Accepted Manuscript Title: Probing the interaction of cefodizime with human serum albumin using multi-spectroscopic and molecular docking techniques Author: Taoying Hu Ying Liu PII: DOI: Reference:
S0731-7085(15)00020-5 http://dx.doi.org/doi:10.1016/j.jpba.2015.01.010 PBA 9893
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
27-9-2014 31-12-2014 6-1-2015
Please cite this article as: http://dx.doi.org/10.1016/j.jpba.2015.01.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Graphical Abstract
IB
Domain I
IIIB
ip t
IA
Domain III
cr
Sudlow’s site I CEF
IIA
us
Sudlow’s site II
Domain I
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IIB
Page 1 of 33
*Highlights (for review)
The interaction of CEF and HSA was studied by multi-spectroscopic and molecular docking techniques.
The binding of CEF with HSA was not only a static quenching
ip t
procedure but also driven mainly by hydrogen bonds and van der Waals force.
Binding constant, number of binding site and binding distance were
cr
us
calculated.
CEF changed the conformation and secondary structure of HSA.
CEF bound to hydrophobic pocket (site I, subdomain IIA) of HSA.
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Page 2 of 33
*Revised Manuscript
Probing the interaction of cefodizime with human serum albumin
2
using multi-spectroscopic and molecular docking techniques
3
Taoying Hu1, Ying Liu 1,2
4
1. College of Life and Environmental Sciences, Minzu University of China, Beijing100081,
5 6 7
China 2. Beijing Engineering Research Center of Food Environment and Public Health, Minzu University of China, Beijing 100081, China
Corresponding author. Tel.:+86 15810128921; fax: +86 10 68450641. E-mail address:[email protected]
cr
8 9
ip t
1
Abstract: To know the interaction of cefodizime (CEF) with human
11
serum albumin (HSA), techniques of different spectroscopies and
12
molecular modeling were used. The inner filter effects were eliminated to
13
get accurate binding parameters. Steady state fluorescence suggested a
14
static type for CEF-HSA interaction, and the complex formation had a
15
high affinity of 105 Lmol1. On the basis of the thermodynamic results
16
and site marker competitive experiments, it was considered that CEF was
17
bound to site I (subdomain IIA) of HSA mainly by hydrogen bonds and
18
van der Waals force. The calculated binding distance (r) indicated that the
19
non-radioactive energy transfer came into being in the interaction
20
between CEF and HSA. Furthermore, molecular modeling was applied to
21
further define that CEF interacted with the Trp214, Lys199, Phe211,
22
Leu238 residues of HSA. In addition, three-dimensional fluorescence and
23
circular dichroism (CD) results showed that the binding of CEF can cause
24
conformational and some microenvironmental changes of HSA. This
25
paper provides reasonable models helping us further understand the
26
transportation and distribution of CEF when it spreads into human blood
27
serum
28
pharmacodynamics.
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10
which
is
of
great
importance
in
pharmacology
and
Corresponding author. Tel.:+8615810128921; fax: +861068450641. E-mail address:[email protected] 1
Page 3 of 33
29
Keywords: Cefodizime (CEF); Human serum albumin (HSA); Multi-
30
spectroscopic techniques; Molecular modeling
31 32
1. Introduction The most abundant serum protein, i.e., albumin, serves as a transport
34
vehicle for several endogenous and exogenous compounds, such as fatty
35
acids, dyes, drugs and steroids [1-4]. The distribution, metabolism,
36
excretion and toxicity of ligands are related to their binding affinities to
37
proteins,
38
approximately 60% of the total protein, HSA is the most abundant protein
39
in blood plasma contributing 80% of the colloid osmotic pressure. HSA is
40
a single-chain, non-glycosylated polypeptide with well-known 585 amino
41
acids sequences and contains three homologous α-helical domains (I, II
42
and III) which assemble to form a heart-shaped molecule. Each domain
43
contains two subdomains (A and B), which are predominantly helical and
44
extensively cross-linked through several disulfide bridges. Its amino acid
45
sequence contains a total of 17 disulfide bridges, one free thiol (Cys-34)
46
and a single tryptophan (Trp-214) [1,2]. It is capable of binding
47
reversibly to a wide variety of drugs, due to which increased solubility in
48
plasma, decreased toxicity, and/or protection against oxidation of bound
49
ligands occur. The existence of two principle binding regions, viz.
50
Sudlow sites I and II, are responsible for the protein’s capability to bind
51
different ligands, which are located within hydrophobic cavities in
52
subdomains IIA and IIIA, respectively. Site I is known as warfarin-
53
azapropazone site, which contains a single tryptophan residue in position
54
214 (Trp-214). Site II is known as the indole-benzodiazepine site, which
55
presents two important amino acid residues (Arg-410 and Tyr-411) [3].
human
serum
albumin
(HSA).
By
constituting
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i.e.,
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33
2
Page 4 of 33
The serum albumin-ligand binding studies have been reported in a
57
number of recent papers [4,5]. Many drugs (antibiotic, anesthetic,
58
antidepressant, etc), exert their activity by interaction with biological
59
membranes. We also have literature related to the thermodynamics,
60
binding characteristics, and conformation properties of several antibiotic
61
drugs (viz, nitrofurazone, tosufloxacin tosylate, methacycline and
62
demeclocycline) interaction with albumins [6,7]. Many drugs and other
63
kinds of small bioactive materials have the potential to bind reversibly to
64
serum
65
hydrophobicity and efficiency of the delivery process to the targeted
66
tissues. Binding studies have provided information of the structural
67
features determining the therapeutic effectiveness of drug and developed
68
as an interesting research field in life-sciences, chemistry, and clinical
69
medicine [8].
which,
in
turn,
cannot
ably
modulate
their
M
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albumins
cr
ip t
56
Cefodizime (CEF) (Fig. 1) is an extended-spectrum third-generation
71
cephalosporin antibiotic that is widely used in the treatment of severe
72
infections of the respiratory and urinary tracts. Compared with the other
73
third generation cephalosporin, it has some peculiar merits such as no
74
renal toxic effect, good tolerance and even the activity of immune
75
regulation [9], which a lot of the same kind of antibiotics can’t match.
76
There have been some studies to study the interaction of cephalosporin
77
with protein. For example, Shao et al. [10] studied the binding
78
mechanism of cefodizime sodium to bovine serum albumin (BSA) by
79
fluorescence spectra, and Pan et al. [11] reported the interaction of
80
ceftriaxone sodium to BSA using spectroscopic and circular dichroism
81
(CD) methods. However, these studies are insufficient in terms of
82
elimination of the inner filter effects, site marker competitive experiments
83
and molecular docking, which are of great importance for perfectly
84
demonstrating the interaction of cephalosporin with protein. Thus, in this
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3
Page 5 of 33
85
paper, our probing the interaction of cefodizime with HSA by using
86
multi-spectroscopic methods is based on the aforesaid what are left out in
87
the previous study. In the present work we seek to discuss about the biophysical
89
interactions of CEF with HSA that play an important role in drug
90
transport and storage. The study was established in which the multi-
91
spectroscopic and molecular modeling techniques were utilized to obtain
92
the interaction information of CEF with HSA, i.e., the quenching
93
mechanism of fluorescence, the binding parameters, the effect of CEF on
94
the conformational and secondary structural changes of HSA and the
95
specific binding site. The investigation of the binding interaction between
96
drugs and serum albumins is of much importance in pharmacology and
97
pharmacodynamics. Therefore, such interactions in vitro have been
98
considered as models in protein chemistry to study their binding behavior.
99
2. Experimental
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2.1. Materials
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100
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88
HSA (Sigma, Missouri, USA) stock solution was prepared with the
102
concentration of 1.0 104 molL1. CEF (National Institutes for Food
103
and Drug Control, Beijing, China) was dissolved and diluted to 1.0 103
104
molL1 with ultrapure water. The stock solutions of phenylbutazone and
105
ibuprofen were prepared by dissolving them in a small amount of ethanol,
106
then diluting to 1.0 103 molL1 with water. Phosphate buffer solution
107
(NaH2PO4 (75 mM)Na2HPO4 (75 mM) (19:81, v/v)(pH 7.40)) was used
108
for CD measurement, while Tris (0.2 M)HCl (0.1 M) buffer (25:40,
109
v/v)( pH 7.40) and 0.1 molL1 NaCl solution were used for other
110
measurements. All of the solutions were kept in the dark at 04C. All
Ac
101
4
Page 6 of 33
111
reagents were of analytical reagent grade and ultrapure water was used
112
throughout the experiment.
113
2.2. Measurements Steady state fluorescence measurements were carried out through an F-
115
4500 spectrophotometer (Hitachi, Tokyo, Japan) equipped with a 1.0 cm
116
quartz cell at an excitation wavelength of 280 nm and emission
117
wavelength of 200500 nm. The excitation and emission slits were set at
118
5 nm while the scanning rate was 1200 nm/min and Photomultiplier tube
119
(PMT) voltage 700 V. The three-dimensional fluorescence spectra were
120
performed at an excitation wavelength of 200400 nm and emission
121
wavelength of 200500 nm, and the increments were 5 nm.
cr
us
The
absorption
spectra
were
an
122
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114
recorded
on
a
UV-2800
spectrophotometer (Hitachi, Tokyo, Japan) equipped with a 1.0 cm quartz
124
cell. The wavelength range was 190500 nm with slit width of 2.0 nm.
M
123
Competitive bindings between CEF and HSA in the presence of two
126
site markers (phenylbutazone and ibuprofen) were performed. The ratio
127
of concentrations of CEF and HSA was kept at 5:1, then site markers
128
were gradually added to CEF-HSA mixtures, the fluorescence spectra
129
were recorded upon excitation at 280 nm.
ce pt
ed
125
The CD measurements were obtained over a wavelength range of 190-
131
250 nm at 0.2 nm intervals on a JASCO J-810 CD spectrometer (JASCO,
132
Tokyo, Japan) using a 0.1 cm cell at room temperature. Each spectrum
133
was scanned three times and finally averaged for plots and analyses.
Ac
130
134
Docking calculations were performed with Docking Server on a HSA
135
protein model (PDB-2BXM) [12]. The MMFF94 force field was used for
136
energy minimization of the ligand molecule (CEF). Gasteiger partial
137
charges were added to the ligand atoms. Nonpolar hydrogen atoms were
138
merged, and rotatable bonds were defined. Essential hydrogen atoms, 5
Page 7 of 33
Kollman united atom type charges, and solvation parameters were added
140
with the aid of AutoDock tools [13]. Affinity (grid) maps of 20 20 20
141
Å grid points and 0.375 Å spacing were generated using the Autogrid
142
program [14]. The AutoDock parameter set- and distance-dependent
143
dielectric functions were used in the calculation of the van der Waals and
144
electrostatic terms, respectively.
ip t
139
Docking simulations were performed using the Lamarckian genetic
146
algorithm (LGA) and the Solis and Wets local search method [15]. Initial
147
positions, orientations, and torsions of the ligand molecules were set
148
randomly. Each run of the docking experiment was set to terminate after a
149
maximum of 250,000 energy evaluations. The population size was set to
150
150. During the search, a translational step of 0.2 Å and quaternion and
151
torsion steps of 5 were applied.
