A spectral deciphering the perturbation of model transporter protein (HSA) by antibacterial pyrimidine derivative: Pharmacokinetic and biophysical insights

Article abstract of DOI:10.1016/j.jphotobiol.2012.09.010

Steady state fluorescence and UV-vis absorption spectroscopic techniques have been exploited to explore the binding interaction of a antibacterial pyrimidine derivative 2-amino-6-hydroxy-4-(4-hydroxyphenyl)-pyrimidine-5- carbonitrile (AHHPPC) with the model transporter protein, human serum albumin (HSA) under the physiological conditions. It exhibits antibacterial activity against Escherichia coli and Staphylococcus aureus. Analysis of fluorescence quenching data of HSA at different temperatures using Stern-Volmer methods revealed the formation of AHHPPC-HSA complex with binding affinities of the order 10⁴ M^-1. The binding site number (n ≈ 1) and corresponding thermodynamic parameters (ΔG), (ΔH) and (ΔS) were calculated, indicated that binding reaction was endothermic and the hydrophobic interactions plays a major role in stabilizing the complex. The binding distance (r = 3.13 nm) between donor (HSA) and acceptor (AHHPPC) was obtained according to FRET. Changes in the albumin secondary structure imparted by the compound was confirmed using synchronous fluorescence, electronic absorption, circular dichroism (CD) and three-dimensional (3D) fluorescence spectroscopy. All these experimental results clarified that AHHPPC could bind to HSA and be effectively transported and eliminated in body, which could be a useful guideline for further drug design.

Full text of DOI:10.1016/j.jphotobiol.2012.09.010

Journal of Photochemistry and Photobiology B: Biology 118 (2013) 1–8
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
A spectral deciphering the perturbation of model transporter protein (HSA) by
antibacterial pyrimidine derivative: Pharmacokinetic and biophysical insights
⇑
Vishwas D. Suryawanshi, Prashant V. Anbhule, Anil H. Gore, Shivajirao R. Patil, Govind B. Kolekar
Fluorescence Spectroscopy Research Laboratory, Department of Chemistry, Shivaji University, Kolhapur 416 004, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2012
Received in revised form 16 September
Steady state fluorescence and UV–vis absorption spectroscopic techniques have been exploited to explore
the binding interaction of a antibacterial pyrimidine derivative 2-amino-6-hydroxy-4-(4-hydroxy-
phenyl)-pyrimidine-5-carbonitrile (AHHPPC) with the model transporter protein, human serum albumin
2
012
(HSA) under the physiological conditions. It exhibits antibacterial activity against Escherichia coli and
Accepted 25 September 2012
Available online 27 October 2012
Staphylococcus aureus. Analysis of fluorescence quenching data of HSA at different temperatures using
Stern–Volmer methods revealed the formation of AHHPPC–HSA complex with binding affinities of the
4
ꢀ1
order 10 M . The binding site number (n ꢁ 1) and corresponding thermodynamic parameters (
DG),
Keywords:
Fluorescence
(D
H) and ( S) were calculated, indicated that binding reaction was endothermic and the hydrophobic
D
interactions plays a major role in stabilizing the complex. The binding distance (r = 3.13 nm) between
donor (HSA) and acceptor (AHHPPC) was obtained according to FRET. Changes in the albumin secondary
structure imparted by the compound was confirmed using synchronous fluorescence, electronic absorp-
tion, circular dichroism (CD) and three-dimensional (3D) fluorescence spectroscopy. All these experimen-
tal results clarified that AHHPPC could bind to HSA and be effectively transported and eliminated in body,
which could be a useful guideline for further drug design.
Human serum albumin
Fluorescence quenching
Thermodynamic parameters
Pyrimidine derivative
Ó 2012 Elsevier B.V. All rights reserved.
1
. Introduction
Heterocyclic compounds particularly five or six membered ring
that contains disease causing agents like bacteria, viruses and par-
asites. E. coli bacteria are responsible for Hemorrhagic colitis and S.
aureus causes staphyloenterotoxicosis, staphyloenterotoxemia.
Human serum albumin (HSA) has been used as a model protein
for many years. It is the most abundant protein in blood plasma,
which constitutes up to 60% of the total protein corresponding to
compounds have occupied the first place among various classes of
organic compounds for their biological and pharmacological activ-
ities. As pyrimidine is a basic nucleus in DNA and RNA, it has been
found to be associated with diverse biological activities [1,2].
Pyrimidine moiety is an important class of N-containing heterocy-
cles widely used as key building blocks for pharmaceutical agents.
