Study of the interaction between sodium salts of (2E)-3-(4'-halophenyl) prop-2-enoyl sulfachloropyrazine and bovine serum albumin by fluorescence spectroscopy

Article abstract of DOI:10.1002/bio.2364

Three sodium salts of (2E)-3-(4'-halophenyl)prop-2-enoyl sulfachloropyrazine (CCSCP) were synthesized and their structures were determined by ¹H and ¹³C NMR, LC-MS and IR. The binding properties between CCSCPs and bovine serum albumi

Full text of DOI:10.1002/bio.2364

Research article
Received: 18 December 2011,
Revised: 9 February 2012,
Accepted: 16 February 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2364
Study of the interaction between sodium
salts of (2E)-3-(4’-halophenyl)prop-2-enoyl
sulfachloropyrazine and bovine serum
albumin by fluorescence spectroscopy
Xuan Luo, Chuanrong Du, Jinrui Wei, Jiang Deng, Yijie Lin and Cuiwu Lin*
ABSTRACT: Three sodium salts of (2E)-3-(4’-halophenyl)prop-2-enoyl sulfachloropyrazine (CCSCP) were synthesized and their
structures were determined by 1H and 13C NMR, LC-MS and IR. The binding properties between CCSCPs and bovine serum al-
bumin (BSA) were studied using fluorescence spectroscopy in combination with UV–vis absorbance spectroscopy. The results
indicate that the fluorescence quenching mechanisms between BSA and CCSCPs were static quenching at low concentrations
of CCSCPs or combined quenching (static and dynamic) at higher CCSCP concentrations of 298, 303 and 308 K. The binding
constants, binding sites and corresponding thermodynamic parameters (ΔH, ΔS, ΔG) were calculated at different tempera-
tures. All ΔG values were negative, which revealed that the binding processes were spontaneous. Although all CCSCPs had
negative ΔH and positive ΔS, the contributions of ΔH and ΔS to ΔG values were different. When the 4’-substituent was fluorine
or chlorine, van der Waals interactions and hydrogen bonds were the main interaction forces. However, when the halogen was
bromine, ionic interaction and proton transfer controlled the overall energetics. The binding distances between CCSCPs and
BSA were determined using the Förster non-radiation energy transfer theory and the effects of CCSCPs on the conformation
of BSA were analyzed by synchronous fluorescence spectroscopy. Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: halo-cinnamic acid; sulfachloropyrazine; bovine serum albumin; fluorescence spectroscopy; interaction
and metal ions in the bloodstream (6,7). Because bovine serum
Introduction
albumin (BSA) and human serum albumin (HSA) display approx-
Since 2006, our laboratory has been interested in an herbal
imately 76% sequence homology and the 3D structure of BSA is
medicine with procoagulant and haemostatic activities, namely
believed to be similar to that of HSA (8,9), BSA was selected as
Blumea riparia (BL) DC. (Compositae). It has been used in China
to treat gynaecological diseases including menorrhagia, puer-
peral metrorrhagia, peripheral oedema, infertility and vulvar
ulcer (1). According to our previous research, its major procoagu-
lant and haemostatic components are phenolic acids, including
vanillic acid, syringic acid, p-coumaric acid, caffeic acid and pro-
our protein model.
In the present work, three sodium salts chosen from: (2E)-3-(4’-
halophenyl)prop-2-enoyl sulfachloropyrazine (CCSCP); (2E)-N-{4’’-
[(6’’’-chloropyrazin-2’’’-yl)sulfamoyl]phenyl}-3-(4’- fluorophenyl)prop-
2-enamide sodium salt (PFCCSCP); (2E)-3-(4’-chlorophenyl)-N-{4’’-
[(6’’’- chloropyrazin-2’’’-yl)sulfamoyl]phenyl}prop-2-enamide sodium
tocatechuic acid (2). These phenolic acid compounds had (2E)-3-
salt (PCCCSCP); and (2E)-3-(4’- bromophenyl)-N-{4’’-[(6’’’-chloropyra-
phenylprop-2-enoyl or benzoyl frameworks. Our goal was to
make structure modifications of (2E)-3-phenylprop-2-enoic or
benzoic acids to find other compounds with better
zin-2’’’-yl)sulfamoyl]phenyl}prop-2-enamide sodium salt (PBCCSCP)
(Figure 1) were synthesized and the interaction with BSA was
studied using fluorescence and absorption spectroscopy. The
procoagulant and haemostatic activities. At the same time, we
binding constants, basic thermodynamic parameters and binding
introduced sulphonamides (SAs) onto (2E)-3-phenylprop-2-enoyl
or benzoyl frameworks to give these compounds anti-
inflammatory or antimicrobial activities.
