Protein-binding properties of a designed steroidal lactam compound

Article abstract of DOI:10.1016/j.steroids.2013.11.021

Introducing amide bonds into a steroid nucleus or its side chain may reduce the acute toxicity and enhance the pharmaceutical activity. In this work, a designed steroidal amide compound, named 3β-hydroxy-17-aza-d-homo-5- androsten-17-one (HAAO), was synthesized and identified. The interactions between HAAO and human serum albumin (HSA) were studied by multiple spectroscopic methods and molecular modeling procedures. It was found that HAAO locates in Sudlow's site I in subdomain IIA of HSA molecules, relying on hydrogen bonds and van der Waals power to form HAAO-HSA complexes at ground state. The number of binding sites, binding constants, enthalpy change (ΔH^θ), Gibbs free energy change (ΔG ^θ) and entropy change (ΔS^θ) were calculated at different temperatures based on fluorescence quenching theory and classical thermodynamic equation. The percentages content of the HSA's secondary structures in presence of HAAO were detected by circular dichroism (CD) spectra and compared with those in no presence of HAAO. In addition, the experimental results of both binding site and conformational change were further confirmed by molecular modeling investigation, in which more details of the binding were visually unfolded. The information provided by the study may be useful for designing novel chemotherapeutic drugs and be helpful both in the early stages of drug discovery and in clinical practice.

Full text of DOI:10.1016/j.steroids.2013.11.021

Steroids 80 (2014) 30–36
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Protein-binding properties of a designed steroidal lactam compound
⇑
Hua-xin Zhang , Y. Liu
College of Chemical and Pharmaceutical Engineering, Jingchu University of Technology, Jingmen, Hubei 448000, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 August 2013
Received in revised form 19 October 2013
Accepted 22 November 2013
Available online 6 December 2013
Introducing amide bonds into a steroid nucleus or its side chain may reduce the acute toxicity and
enhance the pharmaceutical activity. In this work, a designed steroidal amide compound, named 3b-
hydroxy-17-aza-D-homo-5-androsten-17-one (HAAO), was synthesized and identified. The interactions
between HAAO and human serum albumin (HSA) were studied by multiple spectroscopic methods and
molecular modeling procedures. It was found that HAAO locates in Sudlow’s site I in subdomain IIA of
HSA molecules, relying on hydrogen bonds and van der Waals power to form HAAO–HSA complexes at
Keywords:
ground state. The number of binding sites, binding constants, enthalpy change (
D
Hh), Gibbs free energy
Steroidal derivates
Human serum albumin
Fluorescence probe
Binding site
change (
D
Gh) and entropy change ( Sh) were calculated at different temperatures based on fluorescence
D
quenching theory and classical thermodynamic equation. The percentages content of the HSA’s secondary
structures in presence of HAAO were detected by circular dichroism (CD) spectra and compared with
those in no presence of HAAO. In addition, the experimental results of both binding site and conforma-
tional change were further confirmed by molecular modeling investigation, in which more details of the
binding were visually unfolded. The information provided by the study may be useful for designing novel
chemotherapeutic drugs and be helpful both in the early stages of drug discovery and in clinical practice.
Ó 2013 Elsevier Ltd. All rights reserved.
Molecular modeling
1. Introduction
indicated that many synthesized steroidal amide compounds
exhibited excellent bioactivity in anti-tumor, antibacterial, inhibi-
Steroids are a class of organic compounds with a chemical
structure that contains the core of gonane or a skeleton derived
there from, which are playing important roles in life activities of
animals and plants for their good performance in cell penetration
[1–3]. For example, sex steroids including androgens, estrogens
and progestogens produce sex differences and support reproduc-
tion, glucocorticoids regulate metabolism and immune function,
mineralocorticoids help maintain blood volume and control renal
excretion of electrolytes, and anabolic steroids interact with
androgen receptors to increase muscle and bone synthesis [4–6].
