Spectroscopic analyses on interaction of Amantadine-Salicylaldehyde, Amantadine-5-Chloro-Salicylaldehyde and Amantadine-o-Vanillin Schiff-Bases with bovine serum albumin (BSA)

Article abstract of DOI:10.1016/j.saa.2011.08.076

In this work, three Tricyclo [3.3.1.1(3,7)] decane-1-amine (Amantadine) Schiff-Bases, Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro- Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin (AS-o-V), were synthesized by direct heating reflux method in et

Full text of DOI:10.1016/j.saa.2011.08.076

Spectrochimica Acta Part A 83 (2011) 511–517
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectroscopic analyses on interaction of Amantadine-Salicylaldehyde,
Amantadine-5-Chloro-Salicylaldehyde and Amantadine-o-Vanillin
Schiff-Bases with bovine serum albumin (BSA)
Zhiqiu Wang, Jingqun Gao, Jun Wang∗, Xudong Jin, Mingming Zou, Kai Li, Pingli Kang
College of Chemistry, Liaoning University, Shenyang 110036, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2011
Received in revised form 29 August 2011
Accepted 31 August 2011
In this work, three Tricyclo [3.3.1.1(3,7)] decane-1-amine (Amantadine) Schiff-Bases, Amantadine-
Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin
(AS-o-V), were synthesized by direct heating reflux method in ethanol solution and characterized by
infrared spectrum and elementary analysis. Fluorescence quenching was used to study the interaction
of these Amantadine Schiff-Bases (AS, AS-5-C and AS-o-V) with bovine serum albumin (BSA). According
to fluorescence quenching calculations the bimolecular quenching constant (Kq), apparent quenching
constant (KSV), effective binding constant (KA) and corresponding dissociation constant (KD), binding site
number (n) and binding distance (r) were obtained. The results show that these Amantadine Schiff-Bases
can obviously bind to BSA molecules and the binding strength order is AS < AS-5-C = AS-o-V. Synchronous
fluorescence spectroscopy reveals that these Amantadine Schiff-Bases adopt different way to bind with
BSA molecules. That is, the AS and AS-5-C are accessibility to tryptophan (Trp) residues more than the
tyrosine (Tyr) residues, while the AS-o-V is equally close to the Tyr and Trp residues.
Keywords:
Spectroscopic analysis
Interaction
Bovine serum albumin (BSA)
Amantadine Schiff-Base
Salicylaldehyde derivant
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
activities [16–18]. Aromatic Schiff-Bases have also been used
as efficient herbicide, pesticide and bactericide [19,20]. In order
Tricyclo [3.3.1.1(3,7)] decane-1-amine (Amantadine) is an
antivirotic drug that has been used to treat influenza and Parkinson
disease [1–6]. However, in recent years, influenza is often caused by
viruses and germs at the same time [7,8]. Because it has only antivi-
rotic activity, Amantadine cannot be used to treat current influenza.
Therefore, it is necessary to develop the difunctional or multifunc-
tional anti-influenza drug, which not only has antiviral activity but
also does antibacterial action [9].
Aromatic Schiff-Bases are a large class of organic compounds
with imino (–HC N–) group and simple structures [10–12].
The aromatic plane structure and strong binding ability lead
to bacteriostatic, fungicidal, anti-tumour, and anti-viral biolog-
ical activities. Hence, many aromatic Schiff-Base compounds
have developed as antibacterial, antitumour and antivirotic drugs
in recent years [13–15]. 2-Hydroxybenzaldehyde (Salicylalde-
hyde), which has frequently been used to synthesis aromatic
Schiff-Base compounds, has many biological activities, such as anal-
gesic, anti-inflammatory, antibacterial, bactericidal and antiviral
to combine bacteriostatic ability and anti-viral activity in aro-
matic Schiff-Bases, in this work, the Salicylaldehyde and its
two analogous compounds (5-Chloro-2-hydroxybenzaldehyde (5-
Chloro-Salicylaldehyde) and 2-Hydroxy-3-methoxybenzaldehyde
(o-Vanillin)) were used to react with Amantadine, so that
three Amantadine Schiff-Bases (Amantadine-Salicylaldehyde (AS),
Amantadine-5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-
o-Vanillin (AS-o-V)) were synthesized [21]. There identify was
verified by elemental analysis and IR spectroscopy. The molecu-
lar structures of three synthesized Amantadine Schiff-Bases (AS,
AS-5-C and AS-o-V) are given in Fig. 1.