Drug properties such as absorption, distribution, metabolism,
distances were obtained. At the same time, the effects of differ-
ent halogens of the benzene ring on the binding properties of
CCSCPs to BSA were explored. We believe that the study pro-
vides valuable information for drug design and pharmaceutical
excretion, stability and toxicity during the chemotherapeutic
research.
process are very important. These properties are strongly af-
fected by drug–protein complexes. Therefore, the interaction
between drug and protein and the study of their biological
mechanism have recently become a focus of research (3–5).
* Correspondence to: Cuiwu Lin, 100 Daxue Dong Road, School of Chemis-
try and Chemical Engineering, Guangxi University, Nanning, Guangxi,
530005, P. R. China. E-mail: cuiwulin@163.com
Choosing a good protein model is crucial. Serum albumin, the
major soluble protein constituent in the circulatory system of a
wide variety of organisms, has many important biological
functions. One of them is to reversibly bind to a large variety
of endogenous and exogenous ligands such as fatty acids, drugs
School of Chemistry and Chemical Engineering, Guangxi University,
Nanning, Guangxi, 530004, People’s Republic of China
Luminescence 2012
Copyright © 2012 John Wiley & Sons, Ltd.

X. Luo et al.
Figure 1. Structures of sodium salts of (2E)-3-(4’-halophenyl)prop-2-enoyl sulfachloropyrazine
Procedures
Experimental
General procedure for the syntheses of CCSCPs. CCSCPs were
synthesized from p-halogenated cinnamic acids and sodium
[(4-aminophenyl)sulfonyl](6-chloropyrazin-2-yl)azanide. The syn-
thesis route is shown in Scheme 1. After the reaction, the mixture
was transferred to 1 L beakers and made up to 1 L with distilled
water to precipitate the products. The raw products were sepa-
rated by vacuum filtration and recrystallized in methanol–THF
or methanol–EtOAc solvent systems.
Apparatus
Melting points were measured on an X-4 microscopic melting
point apparatus made by Beijing Tech Instruments. All pH mea-
surements were made with a pHs-3C digital pH meter (Shanghai
Leici Device Works, China) with a combined glass-calomel
electrode. The absorption and IR spectra were obtained using
a Unico (China) UV-2102PCS and Bruker Tensor27, respectively.
Fluorescence spectra were recorded on a Shimadzu RF-5301
fluorescence spectrophotometer (Tokyo, Japan) with a 1-cm
quartz cell and thermostatic cuvette holder. The excitation and
emission bandwidths were both 5 nm. The temperature of the
samples was maintained by recycling water throughout the
experiment. 1H and 13C NMR spectra were recorded on a Bruker
Advance III 300 at 300 MHz for 1H and 75 MHz for 13C,
respectively in DMSO-d6. Chemical shifts are given in d (ppm)
and coupling constants in Hz. ESI-MS was obtained on a
Shimadzu LC-MS 2010A.
CCSCP–BSA interaction study. Fluorescence measurements were
carried out by successive addition of 1 x 10–3 M CCSCP stock
solutions to a fixed amount of BSA to give a final concentration
of 1 x 10–5 M in each 10 mL comparison tube. The final volume
was 10.0 mL with the buffer. Thus, a series of solutions contain-
ing different amount of CCSCPs and a definite amount of BSA
were obtained. The solutions were held in a thermostat water
bath for 10 min before fluorescence measurements. Fluores-
cence emission spectra were read in a range of wavelengths
from 300–450 nm using an excitation wavelength of 295 nm.
All experiments were performed at three temperatures (298,
303 and 308 K). Synchronous fluorescence spectra of BSA (1 x
10–5 M) in the presence of CCSCPs (0.2–1.8 x 10–5 M) were
recorded. The wavelength ranges of synchronous scanning were
from 280–350 nm (Δl =15 nm) and 290–400 nm (Δl = 60 nm) at
298 K.
Reagents
A pH 7.4 buffer solution was prepared as follows: 6.0 g tris
(hydroxymethyl)aminomethane and 4.5 g NaCl (Sinopharm
Group Chemical Reagent Co., Ltd, China) were dissolved in 1 L
distilled water and then 3 M hydrochloric acid solution was used
to adjust the pH to 7.4. BSA (Sigma-Aldrich) was dissolved daily
in 0.1 M Tris–HCl buffer (pH = 7.4) to prepare a stock solution
(1.0 x 10ꢀ4 M) and stored in a refrigerator. CCSCP stock solutions
(1 x 10–3 M) were prepared by dissolving 0.025 mmol derivatives
in 15 mL DMSO and diluting in a 25 mL volumetric flask by tris–
HCl buffer. Working solutions were prepared daily as follows:
0–180 mL CCSCP stock solutions and 1 mL BSA solution were
added into a 10 mL comparison tube and diluted with buffer.