It exhibits a wide spectrum of pharmacophore as it acts as bacteri-
cidal, fungicidal, analgesic, anti-hypertensive and anti-tumor
agents [3–6]. Also, preclinical data from literature survey indicate
continuing research in polysubstituted pyrimidine as potential
anti-tumor agents [7]. 2-amino-6-hydroxy-4-(4-hydroxyphenyl)-
pyrimidine-5-carbonitrile (AHHPPC, Structure IV), a pyrimidine
derivative and its structural analogues possessing anti-bacterial
activities were synthesized in our laboratory (Scheme 1) [8]. It is
synthesized by three-component condensation of aromatic alde-
hyde (I), ethyl cyanoacetate (II) and guanidine hydrochloride (III)
in ethanol under alkaline medium. These compounds possess
interesting in vitro antibacterial activities against Escherichia coli
and Staphylococcus aureus which are common food poisoning bac-
teria. Foodborne diseases are caused by eating or drinking food
ꢀ1
a concentration of 42 g L and provides about 80% of the osmotic
pressure of blood [9,10]. HSA plays an important role in the trans-
port and deposition of a wide range of endogenous and exogenous
substances such as fatty acids, amino acids, hormones, dyes and
many diverse drugs [10,11]. Recently, He and Carter [12] have
determined the three-dimensional structure of HSA through X-
ray crystallography. The globular protein consists of 585 amino
acid residues is a heart-shaped tridomain protein with each do-
main subdivided into two identical subdomains A and B. HSA has
two important binding sites located in hydrophobic cavities in sub-
domain IIA and IIIA. Site I is formed as a pocket in subdomain IIA
and involve the lone tryptophan (Trp) of HSA (Trp-214) (Fig. 1).
Aromatic and heterocyclic ligands were found to bind within two
hydrophobic pockets in subdomains IIA and IIIA, site I and site II
[13]. Protein–drug interaction plays an important role in pharma-
cokinetics and pharmacodynamics of the drugs. This interaction
can take place with a variety of agents in blood, including proteins
such as HSA. The information on the interaction of serum albumin
and drug can help us better to understand the absorption and
distribution of the drug. Extensive investigations into the interaction
⇑
Corresponding author. Fax: +91 0231 2692333.
E-mail address: gbkolekar@yhaoo.co.in (G.B. Kolekar).
1
011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2012.09.010

2
V.D. Suryawanshi et al. / Journal of Photochemistry and Photobiology B: Biology 118 (2013) 1–8
Scheme 1. Scheme for synthesis of 2-amino-6-hydroxy-4-(4-hydroxyphenyl)-pyrimidine-5-carbonitrile (AHHPPC).
to calculate the molar concentrations. All HSA solutions were pre-
pared in a pH 7.4 buffer solution and the HSA stock solution was
kept in the dark at 4 °C. Tris (0.1 M)–HCl (0.1 M) buffer solution
containing NaCl (0.1 M) solution (to maintain the ionic strength
at 0.1) was used to keep the pH of the solution at 7.4. Dilutions
ꢀ5
of the HSA stock solution (1.0 ꢂ 10 M) in Tris–HCl buffer solution
were prepared immediately before use. The stock solution
ꢀ5
(
1.0 ꢂ 10 M) of AHHPPC (synthesized) was prepared in (5:95)
ethanol water mixture. Dissolution of compound was enhanced
by sonication in an ultrasonic bath (Spectra lab Model UCB–40).
All chemicals were of analytical reagent grade and were used with-
out further purification. Double distilled water was used through-
out. In order to simulate human body fluid surroundings and to get
the best sensitivity, the pH = 7.4 Tris–HCl solution was chosen as
the buffer solution in this work.
2.2. Equipments and spectral measurements
Fig. 1. Structure of human serum albumin showing the sites and the subdomains.
The UV–visible absorption spectra were measured at room tem-
perature on a Shimadzu UV-3600 UV–VIS NIR Spectrophotometer
equipped with 1.0 cm quartz cell. The wavelength range was from
between the serum albumin and internal compound or pharmaceu-
tical molecule have been made, but the interaction of protein with
the biologically active pyrimidine derivatives has seldom been re-
ported. In a series study methods concerning the interaction of
drugs and protein, fluorescence techniques are great aids in the
study of interactions between drugs and plasma proteins in general
and serum albumin in particular because of their high sensitivity,
rapidity, and ease of implementation [14]. In this paper, we have
studied in vitro interaction of AHHPPC with HSA by the fluorescence
quenching method. The binding constants are obtained at different
temperatures in the medium of Tris–HCl (pH 7.4) buffer solution.