However, the applications of natural steroids were greatly
restricted because of their limited amount and low content in spite
of the excellent bioactivity. So introducing specified functional
groups into the core or the side chains of natural steroids to obtain
new compounds with higher activity or some special function has
become a focus of research on the steroidal chemistry in recent
years [7]. The introduced functional groups include heteroatom,
hydroxyl, sulfhydryl, ester, oximido, hydrazone, acylamino, etc.
[8,9]. In particular, in the decade of the 2000s, considerable interest
was generated in steroidal derivates with acylamino (ANHACOA)
in their molecular structures among chemists, pharmacologists,
biologists and life scientists [10]. On one hand, plenty of literatures
tion of 5a-reductase, and so on [11,12]. On the other hand, only a
handful of steroidal amides, such as estramustine phosphate so-
dium, dutasteride and finasteride, have already been successfully
used in clinic. One of the most important reasons for this phenom-
enon is that the molecular mechanism of pharmacological action of
steroidal amides is unclear.
Drug–plasma-protein binding is critically involved in drug
pharmacokinetics (i.e., absorption, distribution, metabolism, and
elimination) and pharmacodynamics (pharmacological effects)
[13]. It is therefore highly important to estimate drug-binding abil-
ity to macromolecules in the early stages of drug discovery [14].
Human serum albumin (HSA) is the most abundant protein in
human blood plasma with ascribed drug-binding and transport
properties [15], which can bind a wide range of endogenous and
exogenous compounds and serves as an important carrier for an
extraordinarily broad range of drugs [16–18]. In present work,
HSA was selected as a model to detect the drug–protein binding
at the molecular level.
The aim of this paper was (1) to synthesize 3b-hydroxy-17-aza-
D-homo-5-androsten-17-one (HAAO), a steroidal amide compound
which has obvious effects on human cervical carcinoma and
human liver carcinoma; (2) to investigate the interaction between
HAAO and HSA under simulated physiological conditions (ionic
strength = 0.1 mol Lꢀ1; pH 7.40 0.01). In order to attain these
objectives, multiple spectroscopy methods, molecular probe and
molecular modeling techniques were employed.
⇑
Corresponding author. Tel.: +86 18986973258.
E-mail address: h.x.zhang@yeah.net (H.-x. Zhang).
0039-128X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.11.021

H.-x. Zhang, Y. Liu / Steroids 80 (2014) 30–36
31
II). All the ligands and water molecules were removed before the
analysis. H atoms were added and the biopolymer was charged
using AMBER7 FF99 method. The site of the protein was defined
with ligand (A/WRR2001 for site I, A/IBP2001 for site II); the prin-
cipal regions of ligand binding to HSA were analyzed by Sybyl 8.1
software. The structure of HAAO was generated by sybyl8.1 pack-
age and the molecule was optimized (energy minimization) using
Tripos Force Field after being charged with Gasteiger and Marsili
method. The docking mode of HAAO with HSA was conducted by
a Surflex-Dock program in Sybyl 8.1 package. The original ligand
was redocked into HSA by the same procedures and the results
were close to the crystal structure, which verify the docking
program works well.
2. Experimental
2.1. Materials
HSA was purchased from Sigma (USA, 99%). Warfarin and
ibuprofen were obtained from Hubei Biocause Pharmaceutical
Co., Ltd. (Hubei, China) with the purity no less than 99.7%.
3b-hydroxy-5-androsten-17-one, Ac2O, pyridine, NH2OH, SOCl2,
THF, Tris, HCl, NaCl were purchased from Shanghai Chemical
Reagent Company (China). 3b-hydroxy-17-aza-D-homo-5-andros-
ten-17-one (HAAO) had a purity of no less than 99.5%. All other
chemicals were of analytical grade. Stock solutions of HSA
(10ꢀ5 mol Lꢀ1), HAAO (2 ꢁ 10ꢀ3 mol Lꢀ1), NaCl (0.5 mol Lꢀ1) and
Tris–HCl buffer (0.05 mol Lꢀ1 Tris, 0.15 mol Lꢀ1 HCl) of pH
7.40 0.01 were prepared by directly dissolving the original
reagents. Water used to prepare solutions was double-distilled.