All other chemicals were purchased from Nanning Lantian
Experiment Equipment Co., Ltd (Nanning, Guangxi, China) and
were used without purification.
Sodium salt of (2E)-N-{4’’-[(6’’’-Chloropyrazin-2’’’-yl)sulfamoyl]
phenyl} -3-(4’-fluorophenyl) prop-2-enamide (PFCCSCP)
Light yellow needle crystals from the above recrystallization had
a melting point of 278–279ꢁC, an ESI-MS of m/z 431.1 (calcd 431.8
for C19H13ClFN4O3S); IR (KBr) nmax 3267 (vw, –NH–SO2–), 3115 (w,
–NH–CO–), 1690 (s, >C = O), 1633 (s, >C = C–H), 1600–1300 (m to
s, Ar–H), 1343 and 1191 (vs, >S = O), 1232 (m, F–Ph) and 1092 (m,
1
Cl–Ar) cmꢀ1 for H and 13C NMR spectroscopic data (DMSO-d6)
(Tables 1 and 2).
Scheme 1. Synthesis route of sodium salts of (2E)-3-(4’-halophenyl)prop-2-enoyl sulfachloropyrazine
wileyonlinelibrary.com/journal/luminescence
Copyright © 2012 John Wiley & Sons, Ltd.
Luminescence 2012

Interaction between halocinnamoyl sulfachloropyrazine Na salts and BSA
1
Table 1. Assignments of CCSCPs H NMR data
Position
PFCCSCP (dH, mult., J, Hz)
PCCCSCP (dH, mult., J, Hz)
PBCCSCP (dH, mult., J, Hz)
2
6.767 (d, 16.2)
6.821 (d, 15.7)
6.833 (d, 15.8)
3
7.632 (d, 16.2)
7.668 (d, 15.7)
7.597 (d, 15.8)
2′
3′
5′
6′
2”
3”
5”
6”
7.706 (m, 8.9, 5.5, 2.0)
7.288 (m, 8.9, 2.0)
7.288 (m, 8.9, 2.0)
7.706 (m, 8.9, 5.5, 2.0)
7.953 (ddd, 9.3, 2.2, 1.8)
7.887 (ddd, 9.3, 2.2, 1.8)
7.887 (ddd, 9.3, 2.2, 1.8)
7.953 (ddd, 9.3, 2.2, 1.8)
8.332 (d, 0.6)
7.655 (ddd, 8.6, 2.4, 1.9)
7.490 (ddd, 8.6, 2.4, 1.9)
7.490 (ddd, 8.6, 2.4, 1.9)
7.655 (ddd, 8.6, 2.4, 1.9)
7.961 (ddd, 9.1, 2.1, 1.9)
7.892 (ddd, 9.1, 2.1, 1.9)
7.892 (ddd, 9.1, 2.1, 1.9)
7.961 (ddd, 9.1, 2.1, 1.9)
8.321 (d, 0.37)
7.632 (ddd, 8.7, 2.0, 1.9)
7.578 (ddd, 8.7, 2.0, 1.9)
7.578 (ddd, 8.7, 2.0, 1.9)
7.632 (ddd, 8.7, 2.0, 1.9)
7.957 (ddd, 9.1, 2.2, 2.0)
7.888 (ddd, 9.1, 2.2, 2.0)
7.888 (ddd, 9.1, 2.2, 2.0)
7.957 (ddd, 9.1, 2.2, 2.0)
8.315 (d, 0.37)
3”’
5”’
8.292 (d, 0.6)
8.294 (d, 0.37)
8.289 (d, 0.37)
-CO-NH-
10.632 (s)
10.668 (s)
10.671 (s)
Table 2. Assignments of CCSCPs 13C NMR data
Results and discussion
Fluorescence quenching study
Position
PFCCSCP
PCCCSCP PBCCSCP
Fluorescence quenching refers to any process that decreases the
fluorescence intensity of a fluorophore by a variety of molecular
interactions with a quencher molecule, including excited-state
reactions, molecular rearrangements, energy transfer, ground-
state complex formation and collisional quenching processes.