The binding sites and main sorts of binding force have been sug-
gested. In addition, the conformational changes of HSA are discussed
on the basis of synchronous fluorescence (SF), circular dichroism
2
50 to 450 nm. All Fluorescence emission spectra were recorded on
PC based Spectrofluorometer (JASCO Japan FP-750) equipped with
a Xenon lamp and 1.0 cm quartz cell. Fluorescence emission spec-
tra were recorded at three different temperatures 298, 308 and
3
18 K. Excitation and emission slit width was fixed to 10 nm. An
excitation wavelength of 295 nm was chosen because it is exclu-
sively due to the intrinsic Trp fluorophore. All pH values were mea-
sured by a digital pH-meter with magnetic stirrer (Equip-Tronics
EQ-614 A). For synchronous fluorescence measurements, the exci-
tation range was 280–360 nm,
Dk was set at 15 and 60 nm. Circu-
lar dichroism (CD) spectra were measured with a Jasco J-815
Spectropolarimeter (Jasco, Tokyo, Japan) at room temperature over
wavelength range of 200–300 nm, using a 1 cm quartz cell. The
three-dimensional fluorescence spectra were performed under
the following conditions: the emission wavelength was recorded
between 250–500 nm, the initial excitation wavelength was set
to 250 nm with increment of 10 nm for each scanning curves and
other scanning parameters were identical to those of the fluores-
cence emission spectra. Appropriate blanks corresponding to the
buffer were subtracted to correct the absorbance or fluorescence
background.
(
CD) and three-dimensional spectroscopy.
The aim of the present investigation was to study the affinity of
pyrimidine derivative (AHHPPC) for HSA using UV–visible and
fluorescence spectroscopy to understand the carrier role of serum
albumin for such compound in blood under physiological condi-
tions. It is widely accepted that the distribution, metabolism, and
efficacy of many drugs can be altered based on their affinity to ser-
um albumin. Also efficacy of a drug essentially depends on the
effective concentration of the drug in blood plasma. Significantly,
the determination and understanding of drug interacting with ser-
um albumin are important for the therapy and design of the drug
[
15]. The knowledge of the interaction and binding to HSA may
3. Results and discussion
open new avenues for the design of the most suitable pyrimidine
derivatives without side-effects.
3.1. HSA fluorescence characteristics and quenching mechanism
2
. Materials and methods
Fluorescence is the process of photon emission as a result of the
return of an electron in a higher energy orbital back to a lower
orbital, a variety of molecular interactions can results in quench-
ing, including excited-state reactions, molecular rearrangements,
energy transfer, ground- state complex formation and collisional
quenching. The intensity of fluorescence can be decreased by a
2.1. Materials
HSA was purchased from Fluka Chemical Company (essentially
fatty acid free) and its molecular weight was assumed to be 68,000

V.D. Suryawanshi et al. / Journal of Photochemistry and Photobiology B: Biology 118 (2013) 1–8
3
2
1
1
0
0
.0
.5
.0
.5
.0
2
98K
308K
318K
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
[
Q] (10-6 mol L-1)
Fig. 3. Stern–Volmer plots describing HSA quenching caused by AHHPPC at three
different temperatures. CHSA and CAHHPPC are the same as those in Fig. 2.
Fig. 2. The fluorescence quenching spectra of HSA in the presence of AHHPPC,
–
6
–1
–6
–1
C
(
HSA = 2.0 ꢂ 10 mol L ; CAHHPPC/(10 mol L ) a–g: 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0
Table 1
T = 298 K, pH = 7.40, kex = 295 nm).
Stern–Volmer quenching constants and bimolecular quenching rate constants for the
interaction of AHHPPC with HSA at various temperatures.
pH
T (K)
10–5
K
SV/(L mol–1
)
10–13
k
q
/(L mol–1 s–1
)
R
wide variety of processes; such decrease in intensity is called
quenching. In the experiment, the concentration of HSA solutions
7.40
298
1.014
0.732
0.527
1.014
0.732
0.527
0.9972
0.9953
0.9987
ꢀ5
ꢀ1
308
were stabilized at 2.0 ꢂ 10 mol L and the concentrations of
ꢀ5
ꢀ1
318
AHHPPC varied from 0 to 7.0 ꢂ 10 mol L . In order to investi-
gate the binding of AHHPPC to HSA, the fluorescence emission
spectra of HSA in the presence of various concentration of AHHPPC
were recorded upon excitation at 295 nm and 298 K. It can be seen
in Fig. 2 that the fluorescence intensity of HSA decreased regularly
with the increasing concentration of AHHPPC, accompanied by an
increase of wavelength emission maximum kmax (a red shift, from
R is the correlation coefficient.
investigated concentration, the results exhibited a good linear rela-
tionship. Table 1 shows that KSV values were inversely correlated
with temperatures, which suggested that the fluorescence quench-
ing of HSA was initiated by the formation of ground-state complex
3
43 to 347 nm) in the albumin spectrum. It is suggested that the
polarity around the tryptophan residue was also increased [16].