152
3. Results and discussions
153
3.1. Elimination of the inner filter effects
ed
M
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cr
145
The system of CEF-HSA has absorbance at excitation and emission
155
wavelengths of the fluorescence measurements, which will impact the
156
accuracy of fluorescence data. To eliminate the inner filter effects,
157
absorbance measurements were carried out at the fluorescence excitation
158
and emission wavelengths. The extent of this effect can be roughly
159
evaluated with the following relationship [16]:
160
Fcor Fobs e
161
where Fcor and Fobs are the fluorescence intensities corrected and observed,
162
respectively, and Aex and Aem are the absorption of HSA and CEF at
163
excitation and emission wavelengths, respectively. All the fluorescence
164
intensity used in this study was corrected.
165
3.2. Analysis of fluorescence quenching of HSA
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154
Aex Aem /2
(1)
6
Page 8 of 33
Fluorescence method is an important tool for investigating the
167
interaction between small probe molecules and proteins to get
168
information about the binding mechanism, binding constants, binding
169
mode, binding sites, and intermolecular distances. Fluorescence of HSA
170
originates from tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe)
171
residues, whereas the intrinsic fluorescence of HSA is mainly attributed
172
to the Trp-214 residue alone. As the Phe residue has very low quantum
173
yield and the fluorescence of Tyr residue is almost entirely quenched
174
when it is ionized or close to a carboxyl group, an amino group or a Trp
175
[17]. Fluorescence quenching is the decrease of the quantum yield of
176
fluorescence from a fluorophore, which is induced by a wide variety of
177
molecular interactions with a quencher molecule, viz., excited-state
178
reactions, energy transfer, molecular rearrangements, ground-state
179
complex formation and collisional quenching. If the small molecule can
180
quench the fluorescence of the Trp residues, the Trp residues must be
181
located in or near the binding position [18].
ed
M
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166
A valuable feature of intrinsic fluorescence of a protein is the high
183
sensitivity of Trp to its local environment. Changes in emission spectra of
184
Trp are common in response to the protein conformational transition,
185
subunit association, substrate binding, or denaturation [19]. To
186
comprehend how the structure of HSA is affected by CEF, Fig. 2 was
187
used to indicate the fluorescence emission spectra for HSA with various
188
molar ratios of CEF at the excitation wavelength of 280 nm. The HSA
189
had a strong fluorescence emission with a peak at 351 nm while CEF was
190
almost non-fluorescent. It can be seen that the fluorescence intensities of
191
HSA decreased clearly with increasing CEF concentrations indicating
192
that CEF interacted with HSA and the binding site must be located in or
193
near Trp residues.
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182
7
Page 9 of 33
The various mechanisms of quenching are usually classified as either
195
dynamic or static mechanism. Static quenching refers to the formation of
196
a ground-state complex between the fluorophore (protein) and the
197
quencher, while dynamic quenching refers to a process which involves
198
the fluorophore and the quencher coming into contact during the transient
199
existence of the excited state. In general, static and dynamic quenching
200
can be distinguished by their dependence on temperature. For dynamic
201
quenching, the quenching constant is expected to increase with increasing
202
temperature because higher temperature results in larger diffusion
203
coefficient. On the contrary, higher temperature is likely to lead to the
204
decreased stability of complex and thus smaller values of the static
205
quenching constant [20].
an
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194
In order to confirm the quenching mechanism, the fluorescence
207
quenching data were analysed according to the well-known Stern-Volmer
208
equation [21]:
ed
209
M
206
F0 1 kq 0 Q 1 K SV Q F
(2)
where F0 and F are the steady-state fluorescence intensities in the absence
211
and presence of quencher (CEF), respectively, KSV is the Stern-Volmer
212
quenching constant, which is determined by linear regression of Stern-
213
Volmer equation, [Q] is the initial concentration of quencher, kq is the
214
bimolecular quenching rate constant, and τ0 is the average lifetime of
215
fluorophore without quencher and its value is 108 s. As evident from Fig.
216
3 and Table 1, it can be found that there were good linearity relationship
217
between F0/F and [Q]CEF and the KSV values decreased with increasing
218
temperature, indicating that the likely quenching process was static
219
quenching mechanism rather than dynamic quenching mechanism. As the
220
kq values were far greater than the upper limit of 2.0 1010 Lmol1s1
Ac
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210
8
Page 10 of 33
221
[21], the fluorescence quenching of HSA in the presence of CEF was a
222
static quenching process. UV-vis absorption measurement is a simple but effective method to
224
distinguish static and dynamic quenching. Collisional quenching only
225
affects the excited states of the fluorophores, and thus no changes in the
226
absorption spectra are expected. In contrast, ground-state complex
227
formation will frequently result in perturbation of the absorption
228
spectrum of the fluorophores [22]. The UV-vis absorption spectra of HSA
229
in the presence of different concentrations of CEF were shown in Fig. 4.
230
HSA had two absorption peaks, the intensity of the strong absorption
231
peak at about 208 nm, which reflected the framework conformation of the
232
protein continuously dropped with the concentration of CEF accompanied
233
by a remarkable red shift (from 208 to 213 nm), and the intensity of the
234
weak absorption peak at about 276 nm reflecting the aromatic amino
235
acids (Trp, Tyr, Phe) increased slightly with CEF concentration [22]. The
236
results reconfirmed that the probable quenching mechanism of the
237
intrinsic fluorescence of HSA was not initiated by dynamic collision but
238
by CEF-HSA complex formation.
239
3.3. Analysis of binding equilibria
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223
For the equilibrium between free and bound molecules, when small
241
molecules bind independently to a set of equivalent sites on a
242
macromolecule, the relationship between the binding constant (Ka) and
243
the number of binding sites (n) could be represented by the equation [2]:
244
log
245
where F0, F and [Q] are the same as in Eq. (2). According to Eq. (3), the
246
slope and intercept value of the plot, log[(F0 F)/F] versus log[Q] gives
247
the Ka and n values (Table 2). The results showed that the association
Ac
240
F0 F log K F
a
n log Q
(3)
9
Page 11 of 33
constants (Ka) were decreased with the increasing temperature, which
249
may hint the formation of an unstable complex in the binding reaction.
250
The complex would be partly decomposed with the increasing
251
temperature, therefore, the values of Ka decreased. Furthermore, the
252
values of n were approximately equal to 1, manifesting the existence of
253
just a single binding site in HSA towards CEF.
254
3.4. Thermodynamic parameters and binding forces
cr
ip t
248
The interaction forces between macromolecules and drugs mainly
256
include four acting forces: hydrogen bonds, van der Waals force,
257
hydrophobic force, and electrostatic interactions. If the enthalpy change
258
(∆H) does not vary significantly over the temperature ranged studied,
259
then its value and that of Gibbs free energy change (∆G) and entropy
260
change (∆S) of the reaction, which could be used to verify the binding
261
mode, can be determined by the Van’t Hoff equation [7]: ln K a
H S RT R
ed
M
an
us
255
(4)
where R is the gas constant, T is the experimental temperature, and Ka is
263
the binding constant at the corresponding temperature. The values of ∆H
264
and ∆S were obtained from the slope and intercept of the linear Van’t
265
Hoff plot based on lnKa versus 1/RT, respectively. The free energy
266
change (∆G) was then evaluated from following relationship [7]:
267
G H T S
Ac
ce pt
262
(5)
268
The temperatures were chosen at 298K and 310K, at which HSA did
269
not undergo any structural degradation. The thermodynamic parameters
270
were obtained and presented in Table 2. According to Ross [23], the
271
negative sign for ∆G meant that association process was spontaneous.
272
The negative ∆H and ∆S values for the association reaction between CEF
10
Page 12 of 33
273
and HSA implied that both hydrogen bonds and van der Waals force were
274
the major driving forces of the interaction.
275
3.5. Energy transfer According to the Fӧrster theory of non-radiative energy transfer, the
277
rate of energy transfer depends upon the following factors: (1) the donor
278
can produce fluorescence light, (2) the fluorescence emission spectra of
279
the donor and UV-vis absorption spectra of the acceptor have enough
280
overlap, (3) the distance between the donor and the acceptor is lower than
281
8 nm. The efficiency of energy transfer between the donor and acceptor,
282
E, could be estimated by the following equation [24]:
283
R06 F E 1 6 6 F0 R0 r
284
where r is the binding distance between donor and acceptor, and R0 is the
285
critical distance between donor and acceptor when their transfer
286
efficiency is 50%:
(6)
ed
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cr
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276
(7)
ce pt
R06 8.79 1025 K 2 N 4 J
where K2 is the orientation factor between the emission dipole of the
288
donor and the absorption dipole of the acceptor, N is the refracted index
289
of the medium, φ is the fluorescence quantum yield of the donor, and J is
290
the overlap integral of the fluorescence emission spectra of the donor and
291
the absorption spectra of the acceptor, which could be calculated by the
292
following:
293
F J F
294
where F(λ) is the fluorescence intensity of the donor at wavelength λ, and
295
ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. In
296
the present study, K2 = 2/3, n = 1.336 and φ = 0.15 for HSA [16].
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287
4
(8)
11
Page 13 of 33
The overlap of the fluorescence emission spectra of HSA and the UV-
298
vis absorption spectra of CEF when the molar ratio was 1:1 and at 298K
299
was represented in Fig. 5. From Eqs. (6)(8), we can calculate that R0 =
300
2.31 nm, E = 0.11, J = 5.57 1015 cm3Lmol1, and r = 3.30 nm. The
301
specific binding distance between Trp-214 (donor) and CEF (acceptor)
302
was smaller than 8 nm, which was the condition for energy transfer
303
phenomenon to occur. This indicated that energy transfer between CEF
304
and HSA can occur with high possibility.
305
3.6. Site marker competitive experiments
us
cr
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297
To identify the CEF binding sites on HSA, site marker competitive
307
experiments were carried out using site probes that specifically bind to a
308
known site or region on HSA. Sudlow et al. [25] have suggested two
309
main distinct binding sites on HSA called site I (subdomains IIA) and site
310
II (subdomains IIIA). Site I shows affinity for bulky heterocyclic anion
311
with a negative charge localized in the middle of the molecule, drugs
312
binding in this site include warfarin, phenylbutazone and diflunisal.
