Investigation of the association behaviors between bovine serum albumin and 2-(4-methylphenyl)-3-(N-acetyl)-5-(2,4-dichlorophenoxymethyl)-1,3,4- oxodiazoline

Article abstract of DOI:10.1016/j.saa.2013.04.013

The study was designed to examine the interaction between 2-(4-methylphenyl)-3-(N-acetyl)-5-(2,4-dichlorophenoxymethyl)-1,3, 4-oxodiazoline (MPNDO) and bovine serum albumin (BSA) under physiological conditions by using fluorescence spectroscopy, ultraviol

Full text of DOI:10.1016/j.saa.2013.04.013

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 111 (2013) 260–265
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Investigation of the association behaviors between bovine serum albumin
and 2-(4-methylphenyl)-3-(N-acetyl)-5-(2,4-dichlorophenoxymethyl)-1,3,4-
oxodiazoline
⇑
Zhenzhong Huang , Ruiling Wang, Erwei Han, Lifan Xu, Yonghai Song
College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
We have synthesized a new
compound MPNDO.
Formation of BSA–MPNDO
bioconjugates were monitored by
atomic force microscope.
The work was studied by several
measurements.
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
The study was designed to examine the interaction between 2-(4-methylphenyl)-3-(N-acetyl)-5-(2,4-
dichlorophenoxymethyl)-1,3,4-oxodiazoline (MPNDO) and bovine serum albumin (BSA) under physio-
logical conditions by using fluorescence spectroscopy, ultraviolet absorption spectroscopy, FT-IR spec-
troscopy and circular dichroism spectroscopy and atomic force microscope. Spectroscopic analysis of
the fluorescence emission quenching and ultraviolet absorption revealed that the quenching mechanism
of bovine serum albumin by MPNDO was static quenching procedure. The binding constant and binding
sites number at different temperatures were measured. The average binding distances between donor
(BSA) and acceptor (MPNDO) was estimated to be 1.46 nm (301 K), based on the Föster non-radioactive
energy transfer theory. An average size of 3.1 nm had a high proportion and these dots might be ascribed
to BSA, some other dots with an average size of 6.6 nm might result from BSA–MPNDO bioconjugates
while the average diameter of MPNDO was 1.6 nm, which was reasonable to conclude that one BSA–
MPNDO bioconjugates consisted of one BSA and one MPNDO. The thermodynamic parameters, enthalpy
Received 25 December 2012
Received in revised form 16 March 2013
Accepted 2 April 2013
Available online 10 April 2013
Keywords:
2-(4-Methylphenyl)-3-(N-acetyl)-5-(2,4-
dichlorophenoxymethyl)-1,3,4-oxodiazoline
Bovine serum albumin
Atomic force microscope
Fluorescence spectroscopy
FT-IR
Circular dichroism spectroscopy
change (DH), entropy change (DS) and free energy change (DG) were calculated, which indicated that the
action force was mainly van der Waals forces. The data collected through synchronous fluorescence, FT-IR
spectroscopy and circular dichroism spectroscopy demonstrated that the conformation of BSA was not
affected obviously in the presence of MPNDO.
Ó 2013 Elsevier B.V. All rights reserved.
⇑
Corresponding author. Tel.: +86 13979135290.
E-mail address: fenxi5407@163.com (Z. Huang).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.04.013

Z. Huang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 111 (2013) 260–265
261
under ambient conditions. FT-IR measurement was carried out at
room temperature on a Nicolet 6700 FT-IR spectrometer with a
germanium attenuated total reflection (ATR) accessory (Thermo
Nicolet, USA). The CD measurements were performed at 1.0 nm
Introduction
1,3,4-Oxadiazolines derivatives have attracted great much
attention mainly due to some of their biological properties such
as insecticidal activity, antifungal activity, anti-HIV and anticon-
vulsive activity [1–3]. Up to now, most papers have focused on
1,3,4-Oxadiazolines derivatives, however, to our best knowledge,
MPNDO, which belongs to 1,3,4-phenoxy oxazoline derivatives
have not been reported. Due to the higher biological properties
compared with 1,3,4-Oxadiazolines, it is significantly meaningful
to obtain further product of higher activity and make a part of
the early work to do some guidance for new drug synthesis. Serum
albumins are the major soluble protein constituents of the circula-
tory system and can play a dominant role in the transport and dis-
position of various compounds such as metabolites, drugs, and
other biologically active substances, mostly through the formation
of noncovalent complexes at specific binding sites [4–6]. Thus
study on the combination of serum albumin with drugs and their
derivatives is of great significance not only in providing basic infor-
mation about the nature of drugs and pharmacokinetics but also in
explaining the relationship between the structures and functions
of drugs [7]. Bovine serum albumin (BSA) is usually selected as
the protein model because of the advantages of abundance, medi-
cal importance, low cost, stability, unusual ligand-binding proper-
ties, and also particularly owing to its structural homology with
human serum albumin [8]. BSA is made up of 637 amino acid res-
idues, two of which are tryptophans located at positions 134 and
212. The structure of albumin at physiological pH is predominantly
interviews with
a MOS-450 spectrometer (Biologic, France)
equipped with a 0.1 cm path-length cell at 301 K. NMR spectra
were recorded on an Avance 400 (Bruker, Germany). A FE20 pH
meter with a combined glass electrode (Mettler Toledo, Shanghai,
China) was used for pH measurements. ESI mass spectrum was re-
corded using a Waters ZQ4000/2695 LC–MS spectrometer (USA).