As a result, we predict that Trp-214 is relatively buried inside the
HSA and AHHPPC can bind to HSA, the binding site was adjacent
to the sole tryptophan residue of HSA .Under the same condition,
no fluorescence of AHHPPC was observed. These results indicated
that the binding of AHHPPC to HSA quenches the intrinsic fluores-
cence of the single tryptophan in HSA (Trp-214). The fluorescence
quenching effect was due to the formation of non-fluorescent com-
plex [17]. Fluorescence quenching can occur by different mecha-
nisms, usually classified as either dynamic or static quenching,
which can be distinguished by their differing dependence on tem-
perature and viscosity [18]. Since higher temperatures result in
large diffusion coefficients for dynamic quenching, and the
quenching constants is expected to increase with increasing tem-
perature. In contrast, a higher temperature may bring about the
decrease in the stability of the complexes, resulting in lower
quenching constant for the static quenching. The Stern–Volmer
equation is often applied to elucidate the quenching mechanism
q
[21]. Furthermore, k values were much greater than the maximum
1
0
ꢀ1 ꢀ1
scatter collision quenching constant (2.0 ꢂ 10
M
S ) for
various quenchers with biomolecule, [22] so it was implied that
the static quenching was dominant in AHHPPC–HSA interaction.
The fluorescence data was further examined using modified
Stern–Volmer equation: [23].
F
0
=
D
F ¼ 1=f
a
K
a
1=½Qꢃ þ 1=f
a
ð3Þ
where f is the fraction of the initial fluorescence that is accessible
a
to quencher; K is the Stern–Volmer quenching constant of the
a
accessible fraction and [Q] is the concentration of quencher. Fig. 4
displays the modified Stern–Volmer plots; the dependence of
0
F =DF on the reciprocal values of the quencher concentration
ꢀ1
[Q] is linear. The plots showed that within the investigated con-
centration, the results exhibited a good linear relationship. The
binding strength of drug to HSA is the main factor for its availability
to diffuse from the circulatory system to target [24]. Most ligands
are bound reversibly and exhibit moderate affinities for protein
[
19].
F
0
=F ¼ 1 þ k
q
s
0
½Qꢃ ¼ 1 þ KSV ½Qꢃ
ð1Þ
18
1
1
1
6
4
2
where F and F are the fluorescence intensities in the absence and
0
298K
308K
presence of quencher respectively. k is the bimolecular quenching
constant; is the lifetime of the fluorophore in the absence of
quencher and the fluorescence lifetime of the biopolymer is
q
s
0
3
18K
10
8
ꢀ8
1
0
s; [20] KSV is the Stern–Volmer quenching constant, and [Q]
is the concentration of quencher. The formation of complex was fur-
ther confirmed from the values of quenching rate constants k ,
q
which are evaluated using the equation:
6
4
2
k
q
¼ KSV
=
s
0
ð2Þ
0
0
200000
400000
600000
/ [Q]
800000
1000000
The Stern–Volmer curves of F
0
/F versus [Q] at different temper-
1
atures were shown in Fig. 3 and the calculated KSV and k
q
values
were presented in Table 1. The plots showed that within the
Fig. 4. Modified Stern–Volmer plot for the binding of HSA–AHHPPC.

4
V.D. Suryawanshi et al. / Journal of Photochemistry and Photobiology B: Biology 118 (2013) 1–8
ꢀ1
4
(
binding constants in the range (1–15 ꢂ 10 M ), [25] so the K val-
HSA(2x10-6 M) + (6x10-6M) AHHPPC
HSA(2x10-6 M) + (5x10-6M) AHHPPC
HSA(2x10-6 M) + (4x10-6M) AHHPPC
HSA(2x10-6 M) + (3x10-6M) AHHPPC
HSA(2x10-6 M) + (2x10-6M) AHHPPC
HSA(2x10-6 M) + (1x10-6M) AHHPPC
0
.30
0.25
.20
0.15
ues show that the binding between AHHPPC and HSA is moderate
which indicates that a reversible AHHPPC–HSA complex formation
and AHHPPC can be stored and carried by HSA in the body.
HSA(2x10-6 M) + (0.0M) AHHPPC
0
3
.2. UV–vis absorption spectral studies
0
0
.10
.05
UV–vis absorption measurement is a very simple method and
applicable to explore the structural change and to know the com-
plex formation [26,27]. The UV absorption spectrum (Figs. 5 and 6)
shows the effect of AHHPPC on the HSA absorption spectrum. As
shown in Fig. 6, strong absorption peak was observed at 278 nm
and the peak intensity increased with the addition of AHHPPC. Fur-
thermore, the formation of AHHPPC–HSA complex resulted in a
slight shift (from 278 nm to 286 nm) of spectrum towards longer
wavelength indicating the interaction between AHHPPC and HSA.