313
While ligands binding to site II are aromatic carboxylic acids with
314
negative charged acidic group at the end of the molecule, ligands tend to
315
this
316
indoxylsulfate. In this work, the competitors were used including
317
phenylbutazone, a characteristic marker for site I, and ibuprofen for site II
318
marker. The ratio of CEF to HSA was kept at 5:1 to keep nonspecific
319
binding probes to a minimum. By means of recording the changes in the
320
fluorescence intensity of CEF bound HSA, which was brought about by
321
site marker, information about the specific binding location of CEF in
322
HSA molecule can be obtained. The changes induced by the site markers
323
were presented in Fig. 6. The fluorescence of the complex was
324
remarkably affected in presence of phenylbutazone, conversely the
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306
Ac
region involve ibuprofen, flufenamic acid, diazepam and
12
Page 14 of 33
addition of ibuprofen to the same solution. The observations
326
demonstrated that phenylbutazone displaced CEF from the binding site,
327
while ibuprofen had a little effect on the binding of CEF to HSA. Hence,
328
it can be concluded that the binding of CEF to HSA mainly located
329
within site I (subdomain IIA), so Trp-214 was near or within the binding
330
site, which also provided a reasonable explanation why the calculated
331
value of binding distance r was small.
332
3.7. Molecular docking
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325
To further define the binding site, molecular docking was employed by
334
setting the simulation box to site I. The crystal structure of HSA was
335
taken from the Protein Data Bank (entry PDB code 2BXM). The best
336
energy ranked result was shown in Fig. 7. The inside wall of the pocket of
337
subdomain IIA was formed by hydrophobic side chains, whereas the
338
entrance to the pocket was surrounded by positively charged residues
339
consisting of Lys199, Leu238, Arg218, Ser202 and Ala215 [26]. Thus we
340
can conclude that CEF was able to fit well within the subdomain IIA
341
hydrophobic cavity (see Fig. 7b). The docking result showed the
342
existence of hydrogen bonds, hydrophobic, and polar interactions
343
between CEF and HSA. Furthermore, the distances were measured
344
between the protons of CEF and adjacent residues around the binding site.
345
Active site residues within 5 Å to CEF included Phe206, Arg484, Trp214,
346
Leu481, Leu198 and Val344. In particular, the calculated binding
347
distance r between Trp-214 and CEF was similar to the apparent distance
348
r (3.30 nm) between the donor and the acceptor evaluated from Fӧrster’s
349
non-radiative energy transfer analysis.
350
3.8. Three-dimensional fluorescence spectra
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13
Page 15 of 33
In order to better understand the conformational changes of HSA upon
352
the addition of CEF, three-dimensional fluorescence spectrum was
353
performed on HSA and CEF-HSA system. Fig. 8 described the three-
354
dimensional fluorescence contour maps (A and B) and three-dimensional
355
fluorescence projections (C and D) of HSA and CEF-HSA. The contour
356
map exhibited a bird’s eye view of the fluorescence spectra and can also
357
provide lots of crucial information. As shown in Fig. 8, peak a is the
358
Rayleigh scattering peak. Peak 1 mainly enunciates the spectral behavior
359
max of Trp and Tyr residues. The maximum emission wavelength (λ em ) and
360
the fluorescence intensity of this peak are closely related to the polarity of
361
the microenvironment [27]. With the addition of CEF, the fluorescence
362
intensity of peak 1 decreased markedly, which indicated that the addition
363
of CEF changed the polarity of the Trp residues microenvironment. The
364
weak fluorescence peak 2 chiefly exhibits the conformation of the peptide
365
backbone associated with the helix-coil [28]. The fluorescence intensity
366
max of peak 2 had an obvious decrease with a red shift of λ em (from 347.0 nm
367
to 350.0 nm). It could be derived that the interaction of CEF with HSA
368
led to a conformational change of the protein.
369
3.9. CD spectroscopy
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cr
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351
CD spectra can further confirm the structural changes of HSA upon
371
addition of CEF and the CD spectrum of HSA has two negative bands at
372
around 209 nm and 222 nm, which are contributed to the transition of
373
nπ* of -helix structure [29]. The CD results were expressed in terms
374
of mean residue ellipticity (MRE) in deg cm2dmol1 according to [30]:
375
MRE
376
where Cp is the molar concentration of HSA, n is the number of amino
377
acid residues (n=585, for HSA), and l is the path length. The -helical
Ac
370
observedCD m deg C p nl 10
(9)
14
Page 16 of 33
378
contents in the secondary structure of HSA were calculated from MRE
379
values at 209 nm using the equation [30]:
380
helix(%)
381
where MRE209 is the observed MRE value at 209 nm, 4000 is the MRE of
382
the β-form and random coil conformation cross at 209 nm, and 33000 is
383
the MRE value of a pure -helix at 209 nm.
MRE209 4000 100 33000 4000
ip t
(10)
As was evident in Fig. 9, when CEF was added to a solution of HSA,
385
the intensity of negative bands at 209 nm and 222 nm increased without
386
any shift of peaks, illustrating that the secondary structure of HSA after
387
binding to CEF was predominantly -helical. The calculated results
388
(Table 3) showed that the content of -helix for HSA increased from
389
44.5% to 47.6% when nCEF/nHSA increased from 0:1 to 20:1, which
390
indicated that binding of CEF to HSA may induce some conformational
391
changes of protein. It can be deduced that the -helix structure was
392
affected probably due to insertion of CEF into hydrophobic cavity of
393
HSA.
394
4. Conclusions
ce pt
ed
M
an
us
cr
384
In this paper, the probable quenching mechanism of fluorescence of
396
HSA initiated by CEF was a static quenching process, the negative values
397
of enthalpy change (H = 6.20 kJmol1) and entropy change (S =
398
75.83 Jmol1K1) indicated that the main driving forces of the
399
interaction between CEF and HSA were hydrogen bonds and van der
400
Waals force. The distance (r) of 3.30 nm between the acceptor (CEF) and
401
donor (Trp-214) revealed that the energy transfer from HSA to CEF
402
occurred. The site marker competitive experiments showed that the
403
specific binding of CEF was located in the vicinity of site I (subdomain
404
IIA) of HSA. The red shift (347.0 nm350.0 nm) of peak 2 of three-
Ac
395
15
Page 17 of 33
dimensional fluorescence and the slightly increase of -helix from 44.5%
406
to 47.6% of CD spectra showed that the conformation and micro-
407
environment of HSA had changed because of the binding of CEF.
408
Furthermore, the binding details between CEF and HSA were further
409
confirmed by molecular modeling, which manifested that CEF was bound
410
at subdomain IIA through multiple interactions, such as hydrophobic
411
interaction, polar force, and hydrogen bonds, etc. This study is expected
412
to provide salient biophysical and biochemical clues on elucidating the
413
transport and storage of CEF.
414
Acknowledgements
an
us
cr
ip t
405
Here the authors thank the National Science Foundation of China
416
(21177163), 111Project B08044, Beijing Engineering Research Center of
417
Food Environment and Public Health, Minzu University of China (10301-
418
01404026), First-class University First Class Academic Program of
419
Minzu University of China (YLDX01013), Graduate Student Scientific
420
Research Innovation Project of Minzu University of China (K2014042).
423 424 425 426 427
ed
ce pt
422
Ac
421
M
415
428 429 430 431 432 16
Page 18 of 33
433
References
434
[1] J. Ghuman, P.A. Zunszain, I. Petitpas, A.A. Bhattacharya, M. Otagiri, S. Curry,
435
Structural basis of the drug-binding specificity of human serum albumin, J. Mol.
436
Biol. 353 (2005) 3852. [2] M. Memarpoor-Yazdi, H. Mahaki, Probing the interaction of human serum
438
albumin with vitamin B2 (riboflavin) and L-Arginine (L-Arg) using multi-
439
spectroscopic, molecular modeling and zeta potential techniques, J. Lumin. 136
440
(2013) 150159.
cr
ip t
437
[3] F. Ding, W. Liu, X. Zhang, L. Zhang, Y. Sun, Fluorescence and circular dichroism
442
studies of conjugates between metsulfuron-methyl and human serum albumin,
443
Colloid. Surface. B 76 (2010) 441448.
an
us
441
[4] I. Matei, S. Ionescu, M. Hillebrand, Interaction of fisetin with human serum
445
albumin by fluorescence, circular dichroism spectroscopy and DFT calculations:
446
binding parameters and conformational changes, J. Lumin. 131 (2011) 16291635.
447
[5] M. Yan, X. Chen, S.T. Sun, H.M. Ma, B. Du, Q. Wei, Study on binding
448
mechanism of meso-tetra-(3,5-dibromo-4-hydroxyphenyl) porphyrin with protein
449
by fluorescence method, Spectroscopy and Spectral Analysis 28 (2008)
450
13221326.
ce pt
ed
M
444
[6] F.Y. Deng, C.Y. Dong, Y. Liu, Characterization of the interaction between
452
nitrofurazone and human serum albumin by spectroscopic and molecular
453
modeling methods, Mol. Biosyst. 8 (2012) 14461451.
Ac
451
454
[7] C.Y. Dong, S.Y. Ma, Y. Liu, Studies of the interaction between demeclocycline
455
and human serum albumin by multi-spectroscopic and molecular docking methods,
456 457
Spectrochim. Acta A 103 (2013) 179186. [8] S.A. Markarian, M.G. Aznauryan, Study on the interaction between isoniazid and
458
bovine
459
dimethylsulfoxide, Mol. Biol. Rep. 39 (2012) 75597567.
serumalbumin
by
fluorescence
spectroscopy:
the
effect
of
17
Page 19 of 33
460
[9] S.H. Auda, Y. Mrestani, D.H. Nies, C. Groβe, R.H.H. Neubert, Preparation,
461
physicochemical characterization and biological evaluation of cefodizime metal
462
ion complexes, J. Pharm. Pharmacol. 61 (2009) 753758.
463 [10] S. Shao, B.Y. Ma, X.J. Wang, J.J. Zhang, X.F. Li, Q. Qin, Study on the interaction
between cefodizime sodium and bovine serum albumin, Acta Phys.-Chim. Sin. 21
465
(2005) 792795.
ip t
464
466 [11] J.W. Pan, Z.T. Ye, X.P. Cai, L.X. Wang, Z. Cao, Biophysical study on the
interaction of ceftriaxone sodium with bovine serum albumin using spectroscopic
468
methods, J. Biochem. Mol. Toxic. 26 (2012) 487492.
us
cr
467
469 [12] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
471
receptor flexibility, J. Comput. Chem. 30 (2009) 27852791.
an
470
473
M
472 [13] T.A. Halgren, Merck molecular force field. I. basis, form, scope, parameterization,
and performance of MMFF94, J. Comput. Chem. 17 (1996) 490519.
ed
474 [14] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. 475
Olson, Automated docking using a Lamarckian genetic algorithm and an empirical
476
binding free energy function, J. Comput. Chem. 19 (1998) 16391662.