Due to different mechanisms, quenching can be classified as
either dynamic or static. Dynamic or collisional quenching results
from collision between a fluorophore and a quencher; static
quenching is due to the formation of ground-state complex be-
tween a fluorophore and a quencher. In general, dynamic and
static quenching can be distinguished by their different
dependence on temperature and viscosity. The quenching rate
constants decrease with increasing temperature for static quench-
ing but the reverse effect is observed for dynamic quenching (10).
The fluorescence spectra of BSA in the presence of different
amounts of PFCCSCP, PCCCSCP and PBCCSCP at 298 K are
shown in Figure 2. As can be seen, the fluorescence intensity
of BSA consistently decreased upon increasing the concentra-
tions of CCSCPs, which indicated that CCSCPs could interact with
BSA. Furthermore, the maximum wavelength of BSA shifted
from 344 to 357 nm regardless of PFCCSCP, PCCCSCP or
PBCCSCP after the addition of each of the CCSCPs. A slight red
shift of fluorescence spectrum indicated that the tyrosine
residue was brought to a more hydrophilic environment (10)
with strengthened hydrogen bonding (11) in the BSA–CCSCP
systems. The structure of the hydrophobic subdomain where
tryptophan was placed was not compact and the segment of
polypeptide changed its conformation to a more extended state
after the addition of CCSCPs (12,13).
1
2
3
1′
2′
3′
4′
5′
164.1
164.0
122.4
143.7
134.6
129.6
129.2
133.5
129.2
129.6
133.0
118.9
129.0
132.3
129.0
118.9
137.1
145.5
147.5
140.0
164.0
122.5
143.7
133.8
132.1
129.9
123.4
129.9
132.1
133.1
118.9
129.0
132.4
129.0
118.9
137.0
145.6
147.6
140.1
121.5, 121.5 (6JC–F = 2.1)
143.8
131.2, 131.1 (4JC–F = 3.2)
130.2, 130.1 (3JC–F = 8.5)
116.2, 116.0 (2JC–F = 22.1)
164.7, 161.5 (1JC–F = 248.2)
116.2, 116.0 (2JC–F = 22.1)
6′
130.2, 130.1 (3JC–F = 8.5)
1’’
2’’
3’’
4’’
5’’
6’’
2’’’
3’’’
5’’’
6’’’
132.9
118.9
129.0
132.3
129.0
118.9
137.1
145.5
147.5
140.2
Sodium salt of (2E)-3-(4’-Chlorophenyl)-N-{4’’-[(6’’’-chloropyrazin-
2’’’-yl)sulfamoyl] phenyl}prop-2-enamide (PCCCSCP)
The white powder from the above recrystallization had a
melting point of 282–283ꢁC; ESI-MS m/z 448.1 (calcd 448.3 for
C19H13Cl2N4O3S); IR (KBr) nmax 3266 (vw, –NH–SO2–), 3116 (m,
–NH–CO–), 1688 (m, > C=O), 1631 (m, > C=C–H), 1600–1300 (m
to vs, Ar–H), 1339 and 1163 (vs, > S = O), 1091 (m, Cl–Ar) cmꢀ1 for
1H and 13C NMR spectroscopic data (DMSO-d6) Tables 1 and 2).
To confirm the quenching mechanism, fluorescence quenching
data were analyzed by the Stern-Volmer equation (equation 1):
Sodium salt of (2E)-3-(4’-Bromophenyl)-N-{4’’-[(6’’’-chloropyrazin-
2’’’-yl)sulfamoyl] phenyl}prop-2-enamide (PBCCSCP)
F0
¼ 1 þ KSV½Qꢂ ¼ 1 þ kqt0½Qꢂ
(1)
F
The white powder from the above recrystallization had a melting
point of 282–283ꢁC; ESI–MS m/z 493.1 (calcd 492.8 for
C19H13BrClN4O3S); IR (KBr) nmax 3267 (vw, –NH–SO2–), 3115 (m, –
NH–CO–), 1689 (m, > C = O), 1632 (m, > C = C–H), 1600–1300 (m
to vs, Ar–H), 1339 and 1163 (vs, > S = O), 1093 (m, Cl–Ar) cmꢀ1
for 1H and 13C NMR spectroscopic data (DMSO-d6) (Tables 1 and 2).
where F0 and F are the fluorescence intensity of BSA in the absence
or presence of quencher, respectively; KSV is the Stern-Volmer
quenching constant; [Q] is the quencher concentration; kq is the
bimolecular quenching constant; t0 is the average lifetime of
the molecule without quencher and its value is considered to
be 10–8 s (4,10).