Element analysis was performed on the Vario EL III (Elementar).
Materials
The compound MPNDO was prepared according to the refer-
ence [11] and the route for synthesis is shown in Scheme 1. Com-
pound 2 (0.005 mol) was dissolve in acetic anhydride (20 mL) in
50 mL round bottom flask. The mixture was heated to reflux for
9 h with stirring, and then transferred into 250 mL ice water, stir-
red for 1 h. The precipitation was separated from the solution by
filtration and recrystallized with N,N-dimethylformamide, finally,
white crystals were obtained. The compound MPNDO was charac-
terized by 1H NMR spectrum, 13C NMR spectrum, IR spectrum,
which were consistent with proposed formulation and the struc-
ture of MPNDO was shown in Scheme 1. The melting point (mp)
was measured as 176 °C. 1H NMR (CDCl3, TMS): d 2.420 (s, 3H,
PhACH3), 5.239 (s, 2H, COCH2), 2.524 (s, 3H, OCH3), 6.788–6.810
(d, 1H, J = 8.8 Hz, ArAH), 7.145–7.167 (t, 1H, J = 8.8 Hz, ArAH),
7.261–7.277 (d, 2H, J = 64 Hz, Ar–H), 7.387–7.393 (t, 1H,
J = 24 Hz, ArAH), 7.656–7.675 (d, 2H, J = 76 Hz, ArAH), 8.574 (s,
H, CAH). 13C NMR (CDCl3): d 21.67, 21.86, 25.97, 69.89, 114.82,
124.17, 126.82, 127.48, 128.54, 129.70, 129.85, 129.99, 130.30,
a-helical (67%) with the remaining polypeptide occurring in turns
and extended or flexible regions between sub-domains with no b-
sheets [9]. Bovine serum albumin (BSA) is composed of three struc-
turally homologous domains (I, II and III), each domain contains
two sub-domains (A and B). Trp-134 is in the first domain and lo-
cated on the surface of the molecule, while Trp-212 is in the second
domains and located within a hydrophobic binding pocket [10].
The spectroscopic technique, including UV–vis absorption spec-
troscopy, fluorescence spectroscopy, FT-IR spectroscopy and circu-
lar dichroism spectroscopy, is of great help in studying interactions
of small molecules with protein. Fluorescence assay has been
widely used to investigate proteins, and because of the sensitivity
of the fluorescence to the change of the biomoleculars, researchers
can use fluorescence measurements to understand molecular
interaction. In this paper, measurements including atomic force
microscope, fluorescence quenching spectroscopy and ultraviolet
absorption spectroscopy serve as aids for studying the binding
mechanism and the effect of energy transfer. The conformation
change of BSA was further discussed on the basis of synchronous
fluorescence spectroscopy, FT-IR spectroscopy and circular dichro-
ism spectroscopy.
143.10, 152.62, 165.27, 168.37, 172.00. FTIR (KBr):
t
/cmꢁ1 1721
(s, C@O), 1642 (m, C@N), 1566, 1606 (m, Ar), 1278, 1067 (w,
CAOAC). EIMS: m/z (%) = 379 [M+]. Elemental analyses: C18H16Cl2-
N2O3, calculated C (57.01%), H (4.25%), N (7.39%), measured C
(56.86%), H (4.34%), N (7.31%).