478
ce pt
477 [15] F.J. Solis, R.J.B. Wets, Minimization by random search techniques, Math. Oper.
Res. 6 (1981) 1930.
479 [16] F. Ding, L. Zhang, J.X. Diao, X.N. Li, L. Ma, Y. Sun, Human serum albumin
481
stability and toxicity of anthraquinone dye alizarin complexone: an albumin–dye
Ac
480
model, Ecotox. Environ. Safe. 79 (2012) 238246.
482 [17] J.R. Lakowicz, Principles of fluorescence spectroscopy, third ed., Kluwer 483
Academic Publishers/Plenum Press, New York, 2006.
484 [18] G.W. Zhang, N. Zhao, X. Hu, J. Tian, Interaction of alpinetin with bovine serum 485
albumin: probing of the mechanism and binding site by spectroscopic methods,
486
Spectrochim. Acta A 76 (2010) 410417.
487 [19] F. Ding, W. Liu, L. Zhang, B. Yin, Y. Sun, Sulfometuron-methyl binding to 488
human serum albumin: evidence that sulfometuron-methyl binds at the Sudlow’s 18
Page 20 of 33
489
site I, J. Mol. Struct. 968 (2010) 5966.
490 [20] J. Zhang, Q.S Yan, J.P. Liu, X.H. Lu, Y.S. Zhu, J. Wang, S.J. Wang, Study of the 491
interaction between 5-sulfosalicylic acid and bovine serum albumin by
492
fluorescence spectroscopy, J. Lumin. 134 (2013) 747753.
493 [21] J.G. Xu, Z.B. Wang, Fluorescence analysis, third ed., Science Press, Beijing, 2006.
495
ip t
494 [22] M.N. Zhao, H.M. Zhou, Biophysics, first ed., Higher Education Press, Beijing,
2000.
forces contributing to stability, Biochemistry 20 (1981) 30963102.
us
497
cr
496 [23] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions:
498 [24] P.N. Naik, S.A. Chimatadar, S.T. Nandibewoor, Study on the interaction between
antibacterial drug and bovine serum albumin: a spectroscopic approach,
500
Spectrochim. Acta A 73 (2009) 841845.
an
499
502
M
501 [25] G. Sudlow, D.J. Birkett, D.N. Wade, The characterization of two specific drug
binding sites on human serum albumin, Mol. Pharmacol. 11 (1975) 824832.
ed
503 [26] A.J. Ryan, C.W. Chung, S. Curry, Crystallographic analysis reveals the structural 504
basis of the high-affinity binding of iophenoxic acid to human serum albumin,
505
BMC Struct. Biol. 11 (2011) 18.
507
ce pt
506 [27] Z.J. Cheng, Interaction of tetramethylpyrazine with two serum albumins by a
hybrid spectroscopic method, Spectrochim. Acta A 93 (2012) 321330.
508 [28] H.W. Sun, Y.J. Wu, X.H. Xia, Z.H. Shi, Spectroscopic studies on the interaction
510
characteristics between norethisterone and bovine serum albumin, J. Lumin. 134
Ac
509
(2013) 580587.
511 [29] P. Yang, F. Gao, The principle of bioinorganic chemistry, Science Press, Beijing, 512
2003.
513 [30] I. Matei, M. Hillebrand, Interaction of kaempferol with human serum albumin: a 514
fluorescence and circular dichroism study, J. Pharm. Biomed. Anal. 51 (2010)
515
768773.
19
Page 21 of 33
ip t
Table(s)
cr
Table 1 Stern-Volmer quenching constants for the interaction of CEF with HSA at two different temperatures. KSV(Lmol1)
kq(Lmol1s1)
298
3.35 104
3.35 1012
0.9994
310
2.99 104
2.99 1012
0.9996
R
Ac
ce pt
ed
M
an
us
T(K)
Page 22 of 33
cr
ip t
Table(s)
Table 2 Binding constants and relative thermodynamic parameters of CEF-HSA. H (kJmol1)
1.11 1.05
6.20
S (Jmol1K1)
us
n
75.83
an
Ka (10 Lmol1) 1.12 0.50 5
G (kJmol1) 28.80 27.89
Ac
ce pt
ed
M
T (K) 298 310
Page 23 of 33
ip t
Table(s)
5:1 10:1 20:1
44.8 45.2 47.6
14.7 14.5 12.4
β-turn (%) 12.2
Random (%) 28.3
12.2 11.7 11.8
28.2 28.6 28.1
us
β-sheet (%) 15.0
Ac
ce pt
ed
M
an
0:1
α-helix (%) 44.5
nCEF/nHSA
cr
Table 3 Conformational changes of HSA in the absence and presence of CEF.
Page 24 of 33
20 HO 4
5
O 3
19
6 S 7
9
N 8
12 13
18
17N
23
35 NH2
27 O
33 N
24 14 S 15
16 N H 25
26 28
34
32
S 36
37
N 29 O 30
an
H3C 10
S 11
22 O
cr
2
21 O
us
1 HO
ip t
Figure(s)
H3C 31
Ac
ce pt
ed
M
Fig. 1. The chemical structure of cefodizime.
Page 25 of 33
Figure(s)
ip t
5000
cr
1
4000
F
3000 7
us
2000
0 300
8
330
360
an
1000
390
420
450
nm
Ac
ce pt
ed
M
Fig. 2. Fluorescence emission spectra of CEF-HSA system. cHSA = 2.0 106 molL1; cCEF(105 molL1)(1-7): 0, 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8; curve 8: cHSA = 0, cCEF = 3.0 106 molL1; T = 298K.
Page 26 of 33
1.6
ip t
Figure(s)
cr
298K 310K
1.5
us
F0 /F
1.4 1.3
an
1.2 1.1 1.0 0.6
0.9 1.2 1.5 -5 -1 Q( 10 mol·L )
M
0.3
1.8
Ac
ce pt
ed
Fig. 3. Stern-Volmer plots for the CEF-HSA system at different temperatures; data are mean values standard errors of three independent experiments; cHSA = 2.0 106 molL1.
Page 27 of 33
1.8
0.16 4
1
0.14
cr
1.5
0.12
1.2
1
4
0.10
0.9 0.08 260
0.6
270
0.0 250
nm
290
300
350
M
200
280
an
5
0.3
us
A
ip t
Figure(s)
Ac
ce pt
ed
Fig. 4. UV-vis absorption spectra of HSA in presence of different concentrations of CEF. cCEF (105 molL1)(1-4): 0, 2.0, 4.0, 6.0; cHSA = 2.0 106 molL1; curve 5: cHSA = 0, cCEF = 2.0 105 molL1; T = 298K.
Page 28 of 33
Figure(s)
0.04
ip t
5000 a
4000
0.03
3000
A
F
cr
0.02
2000
0.01
us
1000
b
an
0 0.00 320 340 360 380 400 420 440 460 480 /nm
Ac
ce pt
ed
M
Fig. 5. Overlapping between the fluorescence emission spectrum of HSA (a) and absorption spectrum of CEF (b). cCEF = cHSA = 2.0 106 molL1; T = 298K.
Page 29 of 33
ip t
Figure(s)
1.0
cr
0.9
0.7 lbuprofen phenylbutazone
0.6
an
0.5 0.4 1.0
1.5 2.0 2.5 [Probe]/[HSA]
3.0
M
0.5
us
F/F0
0.8
Ac
ce pt
ed
Fig. 6. Effects of site marker probes on the fluorescence of CEF-HSA. Data are mean values standard errors three independent experiments; cHSA = 2.0 106 molL1, cCEF = 1.0 105 molL1; T = 298K.
Page 30 of 33
Figure(s)
IB
Domain I
IIIB
ip t
IA
Domain III
cr
Sudlow’s site I CEF
IIIA
us
Sudlow’s site II
IIA Domain II
IIB
ed
M
an
(a)
(c)
ce pt
(b)
Ac
Fig. 7. (a) The binding site of CEF on HSA. HSA is shown in cartoon and CEF is represented using spheres. (b) Enlarged binding mode between CEF and HSA. HSA is shown in cartoon, the interacting side chains of HSA are displayed in surface mode and CEF is represented using balls and sticks. (c) Molecular modeling of the interaction between CEF and HSA. The atoms of CEF are blue.
Page 31 of 33
Figure(s)
450
peak 1
450
(A)
peak 2
peak 1
peak 2
4.5E2
1.8E3
1.3E3
8.9E2
300
a
250 210
240
270 ex/nm
300
2.4E3
350
1.2E3 8.0E2
210
5000 (C) HSA
m
200
200
peak 1(3981,281.0/350.0) 4000 3000
F
2000 1000
500 400 em
/n
300 m
200
250 200
300
350
ex/nm
ce pt
/n
300 250 ex/nm
350
330
a
300
ed
400
300
5000
2000 1000
500
270 ex/nm
a
(2212,230.0/350.0) peak 2
M
3000
a
4.0E2
1.6E3
(D) CEF-HSA
4000
(2318,230.0/347.0) peak 2
240
an
peak 1(4447,281.0/350.0)
2.8E3
2.0E3
8.0E2
300 250
330
1.2E3
F
3.1E3
2.2E3
cr
8.9E2
us
2.7E3
350
em/nm
em/nm
1.3E3
ip t
400
400
em
(B)
Ac
Fig. 8. Three-dimensional fluorescence contour maps (A and B) and threedimensional fluorescence projections (C and D) of HSA before and after adding CEF. cHSA = 2.0 106 molL1, cCEF = 3.0 106 molL1; T = 298K.
Page 32 of 33
ip t
Figure(s)
10 1 -20
-24 205
210
215
220
225
-10
220 230 /nm
240
250
M
210
an
-20 200
cr
-22
us
CD ( Mdeg)
4
0
Ac
ce pt
ed
Fig. 9. CD spectra of HSA in the absence and presence of CEF in phosphate buffer (pH 7.40). cCEF(105 molL1) (1-4): 0, 1.0, 2.0, 4.0; cHSA = 2.0 106 molL1.