2.4. Synthesis of HAAO
Using 3b-hydroxy-5-androsten-17-one as a starting material,
according to the literature by Huang et al. [12], HAAO was synthe-
sized in four steps and the procedure for its preparation was shown
in Fig. 1. Yield 76%, mp 168–171 °C; 1H NMR (CDCl3, 600 MHz)
d: 1.02 (3H, s, 19-CH3), 1.18 (3H, s, 18-CH3), 2.34–2.31 (1H, m,
2.2. Instrumental methods
All fluorescence spectra were recorded at four different temper-
atures on LS-55 spectrofluorometer (Perkin-Elmer, America)
equipped with 1.0 cm quartz cells and a thermostatic bath. To
obtain smooth emission spectra with moderate intensity between
260 and 400 nm, the widths of the excitation slit and the emission
slit were set to 15 and 4.5 nm with the scanning speed at 1000 nm/
min. An excitation wavelength of 290 nm was chosen and appro-
priate blanks corresponding to the buffer were subtracted to cor-
rect the background. CD measurements were performed at 310 K
C
16–H), 2.40–2.34 (1H, m, C8–H), 2.47 (1H, ddd, J = 18.6, 7.2, 1.2,
16–H), 3.57–3.50 (1H, m, C3–H), 5.38 (1H, t, J = 3.0, C6–H), 5.67
C
(1H, s, –NH). ESI-MS m/z: 306 (M + 1)+.
3. Results and discussion
3.1. Fluorescence spectra
on
a J-810 Spectropolarimeter (Jasco, Japan) equipped with
1.0 cm quartz cells over a wavelength range of 250–200 nm and
under constant nitrogen flush at a scanning speed of 200 nm/
min. IR spectra were obtained by a Nicolet380 FI-IR infrared spec-
trometer (Thermo Electron, America). Nuclear magnetic resonance
spectra were measured on a FX-90Q (JEOL, Japan) instrument. LC–
MS were carried on a TSQ Quantum Access MAX (Thermo Fisher,
America) instrument. The weight measurements were performed
with an AY-120 electronic analytic weighing scale (Shimadzu,
Japan). All pH measurements were made with a pHS-3 digital
pH-meter (Shanghai, China).
Fluorescence is the process of photon emission as a result of the
return of an electron in a higher energy orbital back to a lower
orbit, which in the life sciences is used generally as a non-destruc-
tive way of tracking or analysis of biological molecules that can
either give intrinsic fluorescence themselves or be ‘‘labeled’’ with
some extrinsic fluorophore [19,20]. A variety of molecular interac-
tions can result in quenching, including excited-state reactions,
molecular rearrangements, energy transfer, ground-state complex
formation, and collisional quenching. The intrinsic fluorescence
of HSA molecule was generated by aromatic amino acid residues
in its structure, especially the tryptophan residues [21].
2.3. Molecular modeling
According to experimental procedures, the fluorescence spectra
of HSA as well as HSA–HAAO systems were recorded (Fig. 2). As
shown in Fig. 2, the fluorescence of HSA around 350 nm regularly
decreased with the increasing amount of HAAO, indicating that
HAAO interacted with HSA and acted as a fluorescence quencher.
Molecular modeling calculations were carried out using Syb-
yl8.1. The crystal structure of HSA was from Brookhaven Proteins
Data Bank (PDB) database (entry codes: 1h9z for site I, 2bxg for site
O
O
NH2OH.HCl / NaAc.3H2O
Ac2O / pyridine
HO
95% CH3CH2OH, stirred, 333K, 2 h
reflux, 24 h
AcO
HON
O
O
H
N
H
N
SOCl2 / THF
13% K2CO3 /CH3OH
stirred, reflux, 4 h
stirred, reflux, 2 h
HAAO
AcO
AcO
Fig. 1. Procedure for the preparation of HAAO.
HO

32
H.-x. Zhang, Y. Liu / Steroids 80 (2014) 30–36
Table 1
Stern–Volmer constants.