The BSA was purchased from Shanghai Rich Joint Chemical Re-
agents Company. The solution was prepared into a concentration
of 1.0 ꢂ 10ꢁ5 mol Lꢁ1 by dissolving the solid BSA in a Tris–HCl buf-
fer. The Tris–HCl (0.05 mol Lꢁ1
, pH = 7.40) buffer containing
0.05 mol Lꢁ1 NaCl was selected to keep the pH value constant
and to maintain the ionic strength of the solution. Compound
MPNDO was prepared into a concentration of 1.0 ꢂ 10ꢁ4 mol Lꢁ1
in dimethyl sulphoxide (DMSO). DMSO (Alfa Aesar) is of spectro-
photometric grade. All other chemicals are of analytical grade.
Doubly distilled water was used throughout. All testing aqueous
solution contained 1% DMSO for determination.
Procedures
Experimental
A 10 lL aqueous solution containing appropriate concentration
of BSA (1.0 ꢂ 10ꢁ5 mol Lꢁ1), of MPNDO (7.5 ꢂ 10ꢁ6 mol Lꢁ1), of
BSA–MPNDO conjugates was dropped onto a freshly cleaved mica
surface, respectively. After that, the mica was rinsed with pure
water and dried in air for AFM imaging. Appropriate amounts of
1.0 ꢂ 10ꢁ4 mol Lꢁ1 MPNDO were added to 6 10 mL flasks, respec-
tively, and then 1 mL of BSA solution was added and diluted to
10 mL with Tris–HCl buffer. The final concentrations of MPNDO
Apparatus
All fluorescence measurements were carried out on a LS55 spec-
trofluorimeter (PerkinElmer, USA) equipped with a TB-85 Thermo
Bath (Shimadzu, Japan) and a 1.0 cm quartz cells. The emission
and excitation slits were 5 nm, 10 nm, respectively, and the scan
speed was 500 nm minꢁ1. The UV-via absorption spectra were ob-
tained from a Lambda 35 UV–vis spectrophotometer (PerkinElmer,
USA) using a 1.0 cm quartz cell. AFM measurements were studied
with an AJ-III AFM instrument (Aijian, Shanghai, China) in tapping
mode. Standard silicon (Si) cantilevers (spring constant,
0.6–6 N mꢁ1) were used under its resonance frequency (typically,
60–150 kHz). All AFM images were acquired at room temperature
in BSA solution were 0.0, 1.0, 2.0, 3.0, 4.0 and 5.0 ꢂ 10ꢁ6 mol Lꢁ1
,
respectively. The resultant mixtures were then incubated at
301 K for 0.5 h. The fluorescence spectra of BSA under different
concentrations of MPNDO were tested in the range of 300–
500 nm upon excitation at 280 nm, each spectrum was the average
of three scans. The synchronous fluorescence spectra were noted at
certain scanning intervals
Dk (Dk = 15 nm, 60 nm). 20 lL solution

262
Z. Huang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 111 (2013) 260–265
Scheme 1. The synthesis route for MPNDO.
containing appropriate concentration of BSA (1.0 ꢂ 10ꢁ4 mol Lꢁ1),
of BSA–MPNDO conjugates was dropped on ZnSe window and
dried with dry air. The FT-IR spectra of BSA in presence and ab-
sence of MPNDO taken via the ATR method with resolution of
4 cmꢁ1 and 32 scans were recorded (n = 3 replicates) in the range
of 400–4000 cmꢁ1. The corresponding absorbance contributions
of buffer and free MPNDO solutions were recorded and digitally
substracted with the same instrumental parameters. The CD spec-
tra of BSA (5.0 ꢂ 10ꢁ7 mol Lꢁ1) in presence and absence of MPNDO
(1.0 ꢂ 10ꢁ8 mol Lꢁ1) were corrected for buffer absorption and re-
corded in the range of 190–250 nm, and each spectrum was the
average of three scans.
600
500
400
300
200
100
0
a
f
300
350
400
450
500
Wavelength/nm
Results and discussion
Fig. 2. Fluorescence spectra of BSA with the addition of MPNDO at 301 K.
c(BSA) = 1.0 ꢂ 10ꢁ6 mol Lꢁ1
; 5
c(MPNDO)/(10ꢁ6 mol/L), a–f: 0, 1, 2, 3, 4,
Mechanism of fluorescence quenching
respectively.