Page 33 of 33
S0731-7085(15)00020-5 http://dx.doi.org/doi:10.1016/j.jpba.2015.01.010 PBA 9893
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
27-9-2014 31-12-2014 6-1-2015
Please cite this article as: http://dx.doi.org/10.1016/j.jpba.2015.01.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Graphical Abstract
IB
Domain I
IIIB
ip t
IA
Domain III
cr
Sudlow’s site I CEF
IIA
us
Sudlow’s site II
Domain I
Ac
ce pt
ed
M
an
IIB
Page 1 of 33
*Highlights (for review)
The interaction of CEF and HSA was studied by multi-spectroscopic and molecular docking techniques.
The binding of CEF with HSA was not only a static quenching
ip t
procedure but also driven mainly by hydrogen bonds and van der Waals force.
Binding constant, number of binding site and binding distance were
cr
us
calculated.
CEF changed the conformation and secondary structure of HSA.
CEF bound to hydrophobic pocket (site I, subdomain IIA) of HSA.
Ac
ce pt
ed
M
an
Page 2 of 33
*Revised Manuscript
Probing the interaction of cefodizime with human serum albumin
2
using multi-spectroscopic and molecular docking techniques
3
Taoying Hu1, Ying Liu 1,2
4
1. College of Life and Environmental Sciences, Minzu University of China, Beijing100081,
5 6 7
China 2. Beijing Engineering Research Center of Food Environment and Public Health, Minzu University of China, Beijing 100081, China
Corresponding author. Tel.:+86 15810128921; fax: +86 10 68450641. E-mail address:[email protected]
cr
8 9
ip t
1
Abstract: To know the interaction of cefodizime (CEF) with human
11
serum albumin (HSA), techniques of different spectroscopies and
12
molecular modeling were used. The inner filter effects were eliminated to
13
get accurate binding parameters. Steady state fluorescence suggested a
14
static type for CEF-HSA interaction, and the complex formation had a
15
high affinity of 105 Lmol1. On the basis of the thermodynamic results
16
and site marker competitive experiments, it was considered that CEF was
17
bound to site I (subdomain IIA) of HSA mainly by hydrogen bonds and
18
van der Waals force. The calculated binding distance (r) indicated that the
19
non-radioactive energy transfer came into being in the interaction
20
between CEF and HSA. Furthermore, molecular modeling was applied to
21
further define that CEF interacted with the Trp214, Lys199, Phe211,
22
Leu238 residues of HSA. In addition, three-dimensional fluorescence and
23
circular dichroism (CD) results showed that the binding of CEF can cause
24
conformational and some microenvironmental changes of HSA. This
25
paper provides reasonable models helping us further understand the
26
transportation and distribution of CEF when it spreads into human blood
27
serum
28
pharmacodynamics.
Ac
ce pt
ed
M
an
us
10
which
is
of
great
importance
in
pharmacology
and
Corresponding author. Tel.:+8615810128921; fax: +861068450641. E-mail address:[email protected] 1
Page 3 of 33
29
Keywords: Cefodizime (CEF); Human serum albumin (HSA); Multi-
30
spectroscopic techniques; Molecular modeling
31 32
1. Introduction The most abundant serum protein, i.e., albumin, serves as a transport
34
vehicle for several endogenous and exogenous compounds, such as fatty
35
acids, dyes, drugs and steroids [1-4]. The distribution, metabolism,
36
excretion and toxicity of ligands are related to their binding affinities to
37
proteins,
38
approximately 60% of the total protein, HSA is the most abundant protein
39
in blood plasma contributing 80% of the colloid osmotic pressure. HSA is
40
a single-chain, non-glycosylated polypeptide with well-known 585 amino
41
acids sequences and contains three homologous α-helical domains (I, II
42
and III) which assemble to form a heart-shaped molecule. Each domain
43
contains two subdomains (A and B), which are predominantly helical and
44
extensively cross-linked through several disulfide bridges. Its amino acid
45
sequence contains a total of 17 disulfide bridges, one free thiol (Cys-34)
46
and a single tryptophan (Trp-214) [1,2]. It is capable of binding
47
reversibly to a wide variety of drugs, due to which increased solubility in
48
plasma, decreased toxicity, and/or protection against oxidation of bound
49
ligands occur. The existence of two principle binding regions, viz.
50
Sudlow sites I and II, are responsible for the protein’s capability to bind
51
different ligands, which are located within hydrophobic cavities in
52
subdomains IIA and IIIA, respectively. Site I is known as warfarin-
53
azapropazone site, which contains a single tryptophan residue in position
54
214 (Trp-214). Site II is known as the indole-benzodiazepine site, which
55
presents two important amino acid residues (Arg-410 and Tyr-411) [3].
human
serum
albumin
(HSA).
By
constituting
Ac
ce pt
ed
M
an
i.e.,
us
cr
ip t
33
2
Page 4 of 33
The serum albumin-ligand binding studies have been reported in a
57
number of recent papers [4,5]. Many drugs (antibiotic, anesthetic,
58
antidepressant, etc), exert their activity by interaction with biological
59
membranes. We also have literature related to the thermodynamics,
60
binding characteristics, and conformation properties of several antibiotic
61
drugs (viz, nitrofurazone, tosufloxacin tosylate, methacycline and
62
demeclocycline) interaction with albumins [6,7]. Many drugs and other
63
kinds of small bioactive materials have the potential to bind reversibly to
64
serum
65
hydrophobicity and efficiency of the delivery process to the targeted
66
tissues. Binding studies have provided information of the structural
67
features determining the therapeutic effectiveness of drug and developed
68
as an interesting research field in life-sciences, chemistry, and clinical
69
medicine [8].
which,
in
turn,
cannot
ably
modulate
their
M
an
us
albumins
cr
ip t
56
Cefodizime (CEF) (Fig. 1) is an extended-spectrum third-generation
71
cephalosporin antibiotic that is widely used in the treatment of severe
72
infections of the respiratory and urinary tracts. Compared with the other
73
third generation cephalosporin, it has some peculiar merits such as no
74
renal toxic effect, good tolerance and even the activity of immune
75
regulation [9], which a lot of the same kind of antibiotics can’t match.
76
There have been some studies to study the interaction of cephalosporin
77
with protein. For example, Shao et al. [10] studied the binding
78
mechanism of cefodizime sodium to bovine serum albumin (BSA) by
79
fluorescence spectra, and Pan et al. [11] reported the interaction of
80
ceftriaxone sodium to BSA using spectroscopic and circular dichroism
81
(CD) methods. However, these studies are insufficient in terms of
82
elimination of the inner filter effects, site marker competitive experiments
83
and molecular docking, which are of great importance for perfectly
84
demonstrating the interaction of cephalosporin with protein. Thus, in this
Ac
ce pt
ed
70
3
Page 5 of 33
85
paper, our probing the interaction of cefodizime with HSA by using
86
multi-spectroscopic methods is based on the aforesaid what are left out in
87
the previous study. In the present work we seek to discuss about the biophysical
89
interactions of CEF with HSA that play an important role in drug
90
transport and storage. The study was established in which the multi-
91
spectroscopic and molecular modeling techniques were utilized to obtain
92
the interaction information of CEF with HSA, i.e., the quenching
93
mechanism of fluorescence, the binding parameters, the effect of CEF on
94
the conformational and secondary structural changes of HSA and the
95
specific binding site. The investigation of the binding interaction between
96
drugs and serum albumins is of much importance in pharmacology and
97
pharmacodynamics. Therefore, such interactions in vitro have been
98
considered as models in protein chemistry to study their binding behavior.
99
2. Experimental
cr
us
an
M
ed
2.1. Materials
ce pt
100
ip t
88
HSA (Sigma, Missouri, USA) stock solution was prepared with the
102
concentration of 1.0 104 molL1. CEF (National Institutes for Food
103
and Drug Control, Beijing, China) was dissolved and diluted to 1.0 103
104
molL1 with ultrapure water. The stock solutions of phenylbutazone and
105
ibuprofen were prepared by dissolving them in a small amount of ethanol,
106
then diluting to 1.0 103 molL1 with water. Phosphate buffer solution
107
(NaH2PO4 (75 mM)Na2HPO4 (75 mM) (19:81, v/v)(pH 7.40)) was used
108
for CD measurement, while Tris (0.2 M)HCl (0.1 M) buffer (25:40,
109
v/v)( pH 7.40) and 0.1 molL1 NaCl solution were used for other
110
measurements. All of the solutions were kept in the dark at 04C. All
Ac
101
4
Page 6 of 33
111
reagents were of analytical reagent grade and ultrapure water was used
112
throughout the experiment.
113
2.2. Measurements Steady state fluorescence measurements were carried out through an F-
115
4500 spectrophotometer (Hitachi, Tokyo, Japan) equipped with a 1.0 cm
116
quartz cell at an excitation wavelength of 280 nm and emission
117
wavelength of 200500 nm. The excitation and emission slits were set at
118
5 nm while the scanning rate was 1200 nm/min and Photomultiplier tube
119
(PMT) voltage 700 V. The three-dimensional fluorescence spectra were
120
performed at an excitation wavelength of 200400 nm and emission
121
wavelength of 200500 nm, and the increments were 5 nm.
cr
us
The
absorption
spectra
were
an
122
ip t
114
recorded
on
a
UV-2800
spectrophotometer (Hitachi, Tokyo, Japan) equipped with a 1.0 cm quartz
124
cell. The wavelength range was 190500 nm with slit width of 2.0 nm.
M
123
Competitive bindings between CEF and HSA in the presence of two
126
site markers (phenylbutazone and ibuprofen) were performed. The ratio
127
of concentrations of CEF and HSA was kept at 5:1, then site markers
128
were gradually added to CEF-HSA mixtures, the fluorescence spectra
129
were recorded upon excitation at 280 nm.
ce pt
ed
125
The CD measurements were obtained over a wavelength range of 190-
131
250 nm at 0.2 nm intervals on a JASCO J-810 CD spectrometer (JASCO,
132
Tokyo, Japan) using a 0.1 cm cell at room temperature. Each spectrum
133
was scanned three times and finally averaged for plots and analyses.
Ac
130
134
Docking calculations were performed with Docking Server on a HSA
135
protein model (PDB-2BXM) [12]. The MMFF94 force field was used for
136
energy minimization of the ligand molecule (CEF). Gasteiger partial
137
charges were added to the ligand atoms. Nonpolar hydrogen atoms were
138
merged, and rotatable bonds were defined. Essential hydrogen atoms, 5
Page 7 of 33
Kollman united atom type charges, and solvation parameters were added
140
with the aid of AutoDock tools [13]. Affinity (grid) maps of 20 20 20
141
Å grid points and 0.375 Å spacing were generated using the Autogrid
142
program [14]. The AutoDock parameter set- and distance-dependent
143
dielectric functions were used in the calculation of the van der Waals and
144
electrostatic terms, respectively.
ip t
139
Docking simulations were performed using the Lamarckian genetic
146
algorithm (LGA) and the Solis and Wets local search method [15]. Initial
147
positions, orientations, and torsions of the ligand molecules were set
148
randomly. Each run of the docking experiment was set to terminate after a
149
maximum of 250,000 energy evaluations. The population size was set to
150
150. During the search, a translational step of 0.2 Å and quaternion and
151
torsion steps of 5 were applied.