CHSA =2*10-6mol L-1
C /10-6mol L-1
T=293K pH=7.40
·
·
Q
200
150
100
50
a
a 0
pH
7.4
T (K)
KSV (105L molꢀ1
)
SDa
b 0.5
c 1.0
d 1.5
e 2.0
293
299
305
311
1.445
1.298
1.104
1.016
0.0079
0.0046
0.0055
0.0039
h
f
2.5
a
g 3.0
h 3.5
SD is the standard deviation for KSV
.
H
N
O
was not initiated by dynamic collision and the most probable mech-
anism should be static quenching, with HAAO–HSA complex forma-
tion at ground-state.
HO
320
340
360
380
400
420
440
3.3. Binding equilibrium and thermodynamics
Wavelength (nm)
In the static quenching interaction, when small molecules bind
independently to a set of equivalent sites on a macromolecule, the
binding constant (Kb) and the number of binding sites (n) can be
determined by the following equation [25]:
Fig. 2. The fluorescence spectra of HSA in the absence and in the presence of HAAO.
3.2. Quenching mechanism analysis
ꢀ
ꢁ
F0 ꢀ F
log
¼ log Kb þ n log½Qꢂ
ð2Þ
The quenching mechanisms are usually classified as either
dynamic quenching or static quenching. Dynamic and static
quenching can be distinguished by their different dependence on
temperature and viscosity [22]. In dynamic quenching, interaction
between quencher and phosphor takes place during the lifetime of
the excited state. Dynamic quenching is caused by collision and
higher temperature will result in larger diffusion coefficients, the
dynamic quenching constants are expected to increase with
increasing temperature [19,23].
F
where F0, F, and [Q] are the same as in Eq. (1), Kb is the binding con-
stant, and n is the number of binding sites per HSA molecule. The
value of log (F0/Fꢀ1) is linear with the value of log [Q], in which
the slope is the number of binding sites (n) whereas the intercept
(ordinate at the origin) is the logarithm value of Kb (logKb). The
regression equations (Fig. 4) shows the slopes are closed to 1, which
indicates there is only one binding site with high affinity for HAAO
in HSA molecule, under specified conditions.
The enthalpy change and entropy change of HAAO–HSA interac-
tion are important for confirming binding mechanism. Therefore,
the dependence of binding constant on temperature was studied
using the Eqs. (3) and (4):
Supposing the quenching was dynamic quenching, the fluores-
cence data at different temperatures could be analyzed with the
well-known Stern–Volmer equation [24]:
F0
F
¼ 1 þ KSV½Qꢂ
ð1Þ
ꢀ
D
RT
Hh
D
Sh
R
ln K ¼
þ
ð3Þ
where F0 and F are the fluorescence intensities in the absence and in
the presence of quencher, respectively, KSV is the Stern–Volmer
quenching constant, [Q] is the concentration of the quencher.
Hence, Eq. (1) was applied to determine KSV by linear regression
of a plot of F0/F against [Q]. The Stern–Volmer plots at four different
temperatures (Fig. 3) and the calculation of KSV from Stern–Volmer
plots (Table 1) exhibited the effect on fluorescence quenching by
HAAO at each temperature. It was easy to notice that the value of
KSV was inversely correlated with temperature, which indicated
that the quenching mechanism of the HAAO–HSA binding reaction
D
Gh ¼
D
Hh ꢀ T ꢃ
D
Sh
ð4Þ
where
D
Hh,
D
Gh and
D
Sh represent the change of enthalpy, Gibbs
free energy and entropy, respectively. The plot of logK versus 1/T
(Fig. 5) enables the determination of
Hh, Sh in isobaric processes
and
Gh can be calculated by Eq. (4). The thermodynamic calculat-
ing results are listed in Table 2. The negative
Gh indicated the
D
D
D
D
interaction between HAAO and HSA is spontaneous, under
F0/F=0.9920+0.1433×106[Q]
log(F0/F-1)=5.164+1.0039*log[Q]
-0.3
1.5
log(F0/F-1)=5.080+0.9962*log[Q]
log(F0/F-1)=4.767+0.9501*log[Q]
F0/F=0.9952+0.1292×106[Q]
F0/F=1.0031+0.1107×106[Q]
log(F0/F-1)=4.641+0.9341*log[Q]
-0.6
1.4
F0/F=1.0033+0.1019×106[Q]
F
1.3
F
-0.9
1.2
293K; R=0.9993
299K; R=0.9990
305K; R=0.9984
293K; R=0.9989
299K; R=0.9995
305K; R=0.9991
311K; R=0.9995
-1.2
1.1
311K; R=0.9979
0.5
1.0
1.5
2.0
2.5
3.0
3.5
-6.25
-6.00
log[Q]
-5.75
-5.50
-6
-1
.