The well known serum albumin is the most abundant pro-
teins in the plasma [22–24], and has many physiological functions.
In this work, the bovine serum albumin (BSA) was chosen as a
target protein molecule because of its low cost, ready availabil-
ity, ligand-binding properties [25–27]. Moreover, the molecular
structure of BSA is similar to that of human serum albumin
(HSA) with 76% identify, so the results of the studies con-
ducted here are applicable to HSA [28]. Hence, the quenching
of intrinsic fluorescence is used to research the interaction
between three synthesized Amantadine Schiff-Bases (AS, AS-5-
C and AS-o-V) and BSA molecules. The interaction strength and
mode of binding were also studied by synchronous fluorescence
spectroscopy.
∗
Corresponding author. Tel.: +86 024 62207859; fax: +86 024 62202053.
E-mail address: wangjun890@126.com (J. Wang).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.08.076

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Z. Wang et al. / Spectrochimica Acta Part A 83 (2011) 511–517
spectrophotometer (Cary 50, Varian Company, USA). The composi-
tions of synthesized Amantadine Schiff-Bases were determined by
using an elementalanalyser (Perkin-Elmer 2400, PerkinElmer Com-
pany, USA). Their structures were analyzed by Fourier transform
infrared spectrophotometer (Spectrum 100, Perkin-Elmer Com-
pany, USA) and proton nuclear magnetic resonance (1H NMR)
spectroscopy(Mercury-Vx300, Varian Company, USA). The solution
pH value was measured with a pH meter.
2.3. Syntheses of Amantadine Schiff-Bases (AS, AS-5-C and
AS-o-V)
Three Amantadine Schiff-Bases, Amantadine-Salicylaldehyde
(AS, C17H21NO), Amantadine-5-Chloro-Salicylaldehyde (AS-5-C,
Fig. 1. Molecular structure of Amantadine-Salicylaldehyde (AS), Amantadine-5-
Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin (AS-o-V) Schiff-Bases.
C17H20NOCl) and Amantadine-o-Vanillin (AS-o-V, C18H23NO2),
were synthesized, respectively, through refluxing, distillation and
filtration of mixed ethanol solutions of Amantadine and Sal-
icylaldehyde, 5-Chloro-Salicylaldehyde and o-Vanillin. 4.1296 g
(0.022 mol) Amantadine hydrochloride and 1.2343 g (0.022 mol)
KOH were added to 50 mL ethanol in a 150 mL beaker and then
stirred for 24 h. The produced white precipitates (KCl) were filtered
out and the clear liquid was transferred to a 250 mL three-necked
flask. During the refluxing at 95 ◦C in oil bath, 50 mL Salicylalde-
hyde (2.4424 g, 0.020 mol) ethanol solution was added dropwise
to the above Amantadine ethanol solution. After 3.0 h reflux, the
mixed ethanol solution was concentrated to about 10 mL through
reduced pressure distillation. After naturally cooling to room tem-
perature, the AS Schiff-Base (pale yellow) microcrystals appeared
in the bottom of flask. The sample was filtrated and washed with
ethanol three times and dried at 120 ◦C to constant weight.
AS-5-C (yellow microcrystals) and AS-o-V (deep yellow micro-
crystals) Schiff-Bases were synthesized using similar methods
described above. The elemental analysis of these compounds is
given in Table 1, and the FT-IR and 1H NMR results are given in
Fig. 2 and Tables 2 and 3, respectively.