152
3. Results and discussions
153
3.1. Elimination of the inner filter effects
ed
M
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cr
145
The system of CEF-HSA has absorbance at excitation and emission
155
wavelengths of the fluorescence measurements, which will impact the
156
accuracy of fluorescence data. To eliminate the inner filter effects,
157
absorbance measurements were carried out at the fluorescence excitation
158
and emission wavelengths. The extent of this effect can be roughly
159
evaluated with the following relationship [16]:
160
Fcor Fobs e
161
where Fcor and Fobs are the fluorescence intensities corrected and observed,
162
respectively, and Aex and Aem are the absorption of HSA and CEF at
163
excitation and emission wavelengths, respectively. All the fluorescence
164
intensity used in this study was corrected.
165
3.2. Analysis of fluorescence quenching of HSA
Ac
ce pt
154
Aex Aem /2
(1)
6
Page 8 of 33
Fluorescence method is an important tool for investigating the
167
interaction between small probe molecules and proteins to get
168
information about the binding mechanism, binding constants, binding
169
mode, binding sites, and intermolecular distances. Fluorescence of HSA
170
originates from tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe)
171
residues, whereas the intrinsic fluorescence of HSA is mainly attributed
172
to the Trp-214 residue alone. As the Phe residue has very low quantum
173
yield and the fluorescence of Tyr residue is almost entirely quenched
174
when it is ionized or close to a carboxyl group, an amino group or a Trp
175
[17]. Fluorescence quenching is the decrease of the quantum yield of
176
fluorescence from a fluorophore, which is induced by a wide variety of
177
molecular interactions with a quencher molecule, viz., excited-state
178
reactions, energy transfer, molecular rearrangements, ground-state
179
complex formation and collisional quenching. If the small molecule can
180
quench the fluorescence of the Trp residues, the Trp residues must be
181
located in or near the binding position [18].
ed
M
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166
A valuable feature of intrinsic fluorescence of a protein is the high
183
sensitivity of Trp to its local environment. Changes in emission spectra of
184
Trp are common in response to the protein conformational transition,
185
subunit association, substrate binding, or denaturation [19]. To
186
comprehend how the structure of HSA is affected by CEF, Fig. 2 was
187
used to indicate the fluorescence emission spectra for HSA with various
188
molar ratios of CEF at the excitation wavelength of 280 nm. The HSA
189
had a strong fluorescence emission with a peak at 351 nm while CEF was
190
almost non-fluorescent. It can be seen that the fluorescence intensities of
191
HSA decreased clearly with increasing CEF concentrations indicating
192
that CEF interacted with HSA and the binding site must be located in or
193
near Trp residues.
Ac
ce pt
182
7
Page 9 of 33
The various mechanisms of quenching are usually classified as either
195
dynamic or static mechanism. Static quenching refers to the formation of
196
a ground-state complex between the fluorophore (protein) and the
197
quencher, while dynamic quenching refers to a process which involves
198
the fluorophore and the quencher coming into contact during the transient
199
existence of the excited state. In general, static and dynamic quenching
200
can be distinguished by their dependence on temperature. For dynamic
201
quenching, the quenching constant is expected to increase with increasing
202
temperature because higher temperature results in larger diffusion
203
coefficient. On the contrary, higher temperature is likely to lead to the
204
decreased stability of complex and thus smaller values of the static
205
quenching constant [20].
an
us
cr
ip t
194
In order to confirm the quenching mechanism, the fluorescence
207
quenching data were analysed according to the well-known Stern-Volmer
208
equation [21]:
ed
209
M
206
F0 1 kq 0 Q 1 K SV Q F
(2)
where F0 and F are the steady-state fluorescence intensities in the absence
211
and presence of quencher (CEF), respectively, KSV is the Stern-Volmer
212
quenching constant, which is determined by linear regression of Stern-
213
Volmer equation, [Q] is the initial concentration of quencher, kq is the
214
bimolecular quenching rate constant, and τ0 is the average lifetime of
215
fluorophore without quencher and its value is 108 s. As evident from Fig.
216
3 and Table 1, it can be found that there were good linearity relationship
217
between F0/F and [Q]CEF and the KSV values decreased with increasing
218
temperature, indicating that the likely quenching process was static
219
quenching mechanism rather than dynamic quenching mechanism. As the
220
kq values were far greater than the upper limit of 2.0 1010 Lmol1s1
Ac
ce pt
210
8
Page 10 of 33
221
[21], the fluorescence quenching of HSA in the presence of CEF was a
222
static quenching process. UV-vis absorption measurement is a simple but effective method to
224
distinguish static and dynamic quenching. Collisional quenching only
225
affects the excited states of the fluorophores, and thus no changes in the
226
absorption spectra are expected. In contrast, ground-state complex
227
formation will frequently result in perturbation of the absorption
228
spectrum of the fluorophores [22]. The UV-vis absorption spectra of HSA
229
in the presence of different concentrations of CEF were shown in Fig. 4.
230
HSA had two absorption peaks, the intensity of the strong absorption
231
peak at about 208 nm, which reflected the framework conformation of the
232
protein continuously dropped with the concentration of CEF accompanied
233
by a remarkable red shift (from 208 to 213 nm), and the intensity of the
234
weak absorption peak at about 276 nm reflecting the aromatic amino
235
acids (Trp, Tyr, Phe) increased slightly with CEF concentration [22]. The
236
results reconfirmed that the probable quenching mechanism of the
237
intrinsic fluorescence of HSA was not initiated by dynamic collision but
238
by CEF-HSA complex formation.
239
3.3. Analysis of binding equilibria
ce pt
ed
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223
For the equilibrium between free and bound molecules, when small
241
molecules bind independently to a set of equivalent sites on a
242
macromolecule, the relationship between the binding constant (Ka) and
243
the number of binding sites (n) could be represented by the equation [2]:
244
log
245
where F0, F and [Q] are the same as in Eq. (2). According to Eq. (3), the
246
slope and intercept value of the plot, log[(F0 F)/F] versus log[Q] gives
247
the Ka and n values (Table 2). The results showed that the association
Ac
240
F0 F log K F
a
n log Q
(3)
9
Page 11 of 33
constants (Ka) were decreased with the increasing temperature, which
249
may hint the formation of an unstable complex in the binding reaction.
250
The complex would be partly decomposed with the increasing
251
temperature, therefore, the values of Ka decreased. Furthermore, the
252
values of n were approximately equal to 1, manifesting the existence of
253
just a single binding site in HSA towards CEF.
254
3.4. Thermodynamic parameters and binding forces
cr
ip t
248
The interaction forces between macromolecules and drugs mainly
256
include four acting forces: hydrogen bonds, van der Waals force,
257
hydrophobic force, and electrostatic interactions. If the enthalpy change
258
(∆H) does not vary significantly over the temperature ranged studied,
259
then its value and that of Gibbs free energy change (∆G) and entropy
260
change (∆S) of the reaction, which could be used to verify the binding
261
mode, can be determined by the Van’t Hoff equation [7]: ln K a
H S RT R
ed
M
an
us
255
(4)
where R is the gas constant, T is the experimental temperature, and Ka is
263
the binding constant at the corresponding temperature. The values of ∆H
264
and ∆S were obtained from the slope and intercept of the linear Van’t
265
Hoff plot based on lnKa versus 1/RT, respectively. The free energy
266
change (∆G) was then evaluated from following relationship [7]:
267
G H T S
Ac
ce pt
262
(5)
268
The temperatures were chosen at 298K and 310K, at which HSA did
269
not undergo any structural degradation. The thermodynamic parameters
270
were obtained and presented in Table 2. According to Ross [23], the
271
negative sign for ∆G meant that association process was spontaneous.
272
The negative ∆H and ∆S values for the association reaction between CEF
10
Page 12 of 33
273
and HSA implied that both hydrogen bonds and van der Waals force were
274
the major driving forces of the interaction.
275
3.5. Energy transfer According to the Fӧrster theory of non-radiative energy transfer, the
277
rate of energy transfer depends upon the following factors: (1) the donor
278
can produce fluorescence light, (2) the fluorescence emission spectra of
279
the donor and UV-vis absorption spectra of the acceptor have enough
280
overlap, (3) the distance between the donor and the acceptor is lower than
281
8 nm. The efficiency of energy transfer between the donor and acceptor,
282
E, could be estimated by the following equation [24]:
283
R06 F E 1 6 6 F0 R0 r
284
where r is the binding distance between donor and acceptor, and R0 is the
285
critical distance between donor and acceptor when their transfer
286
efficiency is 50%:
(6)
ed
M
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us
cr
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276
(7)
ce pt
R06 8.79 1025 K 2 N 4 J
where K2 is the orientation factor between the emission dipole of the
288
donor and the absorption dipole of the acceptor, N is the refracted index
289
of the medium, φ is the fluorescence quantum yield of the donor, and J is
290
the overlap integral of the fluorescence emission spectra of the donor and
291
the absorption spectra of the acceptor, which could be calculated by the
292
following:
293
F J F
294
where F(λ) is the fluorescence intensity of the donor at wavelength λ, and
295
ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. In
296
the present study, K2 = 2/3, n = 1.336 and φ = 0.15 for HSA [16].
Ac
287
4
(8)
11
Page 13 of 33
The overlap of the fluorescence emission spectra of HSA and the UV-
298
vis absorption spectra of CEF when the molar ratio was 1:1 and at 298K
299
was represented in Fig. 5. From Eqs. (6)(8), we can calculate that R0 =
300
2.31 nm, E = 0.11, J = 5.57 1015 cm3Lmol1, and r = 3.30 nm. The
301
specific binding distance between Trp-214 (donor) and CEF (acceptor)
302
was smaller than 8 nm, which was the condition for energy transfer
303
phenomenon to occur. This indicated that energy transfer between CEF
304
and HSA can occur with high possibility.
305
3.6. Site marker competitive experiments
us
cr
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297
To identify the CEF binding sites on HSA, site marker competitive
307
experiments were carried out using site probes that specifically bind to a
308
known site or region on HSA. Sudlow et al. [25] have suggested two
309
main distinct binding sites on HSA called site I (subdomains IIA) and site
310
II (subdomains IIIA). Site I shows affinity for bulky heterocyclic anion
311
with a negative charge localized in the middle of the molecule, drugs
312
binding in this site include warfarin, phenylbutazone and diflunisal.