AFM study
Formation of BSA–MPNDO bioconjugates were monitored by
AFM (Fig. 1). All samples showed many small dots dispersed on
mica surface uniformly and these dots might be ascribed to BSA,
BSA–MPNDO bioconjugates or MPNDO. From Fig. 1A, an average
size of 3.1 nm had a high proportion and these dots might be as-
cribed to BSA, another dots with an average size of 6.6 nm might
result from BSA–MPNDO bioconjugates (Fig. 1B). Since the size of
the BSA was about 3.1 nm and the size of the MPNDO was about
1.6 nm (Fig. 1C), it was reasonable to conclude that one BSA–
MPNDO bioconjugates consisted of one BSA and one MPNDO. A
few dots with a larger size might result from BSA–MPNDO con-
sisted of many BSA and many MPNDO.
in Fig. 2). The results showed that a gradual decrease in the
fluorescence intensity of BSA was caused by quenching but
there was no emission wavelength shift in the presence of
MPNDO, the same trends were also observed at other temperatures
(307 K, 313 K).
Quenching can be induced by dynamic process resulting from
collisional encounters between fluorophore and quencher, or static
process caused by the formation of a ground-state complex be-
tween the two compounds. Dynamic and static quenching can be
distinguished by their different dependence upon the temperature
of binding constants and viscosity [12]. Generally, the dynamic
quenching constants are expected to increase with increasing tem-
perature, but the reverse effect is for static quenching [12]. For dy-
namic quenching, the fluorescence data at different temperatures
were analyzed by the well-known Stern–Volmer Equation [12]:
Fluorescence titration of BSA against MPDNO
The fluorescence spectra emission (kex = 280 nm, kem = 345.5
nm) of BSA with the addition of MPNDO were obtained (shown
Fig. 1. AFM images of BSA (A); BSA–MPNDO (B); MPNDO (C). c(BSA) = 1.0 ꢂ 10ꢁ5 mol Lꢁ1; c(MPNDO) = 7.5 ꢂ 10ꢁ6 mol Lꢁ1
.

Z. Huang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 111 (2013) 260–265
263
1.24
1.20
1.16
1.12
1.08
1.04
in the presence and absence of MPNDO are shown in Fig. 4. The
absorption of BSA (curve a) is characterized by a strong band at
278 nm and MPNDO has a weak absorption band at 298 nm (curve
b). Compared with curve d (sum of curve a and curve b), the addi-
tion of MPNDO led to increase in the BSA absorption with a new
absorption peak at 320 nm (curve c), which may be result from
the formation of a ground-state complex. The above observations
further confirmed that the probable quenching mechanism be-
tween BSA and MPNDO is initiated ground-state complex
formation.
301K
307K
313K
Binding constant and binding sites
1
2
3
4
5
For static quenching, if it is assumed that there are similar and
independent binding sites in the biomolecule, the binding constant
and the number sites can be determined according to the method
described by Chipman et al., using the following equation [16,17]:
106 [MPNDO]
Fig. 3. The Stern–Volmer plots of the fluorescence quenching of BSA by MPNDO at
different temperatures.
log½ðF0 ꢁ FÞ=Fꢃ ¼ log Ka þ n log½Qꢃ
ð2Þ
F0=F ¼ 1 þ Kqs0½Qꢃ ¼ 1 þ KSV ½Qꢃ
ð1Þ
where Ka is the binding constant and n is the number sites. Accord-
ing to the plot of log(F0 ꢁ F)/F versus log[Q] at different tempera-
tures (Fig. 5.), the binding constant Ka and binding sites n of
MPNDO to BSA are estimated as 2.01 ꢂ 104 L molꢁ1, 0.94 (301 K),
2.32 ꢂ 103 L molꢁ1, 0.77 (307 K), 8.54 ꢂ 102 L molꢁ1, 0.70 (313 K),
respectively. The values of Ka decreased with increasing tempera-
ture, which was assumed to decrease the stability of MPNDO–BSA
complex. And the values of n decreased with increasing tempera-
ture, which might be due to changes in the protein structure with
temperature [18,19].
where F0 and F are the fluorescence intensities in the absence and
presence of drug, respectively, Kq is the biomolecular quenching
rate constant, s0 is the average lifetime of molecule in the absence
of drug and its value is 10ꢁ8 s [13], [Q] is the drug concentration, KSV
is the Stern–Volmer quenching constant.
