HEBID] = 0 M (1) and 1.38×10-5 M (2). pH = 7.4.
0
-40
0
-80
-40
-120
2
-80
1
2
200
225
250
275
-160
λ (nm)
1
200
225
250
275
λ (nm)
2.4. Energy transfer efficiency and binding distance
The performed spectral studies suggested that both HSA and BSA form complexes with HEBID,
with involvement of the Trp residues of the proteins. HSA possesses only one Trp molecule (Trp214),
while BSA contains two such residues (Trp134 and Trp212), though only Trp134 is readily accessible
to the ligand, being located on the exterior surface of the protein [36]. A way of measuring the distance
r between the ligand and these Trp residues is by using the fluorescence resonance energy transfer
(FRET), as proposed by Förster [37]. FRET is a distance dependent interaction in which excitation
energy is transferred nonradiatively from the donor molecule to the acceptor. The prerequisite for the
occurrence of FRET is the existence of a proper overlap between the emission spectrum of the donor

Molecules 2009, 14
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and the absorption spectrum of the acceptor. Figure 6 shows this overlap in the case of the HSA-
HEBID donor-acceptor pair.
Figure 6. Overlap of the fluorescence spectrum of BSA (1) and absorption spectrum of
HEBID (2).
The extent of the spectral overlap, as well as the distance between donor and acceptor, determine
the magnitude of the energy transfer. According to Förster, the energy transfer efficiency, E, is given
by:
R06
R06 + r6
F
E =
=1−
(6)
F0
where r is the distance between donor and acceptor and R0 is the critical distance, for which the
transfer efficiency is 50%. F0 and F are the fluorescence intensities of HSA in absence and presence
(at d/p = 1) of HEBID, respectively. The magnitude of R0 depends on the spectral properties of the
donor and acceptor molecules:
R06 = 8.8×10−25 k2n−4ΦJ (in cm6)
(7)
Here k is a factor expressing the spatial orientation of the dipole of the acceptor molecule, n is the
refractive index of the medium, Φ the fluorescence quantum yield of the donor in absence of the
acceptor and J is the overlap integral of the emission spectrum of the donor and absorption spectrum of
the acceptor (in units of M-1cm3). J is given by the equation:
∞
J = FD (λ)εA (λ)λ4dλ
(8)
∫
0
where FD(λ) is the corrected fluorescence intensity of the donor at wavelengths λ to (λ + Δλ), with the
total intensity normalized to unity, and εA(λ) is the molar extinction coefficient of the acceptor at
wavelength λ.
The efficiency of the dipole-dipole interaction between donor and acceptor depends on the
alignment of the two dipoles. The orientation factor k2 which describes this proximity ranges from 0
(perpendicular dipoles) to 4 (parallel dipoles). Generally, the dipoles are assumed to be rapidly
moving, on timescales similar to the donor excited-state lifetime, and their orientations are therefore
described as random, with an orientation factor of 2/3 [38].