313
While ligands binding to site II are aromatic carboxylic acids with
314
negative charged acidic group at the end of the molecule, ligands tend to
315
this
316
indoxylsulfate. In this work, the competitors were used including
317
phenylbutazone, a characteristic marker for site I, and ibuprofen for site II
318
marker. The ratio of CEF to HSA was kept at 5:1 to keep nonspecific
319
binding probes to a minimum. By means of recording the changes in the
320
fluorescence intensity of CEF bound HSA, which was brought about by
321
site marker, information about the specific binding location of CEF in
322
HSA molecule can be obtained. The changes induced by the site markers
323
were presented in Fig. 6. The fluorescence of the complex was
324
remarkably affected in presence of phenylbutazone, conversely the
ce pt
ed
M
an
306
Ac
region involve ibuprofen, flufenamic acid, diazepam and
12
Page 14 of 33
addition of ibuprofen to the same solution. The observations
326
demonstrated that phenylbutazone displaced CEF from the binding site,
327
while ibuprofen had a little effect on the binding of CEF to HSA. Hence,
328
it can be concluded that the binding of CEF to HSA mainly located
329
within site I (subdomain IIA), so Trp-214 was near or within the binding
330
site, which also provided a reasonable explanation why the calculated
331
value of binding distance r was small.
332
3.7. Molecular docking
us
cr
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325
To further define the binding site, molecular docking was employed by
334
setting the simulation box to site I. The crystal structure of HSA was
335
taken from the Protein Data Bank (entry PDB code 2BXM). The best
336
energy ranked result was shown in Fig. 7. The inside wall of the pocket of
337
subdomain IIA was formed by hydrophobic side chains, whereas the
338
entrance to the pocket was surrounded by positively charged residues
339
consisting of Lys199, Leu238, Arg218, Ser202 and Ala215 [26]. Thus we
340
can conclude that CEF was able to fit well within the subdomain IIA
341
hydrophobic cavity (see Fig. 7b). The docking result showed the
342
existence of hydrogen bonds, hydrophobic, and polar interactions
343
between CEF and HSA. Furthermore, the distances were measured
344
between the protons of CEF and adjacent residues around the binding site.
345
Active site residues within 5 Å to CEF included Phe206, Arg484, Trp214,
346
Leu481, Leu198 and Val344. In particular, the calculated binding
347
distance r between Trp-214 and CEF was similar to the apparent distance
348
r (3.30 nm) between the donor and the acceptor evaluated from Fӧrster’s
349
non-radiative energy transfer analysis.
350
3.8. Three-dimensional fluorescence spectra
Ac
ce pt
ed
M
an
333
13
Page 15 of 33
In order to better understand the conformational changes of HSA upon
352
the addition of CEF, three-dimensional fluorescence spectrum was
353
performed on HSA and CEF-HSA system. Fig. 8 described the three-
354
dimensional fluorescence contour maps (A and B) and three-dimensional
355
fluorescence projections (C and D) of HSA and CEF-HSA. The contour
356
map exhibited a bird’s eye view of the fluorescence spectra and can also
357
provide lots of crucial information. As shown in Fig. 8, peak a is the
358
Rayleigh scattering peak. Peak 1 mainly enunciates the spectral behavior
359
max of Trp and Tyr residues. The maximum emission wavelength (λ em ) and
360
the fluorescence intensity of this peak are closely related to the polarity of
361
the microenvironment [27]. With the addition of CEF, the fluorescence
362
intensity of peak 1 decreased markedly, which indicated that the addition
363
of CEF changed the polarity of the Trp residues microenvironment. The
364
weak fluorescence peak 2 chiefly exhibits the conformation of the peptide
365
backbone associated with the helix-coil [28]. The fluorescence intensity
366
max of peak 2 had an obvious decrease with a red shift of λ em (from 347.0 nm
367
to 350.0 nm). It could be derived that the interaction of CEF with HSA
368
led to a conformational change of the protein.
369
3.9. CD spectroscopy
ce pt
ed
M
an
us
cr
ip t
351
CD spectra can further confirm the structural changes of HSA upon
371
addition of CEF and the CD spectrum of HSA has two negative bands at
372
around 209 nm and 222 nm, which are contributed to the transition of
373
nπ* of -helix structure [29]. The CD results were expressed in terms
374
of mean residue ellipticity (MRE) in deg cm2dmol1 according to [30]:
375
MRE
376
where Cp is the molar concentration of HSA, n is the number of amino
377
acid residues (n=585, for HSA), and l is the path length. The -helical
Ac
370
observedCD m deg C p nl 10
(9)
14
Page 16 of 33
378
contents in the secondary structure of HSA were calculated from MRE
379
values at 209 nm using the equation [30]:
380
helix(%)
381
where MRE209 is the observed MRE value at 209 nm, 4000 is the MRE of
382
the β-form and random coil conformation cross at 209 nm, and 33000 is
383
the MRE value of a pure -helix at 209 nm.
MRE209 4000 100 33000 4000
ip t
(10)
As was evident in Fig. 9, when CEF was added to a solution of HSA,
385
the intensity of negative bands at 209 nm and 222 nm increased without
386
any shift of peaks, illustrating that the secondary structure of HSA after
387
binding to CEF was predominantly -helical. The calculated results
388
(Table 3) showed that the content of -helix for HSA increased from
389
44.5% to 47.6% when nCEF/nHSA increased from 0:1 to 20:1, which
390
indicated that binding of CEF to HSA may induce some conformational
391
changes of protein. It can be deduced that the -helix structure was
392
affected probably due to insertion of CEF into hydrophobic cavity of
393
HSA.
394
4. Conclusions
ce pt
ed
M
an
us
cr
384
In this paper, the probable quenching mechanism of fluorescence of
396
HSA initiated by CEF was a static quenching process, the negative values
397
of enthalpy change (H = 6.20 kJmol1) and entropy change (S =
398
75.83 Jmol1K1) indicated that the main driving forces of the
399
interaction between CEF and HSA were hydrogen bonds and van der
400
Waals force. The distance (r) of 3.30 nm between the acceptor (CEF) and
401
donor (Trp-214) revealed that the energy transfer from HSA to CEF
402
occurred. The site marker competitive experiments showed that the
403
specific binding of CEF was located in the vicinity of site I (subdomain
404
IIA) of HSA. The red shift (347.0 nm350.0 nm) of peak 2 of three-
Ac
395
15
Page 17 of 33
dimensional fluorescence and the slightly increase of -helix from 44.5%
406
to 47.6% of CD spectra showed that the conformation and micro-
407
environment of HSA had changed because of the binding of CEF.
408
Furthermore, the binding details between CEF and HSA were further
409
confirmed by molecular modeling, which manifested that CEF was bound
410
at subdomain IIA through multiple interactions, such as hydrophobic
411
interaction, polar force, and hydrogen bonds, etc. This study is expected
412
to provide salient biophysical and biochemical clues on elucidating the
413
transport and storage of CEF.
414
Acknowledgements
an
us
cr
ip t
405
Here the authors thank the National Science Foundation of China
416
(21177163), 111Project B08044, Beijing Engineering Research Center of
417
Food Environment and Public Health, Minzu University of China (10301-
418
01404026), First-class University First Class Academic Program of
419
Minzu University of China (YLDX01013), Graduate Student Scientific
420
Research Innovation Project of Minzu University of China (K2014042).
423 424 425 426 427
ed
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422
Ac
421
M
415
428 429 430 431 432 16
Page 18 of 33
433
References
434
[1] J. Ghuman, P.A. Zunszain, I. Petitpas, A.A. Bhattacharya, M. Otagiri, S. Curry,
435
Structural basis of the drug-binding specificity of human serum albumin, J. Mol.
436
Biol. 353 (2005) 3852. [2] M. Memarpoor-Yazdi, H. Mahaki, Probing the interaction of human serum
438
albumin with vitamin B2 (riboflavin) and L-Arginine (L-Arg) using multi-
439
spectroscopic, molecular modeling and zeta potential techniques, J. Lumin. 136
440
(2013) 150159.
cr
ip t
437
[3] F. Ding, W. Liu, X. Zhang, L. Zhang, Y. Sun, Fluorescence and circular dichroism
442
studies of conjugates between metsulfuron-methyl and human serum albumin,
443
Colloid. Surface. B 76 (2010) 441448.
an
us
441
[4] I. Matei, S. Ionescu, M. Hillebrand, Interaction of fisetin with human serum
445
albumin by fluorescence, circular dichroism spectroscopy and DFT calculations:
446
binding parameters and conformational changes, J. Lumin. 131 (2011) 16291635.
447
[5] M. Yan, X. Chen, S.T. Sun, H.M. Ma, B. Du, Q. Wei, Study on binding
448
mechanism of meso-tetra-(3,5-dibromo-4-hydroxyphenyl) porphyrin with protein
449
by fluorescence method, Spectroscopy and Spectral Analysis 28 (2008)
450
13221326.
ce pt
ed
M
444
[6] F.Y. Deng, C.Y. Dong, Y. Liu, Characterization of the interaction between
452
nitrofurazone and human serum albumin by spectroscopic and molecular
453
modeling methods, Mol. Biosyst. 8 (2012) 14461451.
Ac
451
454
[7] C.Y. Dong, S.Y. Ma, Y. Liu, Studies of the interaction between demeclocycline
455
and human serum albumin by multi-spectroscopic and molecular docking methods,
456 457
Spectrochim. Acta A 103 (2013) 179186. [8] S.A. Markarian, M.G. Aznauryan, Study on the interaction between isoniazid and
458
bovine
459
dimethylsulfoxide, Mol. Biol. Rep. 39 (2012) 75597567.
serumalbumin
by
fluorescence
spectroscopy:
the
effect
of
17
Page 19 of 33
460
[9] S.H. Auda, Y. Mrestani, D.H. Nies, C. Groβe, R.H.H. Neubert, Preparation,
461
physicochemical characterization and biological evaluation of cefodizime metal
462
ion complexes, J. Pharm. Pharmacol. 61 (2009) 753758.