2. Experimental
2.1. Materials
Commercially prepared bovine serum albumin (BSA, purity
>98%. 4.8 isoelectric point, 16% nitrogen content, sugar content
0.08% and 0.2% fat content) was obtained from Beijing Abxing Bio-
logical Technology Company and stored in refrigerator at 4.0 ◦C. Tri-
cyclo [3.3.1.1(3,7)] decane-1-amine (Amantadine hydrochloride,
analytical reagent grade), 2-hydroxybenzaldehyde (Salicylalde-
hyde, analytical reagent grade), 5-Chloro-2-hydroxybenzaldehyde
(5-Chloro-Salicylaldehyde, analytical reagent grade) and 2-
Hydroxy-3-methoxybenzaldehyde (o-Vanillin, analytical reagent
grade) were purchased from Tianjing Tianhe Chemical Reagent
Co., Ltd. All other reagents were commercial products of ana-
lytical grade and used as received. The Tris (hydroxyl-methyl)
aminomethane (Tris), HCl and NaCl were all of analytical reagent
grade, and double distilled water was used for all solution prepa-
ration.
2.2. Apparatus and instruments
2.4. Measurement of binding parameters
The fluorescence measurements were performed on a flu-
orophotometer (Cary 300, Varian Company, USA) and the
UV–vis absorption spectra were recorded with an UV–vis
For each of the three Amantadine Schiff-Bases (AS, AS-5-C
and AS-o-V), the binding parameters with BSA molecules were
measured by fluorescence spectroscopy. BSA and Amantadine
Table 1
Elementary analyses of Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin (AS-o-V) Schiff-Bases.
Schiff-Bases
Molecular formula Molecular weight Colour
Melting point (◦C) C (%)
H (%)
N (%)
Calcd. 79.96 Calcd. 8.29 Calcd. 5.49
Found 79.94 Found 8.31 Found 5.53
92
–93
Amantadine-Salicylaldehyde
C17
C17
H
21NO
Pale yellow
255.34
289.79
285.37
Calcd. 70.46 Calcd. 6.96 Calcd. 4.83
Found 70.43 Found 6.98 Found 4.89
130
–133
Amantadine-5-Chloro-Salicylaldehyde
Amantadine-o-Vanillin
H20NOCl
Yellow
Calcd. 75.75 Calcd. 8.12 Calcd. 4.91
Found 75.72 Found 8.10 Found 4.96
104
–107
C
18H23NO2
Deep yellow
Fig. 2. Infrared spectra of Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin (AS-o-V) Schiff-Bases (with potas-
sium bromide (KBr) pellet at room temperature (about 25 ◦C)).

Z. Wang et al. / Spectrochimica Acta Part A 83 (2011) 511–517
513
Table 2
Infrared spectrum data (cm−1) of Amantadine (A), Salicylaldehyde (S), 5-Chloro-Salicylaldehyde (S-5-C), o-Vanillin (S-o-V), Amantadine-Salicylaldehyde (AS), Amantadine-
5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin (AS-o-V).
+
+
Schiff-Bases
ꢀ(OH)
ꢀ(C O)
ꢀ(C N)
ꢀas(NH3
)
ꢀs(NH3
)
ꢀ(Ar–Cl)
ꢀ(Ar–OCH3)
Amantadine
2921
2586
Salicylaldehyde
3422
3441
3435
3428
3435
3432
1663
1660
1639
Amantadine-Salicylaldehyde
5-Chloro-Salicylaldehyde
Amantadine-5-Chloro-Salicylaldehyde
o-Vanillin
1629
1627
1625
1276
1277
1258
1250
Amantadine-o-Vanillin
Table 3
1H NMR (ı in ppm and J in Hz) spectral data of Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin (AS-o-V) in
CDCl3.