Molecules 2009, 14
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Förster’s distance (R0) has been calculated assuming such random orientation of the donor and
acceptor molecules in solution. Thus, k2 was taken 2/3, n = 1.333 (water) and Φ = 0.118 [39]. The
obtained data is listed in Table 2.
Table 2. Förster energy transfer parameters.
E
HSA 0.16
BSA 0.18
J × 1015 (M-1cm3) R0 (nm) r (nm)
1.48
1.51
1.78
1.79
2.34
2.31
An average r distance on the 2-8 nm scale indicates that the energy transfer occurs with high
probability [40]. Furthermore, a larger r value than that of the critical distance R0 suggests the presence
of a static quenching mechanism [41].
3. Experimental
3.1. General
The IR spectra were recorded on a Fourier Transform Genesis DTGS in KBr pellets in the range
4000-400 cm-1. The NMR spectra were recorded on a JEOL Lambda 400 (operating at 300 MHz for
1H and 75 MHz for 13C, respectively) in DMSO-d6, using TMS as internal standard. Mass spectra were
obtained on a Finnigan MAT 90 spectrometer using CI technique; Thin Layer Chromatography was
carried out on silicagel plates (Merck) and visualisation was accomplished using iodine vapours or UV
light (λ = 254 nm). The melting points were determined in open capillaries using an electrical melting
point apparatus and are uncorrected. The absorption and fluorescence spectra were recorded on a V-
560 Jasco UV-VIS spectrophotometer and on a FP-6300 Jasco spectrofluorimeter equipped with a
STR-313 Jasco thermostatic cell holder, respectively. Circular dichroism measurements were carried
out with a Jasco J-815 CD spectrometer. All chemicals were obtained from commercial sources and
used without further purification. Human and bovine serum albumins were fatty acids free, 99%.
3.2. Synthesis
The synthesis of 2-(2-hydroxy-3-ethoxybenzylidene)-1,3-indanedione (HEBID) is based on the
direct condensation between 1,3-indanedione and aromatic aldehydes, as depicted in Scheme 1. 2-
Hydroxy-4-ethoxybenzaldehyde (3.32 g, 20 mmoles) and glacial acetic acid (50 mL) were added to
1,3-indanedione (2.9 g, 20 mmoles) [42]. The mixture was heated and when it became clear,
concentrated hydrochloric acid (1 mL) was added. The mixture was heated for 20 minutes at reflux,
then allowed to cool to room temperature. The solid formed was filtered and washed with ethanol (15
mL) to affird 4.7 g of an orange product (yield 80%); Rf = 0.37 (chloroform-methanol-petroleum ether
1
4:1:2 v:v:v). It was recrystallized from ethanol to yield crystals with m.p. 221-222 ºC. H-NMR (δ
ppm): 9.84 (s, 1H, OH); 8.47 (d, 1H, J=8, H14); 8.36 (s, 1H, H10); 7.91-7.98 (m, 4H, H1-H4); 7.20 (d,
13
1H, J=8, H16); 6,68 (t, 1H, J=8, H15); 4.12 (q, 2H, J=7, CH2O); 1.38 (t, 3H, J=7, Me). C-NMR (δ

Molecules 2009, 14
1623
ppm): 189.6, 188.4 (C7, C9); 149.5 (C13); 146.4 (C12); 141.5 (C10), 139.4, 139.0 (C5, C6); 135.5,
135.3 (C2, C3); 127.0, 124.0 (C11, C8); 122.6, 122.4 (C1, C4); 119.9 (C15) 118.2 (C16), 117.8 (C14);
64.2 (CH2O); 14.2 (Me); IR (ν cm-1): 3073, 1719, 1676, 1569, 1465, 1231; MS (m/z, %): calc. 294.3
([M]+); found 294.9 (100, [M+H]+).
Scheme 1. Synthesis of the arylidene derivative.
3.3. Procedures
Protein solutions in phosphate buffer (pH 7.4) 3×10-6 M (for fluorescence studies) and 0.75×10-6 M
(for CD studies) were prepared and equilibrated over night. A stock solution of HEBID in ethanol
(1.2×10-4 M) was diluted with phosphate buffer to yield a 1.2×10-5 M solution used in the titration
experiments. It should be noted that such low amounts of ethanol do not affect the protein structures
[43].
Binding of HEBID to HSA and BSA was studied by the fluorescence quenching titration method,
using the intrinsic fluorescence of the protein as a probe. To a fixed volume (2 mL) of protein solution
were added increasing volumes of HEBID to obtain different complexes in a range of drug to protein
(d/p) ratio of 0 to 10. Steady-state fluorescence spectra were obtained at excitation wavelength of 286
nm (excitation of Trp and Tyr residues). Synchronous fluorescence spectra were obtained using Δλ =
15 nm (Tyr) and 60 nm (Trp), where Δλ = λem - λex. Circular dichroism spectra of SA and HEBID-SA
complexes (d/p = 18) were scanned in the range 200-280 nm, in order to record the characteristic
signal of the α-helix conformation of the albumins. All experiments were conducted at 25±1 ºC.
4. Conclusions
The binding interactions of HEBID with HSA and BSA were studied under simulated physiological
conditions using steady state and synchronous fluorescence and circular dichroism spectroscopies. The
results showed a significant interaction of HEBID with both HSA (K = 6.54×104 M-1) and BSA (K =
5.65×104 M-1), via one binding site for each protein. The effect of HEBID on the conformations of the
SAs was also discussed, evidencing partial defolding of the proteins (2-5% loss in the α-helix content),
caused by a perturbation of the Trp rather than of the Tyr residues. Binding distances of approximate 2
nm were computed, suggesting a high probability for the SA-HEBID energy transfer.

Molecules 2009, 14
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Sample Availability: Samples of the compound are available from the authors.
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