463 [10] S. Shao, B.Y. Ma, X.J. Wang, J.J. Zhang, X.F. Li, Q. Qin, Study on the interaction
between cefodizime sodium and bovine serum albumin, Acta Phys.-Chim. Sin. 21
465
(2005) 792795.
ip t
464
466 [11] J.W. Pan, Z.T. Ye, X.P. Cai, L.X. Wang, Z. Cao, Biophysical study on the
interaction of ceftriaxone sodium with bovine serum albumin using spectroscopic
468
methods, J. Biochem. Mol. Toxic. 26 (2012) 487492.
us
cr
467
469 [12] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
471
receptor flexibility, J. Comput. Chem. 30 (2009) 27852791.
an
470
473
M
472 [13] T.A. Halgren, Merck molecular force field. I. basis, form, scope, parameterization,
and performance of MMFF94, J. Comput. Chem. 17 (1996) 490519.
ed
474 [14] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. 475
Olson, Automated docking using a Lamarckian genetic algorithm and an empirical
476
binding free energy function, J. Comput. Chem. 19 (1998) 16391662.
478
ce pt
477 [15] F.J. Solis, R.J.B. Wets, Minimization by random search techniques, Math. Oper.
Res. 6 (1981) 1930.
479 [16] F. Ding, L. Zhang, J.X. Diao, X.N. Li, L. Ma, Y. Sun, Human serum albumin
481
stability and toxicity of anthraquinone dye alizarin complexone: an albumin–dye
Ac
480
model, Ecotox. Environ. Safe. 79 (2012) 238246.
482 [17] J.R. Lakowicz, Principles of fluorescence spectroscopy, third ed., Kluwer 483
Academic Publishers/Plenum Press, New York, 2006.
484 [18] G.W. Zhang, N. Zhao, X. Hu, J. Tian, Interaction of alpinetin with bovine serum 485
albumin: probing of the mechanism and binding site by spectroscopic methods,
486
Spectrochim. Acta A 76 (2010) 410417.
487 [19] F. Ding, W. Liu, L. Zhang, B. Yin, Y. Sun, Sulfometuron-methyl binding to 488
human serum albumin: evidence that sulfometuron-methyl binds at the Sudlow’s 18
Page 20 of 33
489
site I, J. Mol. Struct. 968 (2010) 5966.
490 [20] J. Zhang, Q.S Yan, J.P. Liu, X.H. Lu, Y.S. Zhu, J. Wang, S.J. Wang, Study of the 491
interaction between 5-sulfosalicylic acid and bovine serum albumin by
492
fluorescence spectroscopy, J. Lumin. 134 (2013) 747753.
493 [21] J.G. Xu, Z.B. Wang, Fluorescence analysis, third ed., Science Press, Beijing, 2006.
495
ip t
494 [22] M.N. Zhao, H.M. Zhou, Biophysics, first ed., Higher Education Press, Beijing,
2000.
forces contributing to stability, Biochemistry 20 (1981) 30963102.
us
497
cr
496 [23] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions:
498 [24] P.N. Naik, S.A. Chimatadar, S.T. Nandibewoor, Study on the interaction between
antibacterial drug and bovine serum albumin: a spectroscopic approach,
500
Spectrochim. Acta A 73 (2009) 841845.
an
499
502
M
501 [25] G. Sudlow, D.J. Birkett, D.N. Wade, The characterization of two specific drug
binding sites on human serum albumin, Mol. Pharmacol. 11 (1975) 824832.
ed
503 [26] A.J. Ryan, C.W. Chung, S. Curry, Crystallographic analysis reveals the structural 504
basis of the high-affinity binding of iophenoxic acid to human serum albumin,
505
BMC Struct. Biol. 11 (2011) 18.
507
ce pt
506 [27] Z.J. Cheng, Interaction of tetramethylpyrazine with two serum albumins by a
hybrid spectroscopic method, Spectrochim. Acta A 93 (2012) 321330.
508 [28] H.W. Sun, Y.J. Wu, X.H. Xia, Z.H. Shi, Spectroscopic studies on the interaction
510
characteristics between norethisterone and bovine serum albumin, J. Lumin. 134
Ac
509
(2013) 580587.
511 [29] P. Yang, F. Gao, The principle of bioinorganic chemistry, Science Press, Beijing, 512
2003.
513 [30] I. Matei, M. Hillebrand, Interaction of kaempferol with human serum albumin: a 514
fluorescence and circular dichroism study, J. Pharm. Biomed. Anal. 51 (2010)
515
768773.
19
Page 21 of 33
ip t
Table(s)
cr
Table 1 Stern-Volmer quenching constants for the interaction of CEF with HSA at two different temperatures. KSV(Lmol1)
kq(Lmol1s1)
298
3.35 104
3.35 1012
0.9994
310
2.99 104
2.99 1012
0.9996
R
Ac
ce pt
ed
M
an
us
T(K)
Page 22 of 33
cr
ip t
Table(s)
Table 2 Binding constants and relative thermodynamic parameters of CEF-HSA. H (kJmol1)
1.11 1.05
6.20
S (Jmol1K1)
us
n
75.83
an
Ka (10 Lmol1) 1.12 0.50 5
G (kJmol1) 28.80 27.89
Ac
ce pt
ed
M
T (K) 298 310
Page 23 of 33
ip t
Table(s)
5:1 10:1 20:1
44.8 45.2 47.6
14.7 14.5 12.4
β-turn (%) 12.2
Random (%) 28.3
12.2 11.7 11.8
28.2 28.6 28.1
us
β-sheet (%) 15.0
Ac
ce pt
ed
M
an
0:1
α-helix (%) 44.5
nCEF/nHSA
cr
Table 3 Conformational changes of HSA in the absence and presence of CEF.
Page 24 of 33
20 HO 4
5
O 3
19
6 S 7
9
N 8
12 13
18
17N
23
35 NH2
27 O
33 N
24 14 S 15
16 N H 25
26 28
34
32
S 36
37
N 29 O 30
an
H3C 10
S 11
22 O
cr
2
21 O
us
1 HO
ip t
Figure(s)
H3C 31
Ac
ce pt
ed
M
Fig. 1. The chemical structure of cefodizime.
Page 25 of 33
Figure(s)
ip t
5000
cr
1
4000
F
3000 7
us
2000
0 300
8
330
360
an
1000
390
420
450
nm
Ac
ce pt
ed
M
Fig. 2. Fluorescence emission spectra of CEF-HSA system. cHSA = 2.0 106 molL1; cCEF(105 molL1)(1-7): 0, 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8; curve 8: cHSA = 0, cCEF = 3.0 106 molL1; T = 298K.
Page 26 of 33
1.6
ip t
Figure(s)
cr
298K 310K
1.5
us
F0 /F
1.4 1.3
an
1.2 1.1 1.0 0.6
0.9 1.2 1.5 -5 -1 Q( 10 mol·L )
M
0.3
1.8
Ac
ce pt
ed
Fig. 3. Stern-Volmer plots for the CEF-HSA system at different temperatures; data are mean values standard errors of three independent experiments; cHSA = 2.0 106 molL1.
Page 27 of 33
1.8
0.16 4
1
0.14
cr
1.5
0.12
1.2
1
4
0.10
0.9 0.08 260
0.6
270
0.0 250
nm
290
300
350
M
200
280
an
5
0.3
us
A
ip t
Figure(s)
Ac
ce pt
ed
Fig. 4. UV-vis absorption spectra of HSA in presence of different concentrations of CEF. cCEF (105 molL1)(1-4): 0, 2.0, 4.0, 6.0; cHSA = 2.0 106 molL1; curve 5: cHSA = 0, cCEF = 2.0 105 molL1; T = 298K.
Page 28 of 33
Figure(s)
0.04
ip t
5000 a
4000
0.03
3000
A
F
cr
0.02
2000
0.01
us
1000
b
an
0 0.00 320 340 360 380 400 420 440 460 480 /nm
Ac
ce pt
ed
M
Fig. 5. Overlapping between the fluorescence emission spectrum of HSA (a) and absorption spectrum of CEF (b). cCEF = cHSA = 2.0 106 molL1; T = 298K.
Page 29 of 33
ip t
Figure(s)
1.0
cr
0.9
0.7 lbuprofen phenylbutazone
0.6
an
0.5 0.4 1.0
1.5 2.0 2.5 [Probe]/[HSA]
3.0
M
0.5
us
F/F0
0.8
Ac
ce pt
ed
Fig. 6. Effects of site marker probes on the fluorescence of CEF-HSA. Data are mean values standard errors three independent experiments; cHSA = 2.0 106 molL1, cCEF = 1.0 105 molL1; T = 298K.
Page 30 of 33
Figure(s)
IB
Domain I
IIIB
ip t
IA
Domain III
cr
Sudlow’s site I CEF
IIIA
us
Sudlow’s site II
IIA Domain II
IIB
ed
M
an
(a)
(c)
ce pt
(b)
Ac
Fig. 7. (a) The binding site of CEF on HSA. HSA is shown in cartoon and CEF is represented using spheres. (b) Enlarged binding mode between CEF and HSA. HSA is shown in cartoon, the interacting side chains of HSA are displayed in surface mode and CEF is represented using balls and sticks. (c) Molecular modeling of the interaction between CEF and HSA. The atoms of CEF are blue.
Page 31 of 33
Figure(s)
450
peak 1
450
(A)
peak 2
peak 1
peak 2
4.5E2
1.8E3
1.3E3
8.9E2
300
a
250 210
240
270 ex/nm
300
2.4E3
350
1.2E3 8.0E2
210
5000 (C) HSA
m
200
200
peak 1(3981,281.0/350.0) 4000 3000
F
2000 1000
500 400 em
/n
300 m
200
250 200
300
350
ex/nm
ce pt
/n
300 250 ex/nm
350
330
a
300
ed
400
300
5000
2000 1000
500
270 ex/nm
a
(2212,230.0/350.0) peak 2
M
3000
a
4.0E2
1.6E3
(D) CEF-HSA
4000
(2318,230.0/347.0) peak 2
240
an
peak 1(4447,281.0/350.0)
2.8E3
2.0E3
8.0E2
300 250
330
1.2E3
F
3.1E3
2.2E3
cr
8.9E2
us
2.7E3
350
em/nm
em/nm
1.3E3
ip t
400
400
em
(B)
Ac
Fig. 8. Three-dimensional fluorescence contour maps (A and B) and threedimensional fluorescence projections (C and D) of HSA before and after adding CEF. cHSA = 2.0 106 molL1, cCEF = 3.0 106 molL1; T = 298K.
Page 32 of 33
ip t
Figure(s)
10 1 -20
-24 205
210
215
220
225
-10
220 230 /nm
240
250
M
210
an
-20 200
cr
-22
us
CD ( Mdeg)
4
0
Ac
ce pt
ed
Fig. 9. CD spectra of HSA in the absence and presence of CEF in phosphate buffer (pH 7.40). cCEF(105 molL1) (1-4): 0, 1.0, 2.0, 4.0; cHSA = 2.0 106 molL1.
Page 33 of 33