The Stern–Volmer plots are presented in Fig. 3 and the values of
KSV were 4.2 ꢂ 104 L molꢁ1 (301 K), 3.5 ꢂ 104 L molꢁ1 (307 K),
2.8 ꢂ 104 L molꢁ1 (313 K), KSV decreased with a rising temperature,
which indicated that the fluorescence quenching mechanism may
be static. The calculated quenching constants at the corresponding
temperature constant Kq obtained were calculated as 4.2, 3.5,
2.8 ꢂ 1012 L molꢁ1 sꢁ1. It was found all the values of Kq were larger
than the maximum diffusion collision quenching rate constant of
various quenchers with the biopolymer [14]. This result indicated
that the quenching mechanism between BSA and MNPDO was
mainly a static quenching process.
The acting force between MPNDO and BSA
The interaction forces between a ligand and protein may in-
clude hydrophobic force, electrostatic interactions, van der Waals
forces and hydrogen bonds. The thermodynamic parameters can
account for main forces contributing to protein stability[20], and
according to the views of Ross and Subramanian [21]: (1)
and S > 0, hydrophobic forces, (2) H < 0 and S < 0, Van der
Waals interaction and hydrogen bond, (3) H < 0 and S > 0, elec-
DH > 0
D
D
D
UV–vis absorption spectrum
D
D
UV–vis absorption measurement is also a simple but effective
method in detecting static quenching and dynamic quenching
[15]. As dynamic collision only affects the excited state of quench-
ing molecules, whereas it has no influence on the absorption spec-
trum of quenching substance. However, ground-stated complex
formation will frequently result in perturbation of the absorption
spectrum of the fluorophore [13]. The absorption spectra of BSA
trostatic interactions. The enthalpy is often taken to be a constant if
the temperature of system is changed a little. Van’t Hoff Equations
[21] are used to obtain the thermodynamic parameters.
ln Ka ¼ ꢁ
D
H=ðR TÞ þ
D
S=R;
D
G ¼
D
H ꢁ T
D
S ¼ ꢁR T lnK
ð3Þ
where Ka is binding constant at the different temperatures and R is
the gas constant. The values of
DH, DS and DG were calculated to be
ꢁ205.2 kJ molꢁ1
,
ꢁ602.6 J molꢁ1 Kꢁ1
,
ꢁ23.1 kJ molꢁ1
.
Obviously,
0.8
0.6
-0.6
-0.8
-1.0
-1.2
301K
307K
313K
c
d
0.4
0.2
0.0
a
b
200
250
300
350
400
Wavelength/nm
-6.0
-5.8
-5.6
-5.4
-5.2
Fig. 4. UV–vis spectrum (a) the absorption spectrum of BSA only; (b) the absorption
lg [MPNDO]
spectrum of MPNDO only; (c) the absorption spectrum BSA–MPNDO system; (d) the
absorption spectrum sum of (a) and (b). c(BSA) = 7.5 ꢂ 10ꢁ6 mol Lꢁ1
;
c(MPNDO) = 2 ꢂ 10ꢁ5 mol Lꢁ1
.
Fig. 5. The plots of log(F0 ꢁ F)/F versus log c(MPNDO) at different temperatures.

264
Z. Huang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 111 (2013) 260–265
0.10
600
400
200
0
20
10
0
0.08
0.06
0.04
0.02
0.00
a
-10
-20
-30
-40
b
b
a
200
210
220
230
240
250
300
350
400
450
500
λ/nm
Wavelength (nm)
Fig. 6. Spectra overlap between the fluorescence emission of BSA (a) and the
Fig. 8. CD spectra of BSA in the absence and presence of MPNDO
absorption spectrum of MPNDO (b). c(BSA) = c(MPNDO) = 1.0 ꢂ 10ꢁ6 mol Lꢁ1
.
c(BSA) = 5.0 ꢂ 10ꢁ7 mol Lꢁ1; c(MPNDO) = 1.0 ꢂ 10ꢁ8 mol Lꢁ1
.
both the entropy and the enthalpy were negative. So it can be con-
cluded that the Van der Waals interaction and hydrogen bond play
major roles in the acting force. However, given the structural for-
mula of MPNDO does not contain –XH (X = O, S, N) group, the pos-
sibility of forming hydrogen bond between BSA and MPNDO is quite
low. Therefore, it can be concluded that the Van der Waals interac-
tion was the main force during the binding of MPNDO to BSA.
of the donor, and J expresses the degree of spectral overlap between
the donor emission and the acceptor absorption. J could be calcu-
lated by the equation:
.