Luminescence 2012
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence

X. Luo et al.
Figure 2. Fluorescence quenching spectra of BSA at different concentrations of CCSCPs at 298 K, lex = 295 nm; c(BSA) = 1.00 ꢄ 10–5 M; c(CCSCP)/( ꢄ 10–5 M), (a–j) 0.00, 0.20,
0.40, 0.60, 0.80, 1.00, 1.20, 1.40, 1.60 and 1.80, respectively
In many instances, the fluorophore could be quenched both
by collisions and by complex formation with the same quencher.
The characteristic feature of the Stern-Volmer plots in such
circumstances had an upward curvature. The modified Stern-
Volmer equation (2) better accounted for the upward curvature
observed when both static and dynamic quenching occurred
for the same fluorophore;
kq reflects the efficiency of quenching or the accessibility of
the fluorophore to the quencher. The diffusion-controlled
quenching typically resulted in values of kq near 1.0 x 1010
Mꢀ1ꢃsꢀ1 (10). The results in Table 3 show that the values for kq
were much greater than the diffusion-controlled value. With
the rising temperature, KSV decreased for PFCCSCP and PCCCSCP
and increased for PBCCSCP. Therefore, the fluorescence quench-
ing of PFCCSCP and PCCCSCP could be a static quenching pro-
cess at low concentrations rather than a dynamic quenching
process. For PBCCSCP, KSV increased with the rising temperature
but the increment was very small (less than 1%). At the same
time, its kq was more than 2500x the maximum diffusion colli-
sion quenching rate constant (1.0 x 1010 Mꢀ1ꢃsꢀ1). Therefore,
PBCCSCP fluorescence quenching could be a static quenching
process at low concentrations as well.
F0
2
¼ ð1 þ KD½QꢂÞð1 þ KS½QꢂÞ ¼ 1 þ ðKD þ KSÞ½Qꢂ þ KDKS½Qꢂ (2)
F
where KD and KS are the dynamic and static quenching con-
stants, respectively. The modified equation was second order
with respect to [Q], which led to the upward curvature observed
at higher [Q] (4,10).
The Stern-Volmer plots of the quenching of BSA fluorescence
by CCSCPs at different temperatures are displayed in Figure 3.
As can be seen, the plots F0/F for [Q] ranging from 0.20 to 1.80 x
10–5 M of CCSCPs concaved toward the y-axis either at low or high
temperatures. The fluorescence quenching data were analyzed
according to equation (2) at all three temperatures and had good
linearity (for PFCCSCP, R2298 K = 0.9998, R2303 K = 0.9992, R2308
K = 0.9996; for PCCCSCP, R2298 K = 0.9998, R2303 K = 0.9996, R2308
K = 0.9990; for PBCCSCP, R2298 K = 0.9994, R3203 K = 0.9986,
R2308 K =0.9995). Meanwhile, the plots (inserts in Figure 3) of F0/F
for BSA versus [Q] at low concentrations of CCSCPs (0.20 to 1.00
x 10–5 M) at three temperatures based on equation (1) exhibited
good linearity and the values of KSV and kq are illustrated in
Table 3. The phenomena could indicate that a single quenching
mechanism, either dynamic or static quenching, occurred at lower
concentrations of CCSCPs at 298, 303 and 308 K. However, a
combined quenching (static and dynamic) process occurred at
higher concentrations of CCSCPs at all three temperatures.
Binding constants and binding sites
When small molecules bind independently to a set of equivalent
sites on a macromolecule, the binding constant (KA) and the
number of binding sites (n) can be determined by the following
equation (3):
ꢀ
ꢁ
F0 ꢀ F
lg
¼ lgKA þ n lg½Qꢂ
(3)
F
However, this equation can only calculate the binding con-
stant and the number of binding sites for static quenching pro-
cesses (14). Double reciprocal curves of fluorescence quenching
of CCSCPs were plotted (Figure 4) in the same concentration
range based on equation (3). The values of KA and n listed in
Table 4 were determined from the slope and the intercept. The
wileyonlinelibrary.com/journal/luminescence
Copyright © 2012 John Wiley & Sons, Ltd.