[Q] /10 mol L
Fig. 3. Stern–Volmer plots for the quenching of HSA by HAAO.
Fig. 4. log (F0/Fꢀ1log[Q] plots at four different temperatures.

H.-x. Zhang, Y. Liu / Steroids 80 (2014) 30–36
33
Negative
gen bond formation.
D
Sh and
D
Hh reflects the van der Waals force or the hydro-
D
Hh ꢄ0 and
D
Sh > 0 suggests the electrostatic
lnK=6572/T-10.46 R=0.9743 SD=0.16
12
11
force. Furthermore, specific electrostatic interactions between ionic
species in aqueous solution are characterized by a positive value of
D
Sh and a negative Hh value. Based on these rules, it can be
D
speculated that hydrogen bond and van der Waals power were
the major contributors in stabilizing the structure of HSA–HAAO
complex. This conclusion was also supported by the modeling
study, in which the hydrogen bonds were validated.
3.4. Site-selective binding geometry
The crystallographic analyses revealed that HSA is a monomer
of 585 amino acid residues which are divided into three homolo-
gous
a-helical domains, named as domain I (residues1–195),
0.00320
0.00328
0.00336
0.00344
T-1 /K-1
domain II (residues 196–383) and domain III (residues 384–585).
Each domain contains 10 helices that are divided into antiparallel
six-helix and four-helix subdomains, marked by A and B, respec-
tively [27,28]. HSA has several typical binding sites that can be
bound reversibly to endogenous and exogenous ligands with the
binding constants ranging from 104 to 108 Mꢀ1. According to Sud-
low’s nomenclature, there are two high affinity binding sites for
drugs in HSA, site I located in subdomain IIA and site II located
in subdomain IIIA [29]. Warfarin, as other bulky heterocyclic
anions, binds to Sudlow’s site I, whereas ibuprofen, as other
aromatic carboxylates with an extended conformation, prefers
Sudlow’s site II [30,31].
In order to identify the binding site of HAAO in HSA, warfarin
and ibuprofen were selected as two probes. In our experiments,
probe was added to HSA solution in advance. Then increasing
amount of HAAO was added into marker-HSA systems to monitor
whether it had a competition with the site marker for the specified
binding site in HSA, by comparing the fluorescence spectra with
that of the same systems without site markers (blank, dotted lines
in Fig. 6). When warfarin was added into the HSA solution, the
Fig. 5. Van’t Hoff plot for the binding of HAAO with HSA.
physiological conditions. The negative
D
Hh suggested the binding
reaction is an exothermic process, which could in turn give reason-
able explanations for the decreasing value of binding constant Kb
and binding sites n in Table 2. Additionally, the binding equilibrium
and thermodynamics of the parent steroid 3b-hydroxy-5-andros-
ten-17-one were recorded as well (the value in bracket in Table 2).
Compared to the parent steroid at the same temperature, the
observed equilibrium constant of HAAO to HSA is bigger, which
implies that the binding power was strengthened after modification
by amide bonds.
Generally speaking, the force between organic micro molecule
and biological macromolecule includes hydrophobic interaction,
hydrogen bond, van der Waals force and electrostatic force. Accord-
ing to the views of Ross and Subramanian [26], the positive
value is frequently taken as evidence for hydrophobic interaction.