X
X
J ¼
ðFDðkÞꢀAðkÞk4DkÞ
ðFDðkÞ
Dk
ð6Þ
where F(k) is the fluorescence intensity of the fluorescent donor of
wavelength k and
acceptor at wavelength k. In the present case, K2 = 2/3, N = 1.336,
and = 0.15 for BSA [23]. The overlap of the fluorescence emission
e(k) is the molar absorption coefficient of the
Energy transfer between MPNDO and BSA
U
Förster’s non-radioactive energy transfer theory is often used to
determine the drug binding distance between the donor and
acceptor under the following conditions: the donor can produce
fluorescence light, the fluorescence emission spectrum of donor
and the UV–vis absorption spectrum of the acceptor have enough
overlap, the distance between the donor and acceptor approaches
is less than 8 nm [22]. The efficiency of energy transfer (E) can be
expressed according to Förster’s energy-transfer theory [22]:
spectrum of BSA and the absorption spectrum of MPNDO are shown
in Fig. 6, together with above equations, we could obtained these
parameters: J = 9.10 ꢂ 10ꢁ14 cm3 L molꢁ1, and the binding distance
r between MPNDO and the BSA is found to be 1.46 nm, indicating
that the energy transfer from BSA to MPNDO occurred.
Conformational investigation
E ¼ R60=ðR06 þ r6Þ ¼ 1 ꢁ F=F0
ð4Þ
Synchronous fluorescence spectral change of BSA
The synchronous fluorescence spectrum can give only the tyro-
sine residues and the tryptophan residues of BSA when the wave-
where F and F0 are the fluorescence intensities of BSA in the pres-
ence and absence of MPNDO, r is the distance between the acceptor
and donor, and R0 is the critical distance when the transfer effi-
ciency is 50%. The value of R0 was evaluated using the following
equation:
length internal (Dk) is 15 nm and 60 nm, respectively [24]. The
shift in position of maximum emission wavelength correspond to
changes of the polarity around the chromospheres molecule, red
shift of fluorescence spectrum suggests the polarity of the sur-
rounding environment increasing, the hydrophobicity decreasing,
and vice versa [18], thus the environment of amino acid residues
can be studied by measuring the possible shift in maximum emis-
sion wavelength [25–26].
R60 ¼ 8 ꢂ 10ꢁ25K2
U
Nꢁ4
J
ð5Þ
where K2 is the spatial orientation factor of the dipole, N is the
refractive index of the medium,
U is the fluorescence quantum yield
1000
300
A
B
a
800
a
f
f
200
100
0
600
400
200
0
260
280
300
320
260
280
300
320
Wavelength/nm
Wavelength/nm
Fig. 7. Synchronous fluorescence spectra of BSA in the present of MPNDO. ((A)
1, 2, 3, 4, 5, respectively.
D
k = 15 nm and (B)
D
k = 60 nm). c(BSA) = 1.0 ꢂ 10ꢁ6 mol Lꢁ1; c(MPNDO)/(10ꢁ6 mol/L), a–f: 0,

Z. Huang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 111 (2013) 260–265
265
The synchronous fluorescence spectra were measured at
k = 15 nm and k = 60 nm and presented in Fig. 7. As it can be
References
D
D
[1] S. Rohas, N. Gulerman, H. Erdeniz, Synthesis and antimicrobial activity of some
new hydrazones of 4-fluombenzoic acid hydrazide and 3-acetyl-2,5-
disubstituted-1,3,4-oxadiazolines, Farmaco 57 (2002) 171–174.
[2] J.H. Michael, P.Y. Chanyaputhipong, Preparation and Spectroscopic of 3-Xcyl-
1,3,4-oxadiazolines, Heterocyclic Chem. 32 (1995) 1647–1649.
[3] F.M. Liu, J.X. Ye, Synthesis of heterocyclic compounds from 2-ohenyi-1,2,3-
triazole-4-formyl-hydrazine, Chin. J. Chem. 17 (1999) 62–68.
[4] X.M. He, D.C. Carter, Atomic structure and chemistry of human serum albumin,
Nature 358 (1992) 209–215.
[5] B.X. Huang, H.Y. Kim, C. Dass, Probing three-dimensional structure of bovine
serum albumin by chemical cross-linking and mass spectrometry, J. Am. Soc.
Mass Spectrom. 15 (2004) 1237–1247.