Luminescence 2012

Interaction between halocinnamoyl sulfachloropyrazine Na salts and BSA
Figure 3. Stern–Volmer plots of the fluorescence quenching of BSA by CCSCPs at different temperatures (■, 298 K; ●, 303 K; ▲, 308 K)
Table 3. Quenching constants of BSA by CCSCP at different temperatures
BSA–PFCCSCP
BSA–PCCCSCP
BSA–PBCCSCP
T = 298 K
T = 303 K
T = 308 K
KSV ꢄ 105 (M–1)
kq ꢄ 1013 (M–1ꢃs–1)
R
2.13(13)
2.13(13)
0.9943
2.13(14)
2.13(14)
0.9935
2.06(14)
2.06(14)
0.9936
2.57(18)
2.57(18)
0.9923
2.54(17)
2.54(17)
0.9935
2.50(18)
2.50(18)
0.9919
2.53(18)
2.53(18)
0.9928
2.56(18)
2.56(18)
0.9925
2.56(19)
2.56(19)
0.9922
KSV ꢄ 105 (M–1)
kq ꢄ 1013 (M–1ꢃs–1)
R
KSV ꢄ 105 (M–1)
kq ꢄ 1013 (M–1ꢃs–1)
R
results showed that there was strong binding between CCSCPs
and BSA, which means that CCSCPs could be stored and carried
by protein in the body. The binding constants of the interaction
followed the order: PBCCSCP > PCCCSCP > PFCCSCP at all three
temperatures. This means that PBCCSCP had the strongest
ability to bind BSA. Furthermore, the binding constants de-
creased with rising temperatures, which confirms that their
fluorescence quenching was static.
Waals interactions, hydrogen bonds (15). The thermodynamic
parameters were determined using van’t Hoff equation (4):
ΔH ΔS
lnKA ¼ ꢀ
þ
(4)
RT
R
where KA is the binding constant at the corresponding temperature
T, R is the gas constant, T is absolute temperature, ΔH is enthalpy
change and ΔS is entropy change. The ln KA versus 1/T plot enabled
the determination of ΔH and ΔS. The value of enthalpy free energy
change (ΔG) was calculated from equation (5):
Thermodynamic analysis and the interaction forces between
BSA and CCSCPs
ΔG ¼ ΔH ꢀ TΔS ¼ ꢀRT lnKA
(5)
On the basis of the thermochemical behaviour of small molecule
interactions, the interaction force between small molecules and
macromolecules include hydrophobic, electrostatic and van der
The binding studies were carried out at 298, 303 and 308 K
and the values are given in Table 5. All ΔG values no matter at
Luminescence 2012
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence

X. Luo et al.
Figure 4. Plots of lg [(F0 – F)/F] versus log [Q] at different temperatures
Table 4. Binding constants and number of binding sites of CCSCPs to BSA
BSA–PFCCSCP
BSA–PCCCSCP
BSA–PBCCSCP
T = 298 K
T = 303 K
T = 308 K
Log (KA)
7.19(20)
1.54
1.37(4)
0.9973
7.13(19)
1.35
1.36(4)
0.9974
6.97(20)
0.94
1.33(4)
0.9969
7.44(26)
2.76
1.41(5)
0.9956
7.31(23)
2.04
1.38(5)
0.9963
7.27(27)
1.86
1.37(5)
0.9950
7.52(19)
3.33
1.43(4)
0.9975
7.55(25)
3.54
1.43(5)
0.9958
7.47(20)
2.93
1.42(4)
0.9974
KA ꢄ 107 (M–1)
n
R
Log (KA)
KA ꢄ 107 (M–1)
n
R
Log (KA)
KA ꢄ 107 (M–1)
n
R
Table 5. Thermodynamic parameters of BSA–CCSCP
System
ΔG (kJꢃmol–1)
ΔH (kJꢃmol–1)
ΔS (Jꢃmol–1ꢃK)
T = 298 K
T = 303 K
T = 308 K
BSA–PFCCSCP
BSA–PCCCSCP
BSA–PBCCSCP
ꢀ41.01
ꢀ42.45
ꢀ42.92
ꢀ41.37
ꢀ42.40
ꢀ43.79
ꢀ41.11
ꢀ42.86
ꢀ44.03
ꢀ37.89
ꢀ30.30
ꢀ9.60
10.82
40.50
112.16
which temperature or for different CCSCPs were around ꢀ42
kJꢃmol–1, which revealed that the binding process was spontane-
ous. Although all CCSCPs had negative ΔH and positive ΔS, the
contributions of ΔH and ΔS to ΔG values differed for the hree
CCSCPs. From PFCCSCP to PBCCSCP, the contributions of ΔS
increased greatly followed by the steep dipping of ΔHꢁ in the
wileyonlinelibrary.com/journal/luminescence
Copyright © 2012 John Wiley & Sons, Ltd.