The obvious enhancement of absorbance intensity also indicated
the formation of a new complex between AHHPPC and HSA, in
addition the structure of HSA changed upon interaction with
AHHPPC. The strong absorption peak at around 278 nm is due to
the aromatic amino acids (Tryptophan, Tyrosine and Phenylala-
nine) [28,29]. With gradual addition of AHHPPC, the peak intensity
of HSA at 278 nm increases with a red shift, which indicate that the
interaction between AHHPPC and HSA leads to the loosening and
unfolding of the protein backbone and decreases the hydrophobic-
ity of the microenvironment of HSA.
0.00
2
40
280
320
360
400
440
480
Wavelength (nm)
Fig. 6. Effect of AHHPPC on the UV–vis absorption of HSA, CHSA = 2.0 ꢂ 10 mol L–1
–6
;
–1
C
AHHPPC/(10–6 mol L ): 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 (T = 298 K, pH = 7.40).
0
.0
0.2
-0.4
-
298K
08K
18 K
3
3
-
-
0.6
0.8
-1.0
-
-
1.2
1.4
3
.3. Binding constant and binding sites
-6.2
-6.0
-5.8
-5.6
-5.4
-5.2
-5.0
log [Q]
When small molecules bind independently to a set of equiva-
lent sites on a macromolecule, the equilibrium between free and
bound molecules is given by the equation: [30,31],
Fig. 7. The plots of log (F
0
ꢀ F)/F versus log [Q] at three different temperatures CHSA
and CAHHPPC are the same as those in Fig. 2.
ðF
0
ꢀ FÞ
log
¼ log K þ n log½Qꢃ
ð4Þ
Table 2
F
Binding constants (K) and number of binding sites (n) of competitive experiment of
AHHPPC–HSA system.
The binding constant (K) and the number of binding sites (n) can
be calculated using the above equation. A plot of log [(F
versus log [Q] (Fig. 7) gives a straight line, whose slope equals to
n and the intercept on Y-axis equals to log K. The values of K and
n at 298, 308 and 318 K are listed in Table 2. The binding site
10–4 K/(L mol–1
)
n
R
0
ꢀF)/F]
T (K)
98
2
1.2033
1.4814
2.3730
0.8228
0.8956
0.9037
0.9893
0.9964
0.9934
308
318
(
n ꢁ 1) increased slightly with the temperature rising, which shows
R is the correlation coefficient.
the interaction of AHHPPC to HSA seems to be the presence of one
high affinity binding site; the interaction between HSA and AHHPPC
accelerated with the temperature, hence the high affinity binding
site of the interaction is slightly strengthened [32]. The results indi-
cate that there is one class of binding site for HSA. The fact that the
binding constant between AHHPPC and HSA increased with
increasing temperature suggested that there was strong interaction
between AHHPPC and HSA. This clearly implied that AHHPPC would
be bound, stored and transported by HSA in the body [33].
3.4. Thermodynamics and nature of binding forces
Essentially, there are four main types of non-covalent interac-
tions that could play a key role in ligand binding to proteins. These
are hydrogen bonds, van der Waals forces, electrostatic and hydro-
phobic bonds interactions. The thermodynamic parameters, en-
thalpy change (DH) and entropy change (DS) of binding reaction
are the main evidence for confirming binding modes. To obtain
such information, the implications of the present result have been
discussed in conjunction with thermodynamic characteristics ob-
tained for AHHPPC–HSA binding, and the thermodynamic parame-
ters were calculated from the Van’t Hoff equation,
D
RT
H
DS
R
ln K
T
¼ ꢀ
þ
ð5Þ
Fig. 5. UV–visible spectra of: (red line) absorption spectrum of AHHPPC only, (blue
line) absorption spectrum of AHHPPC–HSA 1:1 complex, (black line) absorption
spectrum of HSA only. (T = 298 K, pH = 7.40). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
K is the binding constant at temperature T and R is gas constant. The
enthalpy change ( H) is calculated from the slope of the Van’t Hoff
D

V.D. Suryawanshi et al. / Journal of Photochemistry and Photobiology B: Biology 118 (2013) 1–8
5
relationship. The free energy change (
lowing relationship:
D
G) is estimated from the fol-
Table 3
Thermodynamic parameters of AHHPPC–HSA interaction at pH 7.40.
H (k J mol–1
26.691
)
D
G (k J mol–1
)
D
S (J mol–1
167.45
K
ꢀ1
)
R
T (K)
D
D
G ¼
D
H ꢀ T
DS
ð6Þ
2
3
98
08
ꢀ23.2088
ꢀ24.8833
ꢀ26.5578
0.9994
The temperatures chosen were 298, 308 and 318 K at which
HSA did not undergo any structural degradation. According to
the binding constants at the three temperatures, the thermody-
namic parameters were determined from linear Van’t Hoff plot
318
R is the correlation coefficient.
(Fig. 8) and were presented in Table 3. (The plot of ln K versus
1
/T gave a straight line according to the Van’t Hoff equation). Ross
T
Δ
S (KJ/mol)
1.574
and Subramanian [34] have characterized the sign and magnitude
of the thermodynamic parameter associated with various individ-
ual kinds of interaction that may take place in protein association
processes. Accordingly, as shown in Fig. 9, which illustrates the val-
ues of the thermodynamic parameters for the interaction of the
studied compound with HSA, the binding of AHHPPC to HSA is a
spontaneous process, as indicated by the negative (
accompanied by a positive entropy change ( S). This binding in-
volves an endothermic reaction as manifested by the positive en-
thalpy change, ( H) that is consistent with the increase in K
values with temperature. Thus, according to the rules summarized
by Ross and Subramanian, for drug protein interactions, the posi-
tive value of (
play a major role in the binding process. In other words enthalpy
change of H > 0 and entropy change S > 0 obtained in this case
60
Δ
Δ
T
H (KJ/mol)
G (KJ/mol)
ΔS (KJ/mol)
5
Δ
H (KJ/mol)
40
2
6.691
20
0
1
DG) values,
D
-
-
20
40
Δ
G (KJ/mol)
D
-24.882
Fig. 9. Thermodynamic parameters of the interaction of HSA with AHHPPC at
98 K.
2
DH) and (DS) indicate that hydrophobic interaction
D
D
therefore indicates that the hydrogen bonding and hydrophobic
interactions played a role in the binding of AHHPPC to HSA.
3.5. Energy transfer between AHHPPC and HSA
Forster non-radiative energy transfer theory (FRET) has been
used as a ‘‘spectroscopic ruler’’ for measuring molecular distances
in biological and macromolecular systems [35]. It takes place when
the fluorescence emission band of one molecule (donor) overlaps
with an excitation band of a second (acceptor) that is within 2–
8
nm [36,37]. The overlap of the excitation spectrum of the
AHHPPC with the fluorescence emission spectra of HSA is shown
in Fig. 10. Using FRET, the distance r between AHHPPC and HSA
(
Trp) could be calculated by the equation:
6
F
R
0
Fig. 10. Spectral overlap of AHHPPC excitation (curve A) with HSA emission (curve
E ¼ 1 ꢀ F
¼
ð7Þ
6
–6
–1
6
F); T = 298 K, CHSA = CAHHPPC = 2.0 ꢂ 10 mol L
.
0
R þ r
0
where r is the binding distance between donor and receptor, and R
0
is the critical distance when the efficiency of excitation energy
transferred to the acceptor is 50%. It can be calculated from donor
emission and acceptor absorption spectra using the Föster formula:
where K2 is the spatial orientation factor of the dipole, n is the
refractive index of the medium, / is the fluorescence quantum yield
of the donor, and J is the overlap integral of the fluorescence
emission spectrum of the donor with the absorption spectrum of
the acceptor, which can be calculated by the following equation:
6
0
ꢀ25
2
ꢀ4
R ¼ 8:79 ꢂ 10 K n /J
ð8Þ
R
1
4
FðkÞ
0
e
ðkÞ k dk
0
J ¼
R
1
ð9Þ
1
1
1
0.2
0.1
0.0
FðkÞ dk
y = -3209.5x + 20.141
2
R
= 0.999
where FðkÞ is the fluorescence intensity of the fluorescent donor at
9
9
9
9
9
9
9
.9
.8
.7
.6
.5
.4
.3
wavelength k to k þ
Dk; and
e
ðkÞ is the molar absorption coefficient
2
of the acceptor at wavelength k. In the present case K = 2/3,
n = 1.336 and / = 0.118 [38,39]. Hence, from Eqs. (7)–(9), we could
calculate that
r < 7 nm. The values for R
0.5R < r < 1.5R , [40] indicating an interaction between AHHPPC
R
0
= 2.6952 nm; E = 0.2890 and, r = 3.13 nm,
0
and r are on the 2–8 nm scale and
0
0
and HSA (Trp-214). These data presented in Table 4 suggested
that the energy transfer from HSA to AHHPPC could occur with
high probability. In accordance with prediction by Förster’s nonradi-
ative energy transfer theory, these results indicated again a static
quenching interaction between AHHPPC and HSA [41].
0.0031
0.0032
0.0033
0.0034
1/T
Fig. 8. Van’t Hoff plot for the binding of HSA to AHHPPC.

6
V.D. Suryawanshi et al. / Journal of Photochemistry and Photobiology B: Biology 118 (2013) 1–8
Table 4
Energy transfer parameters between protein (HSA) and receptor (AHHPPC).
3
Protein
Receptor Integral spectral overlap J cm
Critical energy transfer distance Efficiency of energy
Distance between donar–
acceptor (r) nm
–
1
(
mol L
Human serum albumin AHHPPC 26.521 ꢂ 10
HSA)
)
R
0
(nm)
transfer (E)
–
15
2.6952
0.2890
3.13
(
Fig. 11. Synchronous fluorescence spectrum of HSA (T = 298 K, pH = 7.40), CHSA = 2.0 ꢂ 10 mol L–1; CAHHPPC/(10–6 mol L–1): 0, 1.0, 2.0, 3.0, 4.0, 5.0 (a)
–6
D
k = 15 nm, and (b)
D
k = 60 nm.
3.6. Conformational investigations
Changes in the albumin secondary structure imparted by the
compound was confirmed using synchronous fluorescence, elec-
tronic absorption, circular dichroism (CD) and three-dimensional
(
3D) fluorescence spectroscopy which may affect the physiological
functions of serum albumin.
3.6.1. Synchronous fluorescence spectra
The conformational changes of HSA due to interactions with
AHHPPC were evaluated by synchronous fluorescence spectros-
copy .This method introduced by Lloyd [42] has been used to char-
acterize complex mixtures providing fingerprints of complex
mixtures. The synchronous fluorescence spectra gives information
about the molecular environment in a vicinity of the tyrosine and
tryptophan residues of the protein respectively and has several
advantages, such as sensitivity, spectral simplification, spectral
band width reduction and avoiding different perturbing effects.
According to the theory of Miller, [43] when the scanning interval
Fig. 12. Circular dichroism (CD) spectra of free HSA and its AHHPPC–HSA complex.
CHSA = 2.0 ꢂ 10 mol L ; CAHHPPC = 2.0 ꢂ 10 mol L ; T = 298 K; pH = 7.4.
–6
–1
–6
–1
Dk between excitation and emission wavelength is stabilized at 15
and 60 nm the synchronous fluorescence gives the characteristic
information of Tyr and Trp residues. The effect of AHHPPC on
HSA synchronous fluorescence spectra is shown in Fig. 11. It can
be seen from Fig. 11 that there is almost no shift in the maximum
which are characteristic of an a-helical structure of protein [45,46].
Thus, to ascertain the possible influence of AHHPPC binding on the
secondary structure of HSA, we measured the far-UV CD spectra of
the free HSA and its AHHPPC–HSA complex at pH = 7.4 and room
temperature in the range of 200–300 nm. The CD results were
expressed in terms of mean residue ellipticity (MRE) in degree
emission wavelength when
D
k ¼ 15 nm, while a faint red shift
(
from 336 to 339 nm) can be observed when
D
k ¼ 60 nm. This shift
effect expresses the change in conformation of HSA and also sug-
gests that the polarity around the Trp-214 residue was increased
and the hydrophobicity was decreased [44]. Furthermore, the fluo-
rescence intensity decreased regularly with the addition of
AHHPPC in both figures, which illustrates the occurrence of fluo-
rescence quenching in the binding process.
2
ꢀ1
cm dmol according to following equation:
Observed CDðm degÞ
MRE ¼
ð10Þ
Cp ꢂ n ꢂ l ꢂ 10
where Cp is the molar concentration of the protein, n is the number
of amino acid residues and l is the path length. The helical content
of the free and combined HSA were calculated from MRE values at
3.6.2. Circular dichroism (CD) spectra
Far ultraviolet CD spectroscopy was performed to examine the
2
08 using the equation shown below [47,48]:
secondary structure of HSA in the presence of AHHPPC. As shown
in Fig. 12, HSA in the absence of AHHPPC exhibits negative absorp-
MRE208 ꢀ 4000
a-helixð%Þ ¼
ꢂ 100
ð11Þ
ꢄ
ꢄ
tion bands with maxima at 222 nm (n ?
p
) and 208 nm (
p
?
p
),
33000 ꢀ 4000

V.D. Suryawanshi et al. / Journal of Photochemistry and Photobiology B: Biology 118 (2013) 1–8
7
Fig. 13. The three-dimensional projections and the corresponding excitation–emission matrix fluorescence diagrams of HSA before (a) and after AHHPPC addition (b).
Table 5
Three-dimensional fluorescence spectral characteristics of HSA and HSA–AHHPPC system.
Peaks
HSA
HSA–AHHPPC
Peak position kex/kem (nm/nm)
Stokes
D
k (nm)
Intensity F
Peak position kex/kem (nm/nm)
Stokes
D
k (nm)
Intensity F
Rayleigh scattering peaks
Fluorescence peaks
280/280
280/343
0
63
65.76
137.54
280/280
280/345
0
65
44.3
72.25
where MRE208 is the observed MRE value at 208 nm, 4000 is the
MRE of the -form and random coil conformation cross at 208 nm
and 33,000 is the MRE value of a pure -helix at 208 nm. From
the above equation, -helicity in the secondary structure of HSA
was determined. In free HSA, the secondary structure consisted of
conformational and microenvironmental changes of HSA were
investigated by comparing the spectral changes in the absence
and presence of AHHPPC, as shown in Fig. 13, with the correspond-
ing parameters shown in Table 5. Peak A is the Rayleigh scattering
peak (kex = kem), whereas the strong peak B mainly reveals the
spectral characteristic of Trp and Tyr residues. Analysis of the
intensity changes of peak B of HSA showed different degrees of dif-
ferences after the addition of AHHPPC. This implies that the inter-
actions of AHHPPC with HSA induce a slight unfolding of the
polypeptide backbone of the protein, resulting in conformational
changes that increase the exposure of some hydrophobic regions
that had been buried [50]. These results are in accordance with
the UV, synchronous fluorescence and CD spectral data. All of these
phenomena indicate that the binding of AHHPPC to HSA induces
conformational and microenvironmental changes in HSA.
a
a
a
5
7.93% of a-helix under experimental conditions.
The CD spectra of HSA exhibited a characteristic of the typical
a-helix structure of the free HSA and its AHHPPC complex with
negative bands at 208 and 217 nm. The binding of AHHPPC to
HSA caused a decrease in band intensity at all wavelength of the
far- UV CD without any significant shift of the peaks, indicating
the decrease of the
a-helical content in protein. However, the CD
spectra of HSA in presence and absence of AHHPPC are similar in
shape, indicating that the structure of HSA is also predominantly
a
-helical. From the above results, it is apparent that the effect of
AHHPPC on HSA causes a conformational change of the protein,
with the loss of -helical stability. The calculating results exhibited
a reduction of -helix structures from 57.93% to 52.33% at molar
a
a
4. Conclusions
ratio AHHPPC/HSA of 1:1. We can conclude that AHHPPC actually
induce little micro-environmental changes around the fluorophore
We used different approaches to explore the interactions be-
tween AHHPPC and HSA under physiological conditions. The inter-
actions were confirmed through alterations in the UV–vis
absorption spectra and quenching of the intrinsic fluorescence of
the protein. The results indicated that the probable mechanism
of interaction is a static quenching process. The binding process
was endothermic, entropy driven and spontaneous, as indicated
by the thermodynamic parameters analyzed, and the major part
of the action force is hydrophobic interactions. Such interactions
seem to slightly induce microenvironmental changes and alter-
ation in protein conformation as shown by synchronous fluores-
cence, circular dichroism (CD) and three-dimensional studies. On
the efficiency of energy transfer between the donor and acceptor,
the distance between the fluorophore (Trp-214) of HSA and
AHHPPC was estimated to be r = 3.13 nm. The biological signifi-
cance of this work was evident since HSA serves as a carrier mole-
cule for multiple drugs and the interaction of AHHPPC and
HSA was not characterized so far. Hence, the report had a great
(
Trp-214) and slightly modify (unfold) the secondary structure of
HSA.
3.6.3. Three-dimensional fluorescence spectra
To obtain more information on the binding of AHHPPC to HSA
and to know conformational change of protein, three-dimensional
spectroscopy were investigated on HSA and HSA–AHHPPC com-
plex. The three-dimensional fluorescence spectra are a rising
analysis technique in recent years. It is well known that three-
dimensional fluorescence spectrum can provide more detailed
information about the configuration of proteins and can be ac-
quired by changing excitation and emission wavelength simulta-
neously [49]. If there is a shift at the excitation or emission
wavelength around the fluorescence peak, or the appearance of a
new peak or disappearance of existing peak, it could be an impor-
tant hint to suggest conformational changes to the protein. The

8
V.D. Suryawanshi et al. / Journal of Photochemistry and Photobiology B: Biology 118 (2013) 1–8
importance in pharmacology and clinical medicine as well as
methodology. Therefore, this is also expected to open the door to
new avenues in the screening and design of appropriate pyrimi-
dine-based drugs that will be of important in modern medical re-
search. All these experimental results and theoretical data clarified
that AHHPPC could bind to HSA and be effectively transported and
eliminated in body, which could be a useful guideline for further
drug design.
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Acknowledgements
The authors (VDS) gratefully acknowledges receiving a fellow-
ship from UGC, New Delhi [University Grant Commission, XIth plan
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for providing funds to department under FIST and SAP programme.
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