D
Sh
Table 2
Observed binding constants, binding sites and thermodynamic parameters for HAAO–HSA interaction.
pH
T/(K)
Kb (L molꢀ1
)
n
SDb
D
Hh (kJ molꢀ1
)
D
Sh (J Kꢀ1
ꢀ86.95 (ꢀ26.67)
)
D )
Gh (kJ molꢀ1
a
7.40
293
299
305
311
105.164 (104.982
105.080 (104.844
104.767 (104.709
104.641 (104.618
)
)
)
)
1.039 (1.031)
0.012 (0.03)
0.014 (0.02)
0.018 (0.03)
0.020 (0.02)
ꢀ54.64 (ꢀ35.72)
ꢀ29.17 (ꢀ27.91)
ꢀ28.65 (ꢀ27.75)
ꢀ28.12 (ꢀ27.59)
ꢀ27.60 (ꢀ27.43)
0.9961 (0.9898)
0.9501 (0.9485)
0.9341 (0.9317)
a
The value in bracket is the corresponding result of the parent steroid.
SD is the standard deviation for Kb.
b
-6
-1
-6
-1
300
250
200
150
100
50
.
250
.
CHSA =2*10 mol L , Cwarfarin =0
=2*10 mol L ,
=0
CHSA
Cibuprofen
A
B
-6
-1
-6
-1
.
.
CHSA =Cwarfarin =2*10 mol L
CHSA =Cibuprofen =2*10 mol L
-6
-1
-6
-1
.
CHAAO /10 mol L
.
CHAAO /10 mol L
a'
h'
200
150
100
50
a
h
a, a'
0
a, a' 0
a'
b, b' 0.5
c, c' 1.0
d, d' 1.5
e, e' 2.0
f, f' 2.5
g, g' 3.0
h, h' 3.5
b, b' 0.5
c, c' 1.0
d, d' 1.5
e, e' 2.0
f, f' 2.5
g, g' 3.0
h, h' 3.5
a
h
h'
-6
-1
-6
-1
.
Ibuprofen only (2*10 mol L )
.
Warfarin only (2*10 mol L )
0
0
320
360
400
440
480
320
360
400
440
480
Wavelength (nm)
Wavelength (nm)
Fig. 6. Site competition experiments between HAAO and markers (T = 293 K).

34
H.-x. Zhang, Y. Liu / Steroids 80 (2014) 30–36
Table 3
maximum fluorescence intensity increased obviously accompanied
with a red shift of the maximum emission wavelength (see Fig. 6A).
The fluorescence was gradually quenched with the addition of
HAAO and the binding constant (105.097 L molꢀ1) significantly
decreased compared to the blank at the same temperature. It
was the sign of competition between HAAO and warfarin for the
same location [22]. On the other hand, when ibuprofen was added
into the HSA solution, both intensity and location of the maximum
emission peak were without significant change compared to blank
(see Fig. 6B). The quenching spectra were almost the same with
blank, which suggested that the binding site of HAAO was different
from that of ibuprofen. So, it is time to reach a conclusion that the
prior binding site of HAAO is Sudlow’s site I in the subdomain IIA of
HSA.
Fractions of different secondary structures determined by SELCON3.a
System
HSA
HSA-warfarin 38.9
HSA–HAAO 36.3
H(r) (%) H(d) (%) S(r) (%) S(d) (%) Trn (%) Unrd (%)
40.2
19.6
19.8
19.6
2.9
3.2
3.8
3.0
3.3
4.0
12.9
13.9
15.7
20.8
20.8
22.0
a
Abbreviations: H(r): regular
a-helix; H(d): distorted a-helix; S(r): regular b-
strand; S(d): distorted b-strand; Trn: b-turns; Unrd: unordered structure.
the secondary structural elements analyzed using SELCON3 soft-
ware are listed in Table 3.
From Table 3, a reduction of the a-helix from 59.8% (free HSA)
to 55.9% was observed (at a molar ratio of 10:1), which indicated
that HAAO had bound with the amino acid residues in the main
polypeptide chain of HSA and hydrogen bonding networks were
changed. The ligand HAAO may be fixed by hydrogen bonds with
polypeptide chain. The content change of b-strand (5.9–7.1%),
b-turns (12.9–15.7%), and unordered structure (20.8–22.0%)
suggested the synthesized compound could be carried by serum
albumin in human body and the secondary structure of protein
could be regulated by HAAO to some extent.
3.5. Conformation investigation
Drugs binding would always make an impact on the secondary
structure of serum albumin. In order to further investigate the
details of conformational changes of HSA, circular dichroism (CD)
spectrum method was performed. CD spectrum is a sensitive
technique to monitor the conformation of peptides and proteins
because the structural characterization of proteins depends greatly
on the remarkable sensitivity of CD in far-UV region [32,33]. CD
spectrum of HSA exhibits two negative bands at 208 and 222 nm
3.6. Molecular modeling
To facilitate the understanding of interaction modes between
HAAO and HSA, molecular modeling method was employed since
it could estimate the participation of specific chemical groups
and present a panorama of the drug–protein interaction at the
molecular level [36,37]. In our work, Sybyl 8.1 software was
selected to evaluate the most reasonable mode of the interactions.
The highest scoring binding site was in the subdomain IIA of HSA
(Fig. 8, Sudlow’s site I), which was in accordance with the result
giving by the fluorescence probe experiments.
In order to further define the binding details, molecule docking
was performed by Suflex-dock program in Sybyl 8.1 and the best
energy ranked result was enlarged and displayed in Fig. 9, where
the residues of HSA adjacent to HAOO were presented by lines.
which reflect the characteristic of
a-helix in the advanced structure
of protein as these two negative peaks are both contributed to
n?p⁄ transfer for the peptide bond of
a-helix. CD results can be
expressed in terms of the mean residue ellipticity (MRE) in deg cm2
d mol–1 according to the following equation [22]:
Obser
v
ed CDðmdegÞ
MRE ¼
ð5Þ
Cpnl ꢁ 10
where Cp is the molar concentration of the protein, n is the number
of amino acid residues and l is the path length (0.1 cm). The -heli-
cal contents of free and combined HSA were calculated from MRE
a
values at 208 nm using the equation [34,35]:
ꢀMRE208 ꢀ 4000
a-helix ð%Þ ¼
ð6Þ
33000 ꢀ 4000
where MRE208 is the observed MRE value at 208 nm, 4000 is the
MRE of the b-form and random coil conformation cross at 208 nm
and 33,000 is the MRE value of a pure
a-helix at 208 nm. Using
Eq. (6), the -helicity in the secondary structure of HSA was deter-
a
mined and the results were pasted on Fig. 7 and the estimates for
5000
0
-5000
-10000
-15000
b
c
a
-20000
-25000
200
210
220
230
240
250
260
Wavelength(nm)
Fig. 8. Binding site of HAAO on HSA. HSA is shown in cartoon. HAAO is represented
using spheres. The atoms of HAAO are color-coded as follows: C, white; H, blue
green; O, red; N, blue. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
Fig. 7. The CD spectrum of HSA(a), and HAAO–HSA system (b)and HAAo-warfarin
system (c).

H.-x. Zhang, Y. Liu / Steroids 80 (2014) 30–36
35
demonstrated the interaction was a spontaneous as well as an exo-
thermic process with a modest degree of reversibility around body
temperature. The molecular modeling study presented a panorama
of the uppermost binding mode including binding site and binding
power, the results of which were in accordance with experimental
results of fluorescence probe and thermodynamic study. All the
evidence suggested HAAO could be transported and stored in blood
plasma. The study provided full spectroscopic, thermodynamic and
geometrical details for clarifying the binding mechanisms of HAAO
with HSA and is helpful for understanding the molecular basis of
HAAO’s pharmacological action.
Acknowledgements
The authors thank Prof. Y. Liu in Wuhan University for the
contribution to molecular modeling. The authors gratefully
acknowledge the financial support of the Research Foundation of
Education Department of Hubei Province, China (Grant No.
Q20134303) and Scientific Research Project of Jingchu University
of Technology (Grant Nos. ZR201202, ZR201302 and ZR201317).
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Fig. 9. Molecular modeling of the interaction between HAAO and HSA. Only
residues within 5 Å are displayed. The residues of HSA are presented by lines and
HAAO is presented by balls and sticks.
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