Luminescence 2012

Interaction between halocinnamoyl sulfachloropyrazine Na salts and BSA
values of their ΔG. This phenomenon indicated that the interac-
tion forces in BSA–PFCCSCP and BSA–PCCCSCP systems were
van der Waals interactions and hydrogen bonds. When 4′-
substituent in CCSCP structure was changed to bromine, the
ionic interaction and proton transfer could control the overall
energetics (15).
where k2 is spatial orientation factor of the dipole, N is the refrac-
tive index of the medium in the wavelength range where the
spectral overlap is significant, Φ is the fluorescence quantum
yield of the donor in the absence of acceptor, and J is the overlap
integral of the fluorescence emission spectrum of the donor and
the absorption spectrum of the acceptor as in equation (8):
P
FðlÞeðlÞl4Δl
P
J ¼
(8)
Energy transfer between BSA and CCSCP
FðlÞΔl
Resonance energy transfer (RET) is another important process
occurring in the excited state. This process occurs when the emis-
sion spectrum of the donor overlaps with the absorption spec-
trum of the acceptor. Unlike other fluorescence phenomena,
RET does not depend on interactions of the fluorophore with
other molecules in the surrounding solvent shell. According to
Förster theory (5,16), non-radioactive energy transfer is effective
over much longer distances and the intervening solvent or mac-
romolecule have few effects on the efficiency of energy transfer,
which depends primarily on the donor-to-acceptor (D–A)
distance. The extent of energy transfer is determined by the
distance between the donor and acceptor and the extent of
spectral overlap. The efficiency of energy transfer (E) was related
to the D–A distance and it could be calculated using equation (6):
where F(l) is the fluorescence intensity of the fluorescence donor
at wavelength l and e(l) is the molar absorbance of the acceptor
at wavelength l. The overlap of the fluorescence emission
spectra of BSA and the absorption spectra of CCSCPs at 298 K
are shown in Figure 5.
Under the above experimental conditions, k2 = 2/3, N was the
average value of water and organic solute (1.366) and Φ = 0.118
(10,16). According to equations (6–8), the values of the parameters
are listed in Table 6. The binding distances of three CCSCPs were
less than 8 nm and 0.5R0 < r < 1.5R0, which indicated that the
energy transfer from BSA to CCSCPs occurred with high probability
(9,16). Furthermore, three CCSCPs had a similar ability to bind with
BSA because of nearly same binding distances.
F
R06
E ¼ 1 ꢀ
¼
(6)
F0 R60 þ r6
Table 6. Distance parameters between BSA and CCSCPs
where R0 is the Förster distance when the efficiency of resonance
energy transfer is 50% and r is the D–A distance. The value of R0
can be calculated using equation (7):
System
J (cm3ꢃM–1)
E
R0 (nm)
r (nm)
BSA–PFCCSCP
BSA–PCCCSCP
BSA–PBCCSCP
1.51 ꢄ 10–14
1.70 ꢄ 10–14
1.75 ꢄ 10–14
0.66
0.70
0.70
2.59
2.64
2.65
2.31
2.29
2.31
-4
R60 ¼ 8:79 ꢄ 10ꢀ25k2ΦN J
(7)
Figure 5. Overlap of fluorescence emission spectrum of BSA (a) and absorption spectrum of CCSCPs (b). c(BSA) = c(CCSCP) = 1.00 ꢄ 10–5 M; T = 298 K
Luminescence 2012
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence

X. Luo et al.
Conformation change
of tryptophan residues stabled at 350 nm. These results indicate
that CCSCPs did not affect the microenvironment of tryptophan
residues during the binding process and the polarity around the
tyrosine residues decreased and hydrophobicity around tyrosine
residues increased (4,9).
Since synchronous fluorescence can give narrow and simple
spectra, it was used to provide information about the molecular
environment in the vicinity of the molecules and evaluate the
conformational changes of BSA. By measuring the shift of
emission maximum, the environment of amino acid residues in
BSA could be studied and the shifts corresponded to the
changes of the polarity around the molecule (9). The character-
istic information for tyrosine or tryptophan residues was given
by the synchronous fluorescence, when the difference between
excitation wavelength and emission wavelength were stabilized
at 15 or 60 nm (17). The effects of CCSCPs on the synchronous
fluorescence spectra of BSA are shown in Figure 6. It is apparent
from Figure 6 that the maximum emission wavelengths of
tyrosine residues slightly shifted towards shorter wavelengths
(left column, Figure 6) and the maximum emission wavelengths
Conclusions
In this paper, the interactions of CCSCPs and BSA were investi-
gated by fluorescence and ultraviolet spectroscopy. The experi-
mental results indicate that the three CCSCPs formed complexes
with BSA at low concentrations at three temperatures. A
combined quenching (static and dynamic) process occurred at
higher concentrations of CCSCPs. Based on thermodynamic
parameters, hydrophobic and electrostatic interactions were
the main binding forces in the BSA–PFCCSCP and BSA–PCCCSCP
Figure 6. Synchronous fluorescence spectra of BSA in the present CCSCPs. (A) Δl = 15 nm, (B) Δl = 60 nm; T = 298 K; c(BSA) = 1.00 ꢄ 10–5 M; c(CCSCP)/( ꢄ 10–5 M), (a–j)
0.00, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20, 1.40, 1.60 and 1.80, respectively
wileyonlinelibrary.com/journal/luminescence
Copyright © 2012 John Wiley & Sons, Ltd.