[6] J. Zhang, X.J. Wang, Y.J. Yan, W.S. Xiang, Comparative Studies on the Interaction
of Genistein, 8-Chlorogenistein, and 30,8-Dichlorogenistein with Bovine
Serum Albumin, J. Agric. Food Chem. 59 (2011) 7506–7513.
[7] S.Y. Shi, Y.P. Zhang, X.Q. Chen, M.J. Peng, Investigation of flavonoids |bearing
different substituents on ring C and their Cu2+ complex binding with bovine
serum albumin: structure affinity relationship aspects, J. Agric. Food Chem. 59
(2011) 10761–10769.
[8] X.L. Han, P. Mei, Y. Liu, Q. Xiao, F.L. Jiang, Ran Li, Binding interaction of
quinclorac with bovine serum albumin: a biophysical study, Spectrochim. Acta
A 74 (2009) 781–787.
[9] D.C. Carter, J.X. Ho, Structure of serum albumin: AdV, Protein Chem. 45 (1994)
153–203.
[10] T. Peter, Serum albumin, AdV. Protein Chem. 37 (1985) 161–245.
[11] J.F. Hu, D.J. Li, Q.H. Fu, Synthesis and characterization of 1,3,4-oxadiazolines
derivatives, J. Chongqing Univ. (Nat. Sci. Ed.) 4 (2006) 129–132.
[12] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, third ed., Springer
Press, New York, 2006.
seen that the emission peak intensity of BSA gradually declines
upon addition of the MPNDO, while no significant shift change
on maximum emission wavelength was observed. These experi-
mental results indicated that the protein conformation was not af-
fected obviously during the binding process.
Fourier Transform Infrared (FT-IR) study
Infrared spectrum of a protein exhibit a number of amide bands,
which represent different vibrations of the peptide moiety. Among
all amide modes of the peptide group, the single most widely used
one in studies of protein secondary structures is the amide I. The
amides I and II peak occur in the region of 1600–1700 cmꢁ1 and
1500–1600 cmꢁ1, respectively. Amide I band is more sensitive to
the changes in protein secondary structure compared to amide II.
Hence, the amide I band is more useful for the study of protein sec-
ondary structure [27–30]. FT-IR spectra of BSA (1.0 ꢂ 10ꢁ4 mol Lꢁ1
)
were recorded with and without the addition of MPNDO. No
changes were observed in the spectra of the amide I and N–H resid-
ual amide II bands, which were present at 1656 and 1557 cmꢁ1
,
respectively (data not shown). This further confirmed that no
changes to the secondary structure of BSA had occurred because
of the interaction with the MPNDO.
[13] J.R. Lakowica, G. Weber, Quenching of fluorescence by Oxygen A probe for
structural fluctuantions in macromolecules, Biochemistry 12 (1973) 4161–
4170.
[14] M.R. Eftink, C.A. Ghiron, Fluorescence quenching studies with proteins, Anal.
Biochem. 114 (1981) 199–212.
[15] S.Y. Bi, D.Q. Song, Y. Tian, X. Zhou, Z.Y. Liu, H.Q. Zhang, Molecular spectroscopic
study on the interaction of tetracyclines with serum albumins,
Spectrochimica. Acta Part A 61 (2005) 629–636.
[16] M. Alain, B. Michel, D. Michel, How to illustrate ligand–protein binding in a
class experiment, Chem. Educ. 63 (1986) 365.
[17] D.M. Chipman, V. Grisaro, N. Sharon, The binding of oligosaccharides
containing N-acetylplucosamine and N-acetylmuramic acid to lysozyme, J.
Biol. Chem. 242 (1967) 4388–4394.
[18] X.J. Guo, X.D. Sun, S.K. Xu, Spectroscopic investigation of the interaction
between riboflavin and bovine serum albumin, J. Mol. Struct. 931 (2009) 55–
59.
[19] L.N. Zhang, F.Y. Wu, A.H. Liu, Study of the interaction between 2,5-di-[2-(4-
hydroxy-phenyl) ethylene]-terephthalonitril and bovine serum albumin by
fluorescence spectroscopy, Spectrochim. Acta A: Mol. Biomol. Spectrosc.
(2011) 1386–1425.
[20] S.S. Lehrer, , Solute perturbation of protein fluorescence. The quenching of the
tryptophyl fluorescence of model compounds and of lysozyme by iodide ion,
Biochemistry 10 (1971) 3254–3263.
Circular Dichroism (CD) study
Additional evidence regarding no conformational changes of
BSA in the presence of MPNDO came from the Circular Dichroism
(CD) spectroscopy, one of the valuable methods for better under-
standing protein secondary structure. To obtain an insight into
the conformational behavior of BSA in presence and absence of
MPNDO, CD spectroscopic measurements were performed. The
CD spectrum of BSA exhibited two negative bands at 210 and
222 nm (curve a in Fig. 8), which is characteristic of
the advanced structure of the protein [31]. The band at 208 nm
corresponds to -helical, whereas the band
pꢄ transition of the
at 222 nm corresponds to -helical
pꢄ transition for both the
a-helical in
p–
a
p
–
a
and random coil [32]. It is clearly evident from Fig. 8 that CD spec-
trum of BSA (curve a) remains essentially similar to that of the
BSA-MPNDO conjugates (curve b) confirming no perturbation in
the secondary structure of the protein after the conjugation with
MPNDO.
[21] P.D. Ross, S. Subramanian, Thermodynamic of protein association reaction:
forces contributing to stability, Biochemistry 20 (1981) 3096–3120.
[22] T. Forster, O. Sinanglu, Modern Quantum Chemistry, vol. 3., Academic Press,
New York, 1996.
Conclusions
[23] Y.Z. Zhang, B. Zhou, Y.X. Liu, C.X. Zhou, X.L. Ding, Y. Liu, Fluorescence study on
the interaction of bovine serum albumin with P-aminoazobenzene, J. Fluoresc.
18 (2008) 109–118.
[24] J.N. Miller, Recent advances in molecular luminescence analysis, Anal. Proc. 16
(1979) 203–209.
[25] L. Stryer, Fluorescence spectroscopy of proteins, Science 162 (1968) 526.
[26] B.S. Liu, F.L. Zhao, C.L. Xue, J. Wang, Y.K. Lu, Studies on the antagonistic action
between chloramphenicol and quinolones with presence of bovine serum
albumin by fluorescence spectroscopy, J. Lumin. 130 (2010) 859–864.
[27] S. Wi, P. Pancoka, T.A. Keiderling, Predictions of protein secondary structures
using factor analysis on Fourier transform infrared spectra: effect of Fourier
self-deconvolution of the amide I and amide II bands, Biospectroscopy 4
(1998) 93–106.
[28] K. Rahmelow, W. Hubner, Secondary structure determination of proteins in
aqueous solution by infrared spectroscopy: a comparison of multivariate data
analysis methods, Anal. Biochem. 241 (1996) 5–13.
[29] S.Y. Lin, Y.S. Wei, M.J. Li, S.L. Wang, Effect of ethanol or and captopril on the
secondary structure of human serum albumin before and after protein
binding, Eur. J. Pharm. Biopharm. 57 (2004) 457–464.
[30] J.W. Brauner, C.R. Flach, R. Mendeisohn, A quantitative reconstruction of the
amide I contour in the IR spectra of globular proteins: from structure to
spectrum, J. Am. Chem. Soc. 127 (2005) 100–109.
In this paper, a new compound, 2-(4-methylphenyl)-3-(N-acet-
yl)-5-(2,4-dichlorophenoxymethyl)-1,3,4-oxodiazoline was syn-
thesized and the interaction between MPNDO and BSA has been
studied. The intrinsic fluorescence of BSA has been quenched by
MPNDO through static quenching mechanism. The average diame-
ter of BSA–MPNDO changed to be 6.6 nm from 3.1 nm (BSA),
1.6 nm (MPNDO), the binding constant, binding sites number and
average binding distances between BSA and MPNDO at different
temperatures were obtained. The thermodynamic parameters indi-
cated that the action force was mainly van der Waals forces. The
microenvironment and conformation of BSA were demonstrated
not to be changed obviously in the presence of MPNDO by synchro-
nous fluorescence spectra, FT-IR spectra, and circular dichroism
spectroscopy.
Acknowledgements
[31] N. Vigneshwaran, A.A. Kathe, P.V. Varadarajan, R.P. Nachane, R.H.
Balasubramanya, Langmuir 23 (2007) 7113–7117.
[32] V.M. Bolanos-Garcia, S. Ramos, R. Castillo, J. Xicohtencatl Cortes, J.J. Mas-Oliva,
Phys. Chem. B 105 (2001) 5757–5765.
We thank the National Natural Science Foundation of China
(No. 20905032) and the Research Program of Jiangxi Province
Department of Education (GJJ12201) for the financial support.