Schiff-Bases
Adamantane ring-H
Benzene ring-H
CH
N
Ar–OH
OCH3
–
Amantadine-Salicylaldehyde
1.67–1.79 (m,6H,CH2); 1.85 (s,6H,CH2);
2.19 (s,3H,CH)
6.84 (t-like,3J = 7.5/8.1,1H); 6.93
(d,3J = 8.1,1H); 7.26
8.32(s,1H)
14.55(s,1H)
(t-like,3J = 7.5/8.1,2H)
Amantadine-5-Chloro-Salicylaldehyde
Amantadine-o-Vanillin
1.67–1.79 (m,6H,CH2); 1.83 (s,6H,CH2);
2.19 (s,3H,CH)
1.66–1.78 (m,6H,CH2); 1.85 (s,6H,CH2);
2.17 (s,3H,CH)
6.87 (d,3J = 9.6,1H); 7.15–7.27 (m,2H)
8.24(s,1H)
8.24(s,1H)
14.51(s,1H)
15.19(s,1H)
–
6.68 (t-like,3J = 7.8/8.1,1H); 6.83
(d,3J = 8.1,1H); 6.84 (d,3J = 7.5,1H)
3.88(s,3H)
Fig. 3. Fluorescence spectra (ꢁex = 280 nm) of BSA solutions with Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-
Vanillin (AS-o-V) concentrations from 0.00 × 10−5 mol/L (o) to 2.50 × 10−5 mol/L (e) at 0.50 × 10−5 mol/L intervals ([BSA] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L,
pH = 7.40, Tsolu = 37.00 0.02 ◦C and Vtotal = 25.00 mL).
Schiff-Bases stock solutions were prepared using Tris–HCl–NaCl
buffer solution (pH = 7.40, [Tris–HCl] = [NaCl] = 0.05 mol/L) and
their concentrations were 2.00 × 10−5 mol/L and 5.00 × 10−5 mol/L,
respectively. To a 25.00 mL volumetric flask, 12.50 mL BSA stock
solution and appropriate volume of Amantadine Schiff-Bases
stock solution were added and then diluted to the mark with
Tris–HCl–NaCl buffer solution. The final BSA concentration was
1.00 × 10−5 mol/L, and the Amantadine Schiff-Base concentra-
tions were varied from 0.00 × 10−5 mol/L to 2.50 × 10−5 mol/L
at 0.50 × 10−5 mol/L intervals. The fluorescence spectra of BSA
solutions along with the increase of Amantadine Schiff-Base con-
centrations were recorded as shown in Fig. 3 in the wavelength of
250–550 nm with excited wavelength at 280 nm and 5.0 nm/5.0 nm
slit widths. All test solutions were incubated for 10 min before mea-
surement, and then the curves of fluorescence quenching spectra
were obtained at 37.00 0.02 ◦C. The maximal intensities of the
intrinsic fluorescence of BSA solutions were recorded at 348 nm for
the calculation of quenching parameters. The corresponding results
are shown in Fig. 4 and Table 3. The binding distances were also cal-
culated according to Förster’s nonradiative energy transfer theory
Fig. 4. Stern–Volmer plot (a), Lineweaver–Burk plot (b) and Double logarithm plot (c) of BSA solutions with Amantadine-Salicylaldehyde (AS), Amantadine-
5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin (AS-o-V) concentrations from 0.00 × 10−5 mol/L to 2.50 × 10−5 mol/L at 0.50 × 10−5 mol/L intervals
([BSA] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 0.02 ◦C and Vtotal = 25.00 mL).

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Z. Wang et al. / Spectrochimica Acta Part A 83 (2011) 511–517
Fig. 5. Spectral overlap of fluorescence (ꢁex = 280 nm) of BSA solution and absorption of Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-5-
C) and Amantadine-o-Vanillin (AS-o-V) solutions ([BSA] = [AS] = [AS-5-C] = [AS-o-V] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 0.02 ◦C and
Vtotal = 25.00 mL).
new peaks at 1627 cm−1 for AS-5-C, and at 1625 cm−1 for AS-o-
V occur. These changes also indicate the formation of the C
bond. For all three Amantadine Schiff-Bases there are broad ꢀ(OH)
absorption bands around 3400 cm−1, revealing the presence of OH
bonds.
(FRET). The spectral overlap of fluorescence emission of BSA solu-
tion and UV–vis absorption of Amantadine Schiff-Base solutions
are all given in Fig. 5. The corresponding parameters are listed in
Table 4.
N
The 1H NMR of the synthesized Amantadine Schiff-Bases was
determined using CCl3D as solvent. The corresponding data are
listed in Table 3. From Table 3 it can be seen that the sin-
gle peaks at 8.32 ppm, 8.24 ppm and 8.24 ppm chemical shifts,
respectively, for AS, AS-5-C and AS-o-V should be attributed to
the proton peaks of –CH N– bond of Amantadine Schiff-Base.
In general, the chemical shift of the proton peak of –CHO in
Salicylaldehyde is about 9.48 ppm. The results indicate that the
imino bond (–CH N–) has already formed between Amantadine
and Salicylaldehyde (5-Chloro-Salicylaldehyde and o-Vanillin). The
benzene ring-H of Salicylaldehyde (5-Chloro-Salicylaldehyde and
o-Vanillin) has a certain degree of change compared with cor-
responding those before chemical reaction. It also illustrates the
formation of imino bond (–CH N–). Because of ꢃ single bond, the
chemical shift of Adamantane ring-H only has a little change. Of
course, due to the existence of oxygen atom, the proton peaks at
14.55 ppm, 14.51 ppm and 15.19 ppm chemical shifts of phenolic
hydroxyl group (Ar–OH), respectively, for AS, AS-5-C and AS-o-V
for as well as the proton peaks of methoxy (OCH3) at 3.88 ppm
chemical shift of AS-o-V also almost unchanged.
2.5. The determination of synchronous fluorescence spectra
In order to confirm the binding sites of the three Aman-
tadine Schiff-Bases (AS, AS-5-C and AS-o-V) to BSA molecules,
the synchronous fluorescence spectra of BSA solutions along
with the increase of Amantadine Schiff-Base concentrations were
researched in wavelength ranges from 250 nm to 350 nm for
ꢂꢁ = 15 nm and from 225 nm to 325 nm for ꢂꢁ = 60 nm. The results
are all given in Fig. 6, and the corresponding ratios of synchronous
fluorescence quenching (RSFQ (%)) are illustrated in Fig. 7.
3. Results and discussion
3.1. Infrared spectra and 1H NMR of Amantadine Schiff-Bases (AS,
AS-5-C and AS-o-V)
The changes of the infrared spectra shown in Fig.
2
can reveal the formation of the three Amantadine Schiff-
Bases expected (Amantadine-Salicylaldehyde (AS), Amantadine-
5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin
(AS-o-V)). From the data in Table 2, it can be seen that the ꢀ(C O)
of Salicylaldehyde at 1663 cm−1 and ꢀas(NH3+) and ꢀs(NH3+) of
Amantadine at 2921 cm−1 and 2586 cm−1 all disappear, and that a
new peak occurs at 1629 cm−1. This indicates that the C N bond
of AS Schiff-Base forms. Similarly, the ꢀ(C O) at 1660 cm−1 for
5-Chloro-Salicylaldehyde and at 1639 cm−1 for o-Vanillin as well
as the ꢀas(NH3+) and ꢀs(NH3+) of Amantadine all disappear, and
3.2. Fluorescence spectra of BSA solutions with Amantadine
Schiff-Bases (AS, AS-5-C and AS-o-V)
It is well known that most of protein molecules naturally fluo-
resce around 348 nm (under 280 nm wavelength excitation), which
mainly attributes to their fluorescent tryptophan (Trp) residues
Table 4
Quenching constants (KSV and Kq), binding constants, stable constants and binding site numbers calculated according to Stern–Volmer plots, Lineweaver–Burk plots and Double
logarithm plots of BSA solutions with Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin (AS-o-V) concentrations
(from 0.00 × 10−5 mol/L to 2.50 × 10−5 mol/L at 0.50 × 10−5 mol/L intervals) ([BSA] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 0.02 ◦C and
Vtotal = 25.00 mL).
System
Stern–Volmer plot
R2
KSV (L/mol)
Kq (L/mol s)
BSA + Amantadine-Salicylaldehyde
BSA + Amantadine-5-Chloro-Salicylaldehyde
BSA + Amantadine-o-Vanillin
F/F0 = 0.24 [Q] + 1
F/F0 = 0.23 [Q] + 1
F/F0 = 0.21 [Q] + 1
0.9990
0.9957
0.9975
2.42 × 104
2.30 × 104
2.11 × 104
2.42 × 1012
2.30 × 1012
2.11 × 1012
System
Lineweaver–Burk plot
R2
f
KLB (L/mol)
KD (mol/L)
BSA + Amantadine-Salicylaldehyde
BSA + Amantadine-5-Chloro-Salicylaldehyde
BSA + Amantadine-o-Vanillin
1/[(F0 − F)/F0] = 3.74/[Q] + 1.22
1/[(F0 − F)/F0] = 4.97/[Q] + 0.70
1/[(F0 − F)/F0] = 5.22/[Q] + 0.76
0.9993
0.9999
0.9981
1.22
0.70
0.76
3.27 × 104
1.42 × 104
1.46 × 104
3.06 × 10−5
7.06 × 10−5
6.84 × 10−5
System
Double logarithm plot
R2
KA (L/mol)
n
ꢂG0 (kJ/mol)
BSA + Amantadine-Salicylaldehyde
BSA + Amantadine-5-Chloro-Salicylaldehyde
BSA + Amantadine-o-Vanillin
log[(F0 − F)/F] = 0.96 log[Q] + 4.21
log[(F0 − F)/F] = 1.03 log[Q] + 4.49
log[(F0 − F)/F] = 1.04 log[Q] + 4.50
0.9967
0.9975
0.9977
1.61 × 104
3.11 × 104
3.15 × 104
0.96
1.03
1.04
−24.98
−26.66
−26.70

Z. Wang et al. / Spectrochimica Acta Part A 83 (2011) 511–517
515
Fig. 6. Synchronous fluorescence spectra of BSA solutions with Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin
(AS-o-V) concentrations from 0.00 × 10−5 mol/L (o) to 2.50 × 10−5 mol/L (e) at 0.50 × 10−5 mol/L intervals ([BSA] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40,
Tsolu = 37.00 0.02 ◦C and Vtotal = 25.00 mL).
[29,30]. Likewise, as shown in Fig. 3 the bovine serum albumin
(BSA) molecules in aqueous solution show the obvious intrin-
sic fluorescence. However, the fluorescences of BSA molecules
are gradually quenched along with the increase of Amantadine
Schiff-Base (BSA + AS, BSA + AS-5-C or BSA + AS-o-V) concentration
compared with that of pure BSA solution. Apparently, this quench-
ing is owing to the interaction of Amantadine Schiff-Bases with BSA
molecules.
equation (1) [31]:
F0
= 1 + KSV[Amantadine Schiff-Base]
F
= 1 + Kqꢄ0[Amantadine Schiff-Base]
(1)
Fig. 4(a) displays the Stern–Volmer plots of BSA solutions in
the presence of Amantadine Schiff-Bases with various concentra-
tions. It can be found that all three plots exhibit a comparatively
good linear relationship. From Stern–Volmer equation (1), for three
systems their KSV could be obtained. In general, for the most of
protein molecules with fluorescence, the ꢄ0 is known to be approx-
imately 10−8 s. Thus, as shown in Table 4, for three courses these
Kq are all greater than the maximum value (2.0 × 1010 L/mol s) for
the diffusion controlled quenching process of biological macro-
molecules. It indicates that the dominating quenching mechanism
is not dynamic but static.
3.3. Interaction mode and binding site number
The interaction of these three Amantadine Schiff-Bases (AS, AS-
5-C and AS-o-V) with BSA molecules in aqueous solution could be
studied by measuring the change of intrinsic fluorescence of BSA
molecules (excited by 280 nm wavelength light) along with the
increases of Amantadine Schiff-Base concentrations. Firstly, as a
hypothetical dynamic quenching process, the data of fluorescence
intensities at 348 nm could be analyzed by using the Stern–Volmer
Fig. 7. Ratios of synchronous fluorescence quenching (RSFQ
) of BSA solutions with Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-
5-C) and Amantadine-o-Vanillin (AS-o-V) concentrations from 0.00 × 10−5 mol/L to 2.50 × 10−5 mol/L at 0.50 × 10−5 mol/L intervals ([BSA] = 1.00 × 10−5 mol/L,
[Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 0.02 ◦C and Vtotal = 25.00 mL).

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Z. Wang et al. / Spectrochimica Acta Part A 83 (2011) 511–517
Table 5
Energy transfer efficiency (E), critical binding distance (R), overlap integral (J) and binding distance (r) calculated according to Foster’s non-radioactive energy transfer theory
([BSA] = [AS] = [AS-5-C] = [AS-o-V] = 1.00 × 10−5 mol/L, [Tris–HCl] = [NaCl] = 50 mmol/L, pH = 7.40, Tsolu = 37.00 0.02 ◦C and Vtotal = 25.00 mL).
System
E (%)
R (nm)
J (cm3 L/mol)
r (nm)
BSA + Amantadine-Salicylaldehyde
BSA + Amantadine-5-Chloro-Salicylaldehyde
BSA + Amantadine-o-Vanillin
16.36
34.84
22.23
2.18
2.28
2.22
3.86 × 10−15
5.11 × 10−15
4.41 × 10−15
2.86
2.53
2.74
For reconfirming the static quenching mechanisms, the data of
fluorescence quenching of BSA solutions along with the increase of
Amantadine Schiff-Base concentrations are also analyzed according
to the Lineweaver–Burk (modified Stern–Volmer or double recip-
rocal) equation (2):
where r is the binding distance between the donor and acceptor,
and R0 is the critical distance when the efficiency (E) of energy
transfer is 50%, which can be calculated by Eq. (6):
R06 = 8.8 × 10−25(K2n−4ϕDJ)
(6)
where K2 is the spatial orientation factor of the dipole, n is the
refractive index of medium, ϕD is the quantum yield of the donor in
the absence of acceptor and J is the overlap integral of the emission
spectrum of the donor and the absorption spectrum of the acceptor.
In the present case, K2, n and ϕD are 2/3, 1.336 and 0.15 for BSA
molecules [39], respectively. And then, the J can be calculated by
Eq. (7):
1
1
1
f
=
+
(2)
(F0 − F)/F0
fKLB[Amantadine Schiff-Base]
Using the plots obtained from Fig. 4(b), the KLB and the cor-
responding dissociation constants KD could be calculated. From
Table 4 it can be found that these Lineweaver–Burk plots all have
a better linear relationship than the corresponding Stern–Volmer
plots. Thus, it could be confirmed again that the fluorescence
quenching mechanism of BSA molecules by these three Amanta-
dine Schiff-Bases are mainly a static quenching procedure indeed.
From Fig. 4(c), the equilibrium constants (KA) and the bind-
ing site numbers (n) could be calculated by using the equation:
(F0 − F)/F = KA[Amantadine Schiff-Base]n, by using logarithmic
treatment the following Double logarithm equation (3) can be
obtained [32–35]:
ꢂ
F(ꢁ)ε(ꢁ)ꢁ4 ꢂꢁ
ꢂ
J =
(7)
F(ꢁ) ꢂꢁ
where ꢁ is the wavelength of corresponding fluorescence spectrum
of BSA solution and absorption spectrum of Amantadine Schiff-
Base (AS, AS-5-C and AS-o-V) solution. The F(ꢁ) and ε(ꢁ) are the
fluorescence intensity of BSA solution and molar absorption coeffi-
cient of Amantadine Schiff-Base solution, respectively, Fig. 5 shows
the spectral overlap of fluorescence emission of BSA solution and
UV–vis absorption of Amantadine Schiff-Base (AS, AS-5-C and AS-o-
V) solutions. The efficiency (E) of energy transfer can be determined
by Eq. (8):
ꢀ
ꢁ
F0 − F
log
= log KA + n log[Amantadine Schif-Base]
(3)
F
Apparently, the KA and n could be measured by the intercept
and slope by plotting log[(F0 − F)/F] against log[Amantadine Schiff-
Base]. It could be seen that the plots also exhibited a good linear
relationship. Maybe, it is because of the 1:1 binding mode of Aman-
tadine Schiff-Base to BSA. All three KA values indicate moderately
strong binding between the BSA molecules and these three Aman-
tadine Schiff-Bases. The KA and n for BSA + AS-5-C and BSA + AS-o-V
systems are slightly bigger than that for BSA + AS system, which
indicates that the presence of chlorine (Cl) atom and methoxy
(OCH3) groups strengthen the interaction of AS-5-C and AS-o-V
with BSA molecules. Moreover, the Amantadine Schiff-Bases have
planar phenol, which is propitious to the binding to BSA molecules
[36]. Three n values are all equal to about 1, indicating that there is
one class of binding site for three Amantadine Schiff-Bases to BSA
molecule.
F
E = 1 −
(8)
F0
According to Eqs. (5)–(8), E, J, R0 and r could be obtained as
shown in Table 5. These distances are all less than 7.0 nm, which
indicates that the energy transfer from BSA molecules to Amanta-
dine Schiff-Bases occurs with high probability [40–43]. Moreover,
it also suggested that the binding of these three Amantadine
Schiff-Bases to BSA molecules was through energy transfer, which
effectively quenched the fluorescence of BSA molecules.
3.5. Synchronous fluorescence spectra of BSA + AS, BSA + AS-5-C
and BSA + AS-o-V solutions
Utilizing KA, the standard free energy (ꢂG0) for binding can be
obtained from the relationship (4):
Synchronous fluorescence spectra can provide much valuable
information about the microenvironment around fluorogens in
biomolecules [44]. In general, for protein molecules, when the
ꢂꢁ = 15 nm is fixed, the spectrum characteristic of Tyr residues
will be observed, while the ꢂꢁ = 60 nm is done, the spectrum
characteristic of Trp residues will be observed [39,40,45]. In this
work, the synchronous fluorescence spectroscopy was used to get
the information about the binding sites of Amantadine Schiff-
Bases (AS, AS-5-C and AS-o-V) to BSA molecules. The synchronous
fluorescence quenching ratios (RSFQ) that express the decreasing
percentages of synchronous fluorescence intensity were calculated
by using the equation: RSFQ = 1 − F/F0, where F and F0 are the syn-
chronous fluorescence intensities of BSA solution in the presence
and absence of Amantadine Schiff-Bases (AS, AS-5-C and AS-o-V),
respectively.
ꢂG0 = −RT ln KA
(4)
The calculated results are all given in Table 4. The negative signs
for ꢂG0 indicate the spontaneity of the binding of three Amanta-
dine Schiff-Bases (AS, AS-5-C and AS-o-V) to BSA molecules under
standard conditions.
3.4. Binding distances between BSA molecules and Amantadine
Schiff-Bases (AS, AS-5-C and AS-o-V)
The binding distance (r) between BSA molecules and these three
Amantadine Schiff-Bases (AS, AS-5-C and AS-o-V) can be calculated
according to the following Förster’s non-radioactive energy transfer
theory (FRET) equation [37,38]:
From Fig. 6 it can be seen that for all three derivative the
fluorescence intensities for ꢂꢁ = 15 nm (upper) and ꢂꢁ = 60 nm
(lower) are quenched with increasing Amantadine Schiff-Base
1
E =
(5)
1 + (r/R0)6

Z. Wang et al. / Spectrochimica Acta Part A 83 (2011) 511–517
517
concentration. Fig. 7 shows that for ꢂꢁ = 15 nm the order of RSFQ
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The authors greatly acknowledge the National Natural Science
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Foundation of Science and Technology Department and Liaoning
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