Luminescence 2012

Interaction between halocinnamoyl sulfachloropyrazine Na salts and BSA
5. Naik PN, Chimatadar SA, Nandibewoor ST. Study on the interaction
systems, but the ionic interactions and proton transfer played
major roles in the binding process of the BSA–PBCCSCP system.
The average binding distances of CCSCPs were around 2.3 nm,
indicating that the energy transfer from BSA to CCSCPs occurred
with high possibility. Moreover, the measured synchronous fluo-
rescence spectroscopy indicated that the conformation of BSA
was changed in the presence of CCSCPs and that binding oc-
curred at tyrosine residues of BSA. The knowledge obtained in
this study can provide information to understand the mode of
action between CCSCPs and BSA and could be beneficial to drug
design and pharmaceutical research.
between antibacterial drug and bovine serum albumin: A spectro-
scopic approach. Spectrochim Acta, Part 2009;A73:841–5.
6. Carter DC, Ho JX. Structure of serum albumin. Adv Protein Chem
1994;45:153–203.
7. Tian J, Liu J, Zhang J, Hu Z, Chen X. Fluorescence studies on the
interactions of barbaloin with bovine serum albumin. Chem Pharm
Bull 2003;51:579–82.
8. He XM, Carter DC. Atomic structure and chemistry of human serum
albumin. Nature (London, U K) 1992;358:209–15.
9. Guo X-J, Sun X-D, Xu S-K. Spectroscopic investigation of the interac-
tion between riboflavin and bovine serum albumin. J Mol Struct
2009;931:55–9.
10. Lakowicz JR. Principles of Fluorescence Spectroscopy. three ed. New
York Springer, 2010.
11. Zhao G-J, Han K-L. Hydrogen bonding in the electronic excited state.
Acknowledgements
Acc Chem Res 2012;45:404–13.
This work was supported by a grant from the National Natural
Science Foundation of China (20962002, 20662001).
12. Yuan T, Weljie AM, Vogel HJ. Tryptophan fluorescence quenching by
methionine and selenomethionine residues of calmodulin: orienta-
tion of peptide and protein binding. Biochemistry 1998;37:3187–95.
13. Bi S, Sun Y, Qiao C, Zhang H, Liu C. Binding of several anti-tumor
drugs to bovine serum albumin: Fluorescence study. J Lumin
2009;129:541–7.
14. Jiang M, Xie MX, Zheng D, Liu Y, Li XY, Chen X. Spectroscopic studies
on the interaction of cinnamic acid and its hydroxyl derivatives with
human serum albumin. J Mol Struct 2004;692:71–80.
15. Ross PD, Subramanian S. Thermodynamics of protein associa-
tion reactions: forces contributing to stability. Biochemistry
1981;20:3096–102.
16. Kang J, Liu Y, Xie MX, Li S, Jiang M, Wang YD. Interactions of human
serum albumin with chlorogenic acid and ferulic acid. Biochim
Biophys Acta, Gen Subj 2004;1674:205–14.
17. Burstein EA, Vedenkina NS, Ivkova MN. Fluorescence and the
location of tryptophan residues in protein molecules. Photochem
Photobiol 1973;18:263–79.
References
1. Huang K, Ding Z, Zhao S, Yan Y. Compendium of Modern Materia
Medica, Beijing, China: Medical Science Press, 2001.
2. Huang L, Lin C, Li A, Wei B, Teng J, Li L. Pro-coagulant activity of
phenolic acids isolated from Blumea riparia. Nat Prod Commun
2010;5:1263–66.
3. Yu M, Ding Z, Jiang F, Ding X, Sun J, Chen S, et al. Analysis of binding
interaction between pegylated puerarin and bovine serum albumin
by spectroscopic methods and dynamic light scattering. Spectro-
chim Acta, Part A 2011;83:453–60.
4. Yu X, Liu R, Yi R, Yang F, Huang H, Chen J, et al. Study of the
interaction between N-confused porphyrin and bovine serum
albumin by fluorescence spectroscopy. Spectrochim Acta, Part A
2011;78:1329–35.
Luminescence 2012
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence