Binding of a potential anti-hepatoma drug cis,cis,trans-[Pt(NH3)2Cl2(O2CCH2CH2COOH)-(OCONHC16H33)] with serum albumin - Thermodynamic and conformational investigations

Article abstract of DOI:10.1039/c5nj01103e

In this paper, a potential anti-hepatoma Pt⁴⁺ drug cis,cis,trans-[Pt(NH₃)₂Cl₂(O₂CCH₂CH₂COOH)-(OCONHC₁₆H₃₃)] was synthesized and selected to investigate its binding to human serum albumin (HSA) by spectroscopy and molecular docking methods. Fluorescence spectra show that the Trp residue, the intrinsic fluorophore in HSA, was induced to a less hydrophobic microenvironment with the addition of the Pt⁴⁺ compound, which induces the denaturation of HSA. Moreover, the fluorescence quenching mechanism was determined to be static quenching. The binding constant (K_A) and the number of binding sites (n) were calculated based on the results of fluorescence measurements. Circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR) and three dimensional fluorescence spectroscopy proved that the Pt⁴⁺ compound could slightly change the secondary structure and induce unfolding of the polypeptides of protein. Thermodynamic parameters indicate that the Pt⁴⁺ compound binds to HSA through electrostatic attraction with one binding site. The molecular docking study indicated that parecoxib is embedded into site I (subdomain IIA) of HSA.

Full text of DOI:10.1039/c5nj01103e

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Binding of a potential anti-hepatoma drug
cis,cis,trans-[Pt(NH3)2Cl2(O2CCH2CH2COOH)-
(OCONHC16H33)] with serum albumin –
thermodynamic and conformational
investigations†
Cite this:DOI: 10.1039/c5nj01103e
Li Qi, Zhong Lu,* Wen-hua Lang, Lu Guo, Chang-geng Ma and Guang-hong Sun
In this paper,
a
potential anti-hepatoma Pt4+ drug cis,cis,trans-[Pt(NH3)2Cl2(O2CCH2CH2COOH)-
(OCONHC16H33)] was synthesized and selected to investigate its binding to human serum albumin (HSA)
by spectroscopy and molecular docking methods. Fluorescence spectra show that the Trp residue, the
intrinsic fluorophore in HSA, was induced to a less hydrophobic microenvironment with the addition of
the Pt4+ compound, which induces the denaturation of HSA. Moreover, the fluorescence quenching
mechanism was determined to be static quenching. The binding constant (KA) and the number of
binding sites (n) were calculated based on the results of fluorescence measurements. Circular dichroism
(CD), Fourier transform infrared spectroscopy (FTIR) and three dimensional fluorescence spectroscopy
proved that the Pt4+ compound could slightly change the secondary structure and induce unfolding of
the polypeptides of protein. Thermodynamic parameters indicate that the Pt4+ compound binds to HSA
through electrostatic attraction with one binding site. The molecular docking study indicated that
parecoxib is embedded into site I (subdomain IIA) of HSA.
Received (in Montpellier, France)
24th July 2015,
Accepted 14th September 2015
DOI: 10.1039/c5nj01103e
www.rsc.org/njc
is less compared to their Pt2+ counterparts. Because Pt4+ is the
most potent member of the Pt anticancer drug family, its
1. Introduction
Platinum-based compounds are among the most active chemo- potential use in liver cancer is attractive.
therapeutic agents available. They are effective against a multi-
As the most abundant protein in the circulatory system,
tude of cancers. Cisplatin is one of the most effective drugs in human serum albumin (HSA) possesses many unique physio-
the treatment of solid tumors including epithelial ovarian logical and pharmacological functions in human body.11–13 The
carcinoma, head and neck squamous carcinoma.1–3 It exerts protein was reported to serve as a transporter for a variety of
its cytotoxic action by forming intra- and interstrand adducts endogenous and exogenous compounds such as drugs, fatty
with DNA, which induce apoptosis thereafter. However, dose- acids and dyes in the bloodstream. The binding between anti-
limited side effects of cisplatin narrow its clinical use. Therefore, it cancer drugs and HSA can significantly affect the absorption,
would be advantageous to increase the therapeutic index of cispla- distribution, metabolism and toxicity of the drugs, and may
tin by increasing its efficacy or decreasing the toxic side effects. In change the functions and structures of the protein. Thus, the
order to do this, nowadays, much attention regarding platinum investigations on the interaction of HSA with anti-cancer drugs
anti-cancer drugs has been paid to Pt4+-based compounds.4–8 There are not only important to comprehend their transport and
are many Pt4+ compounds that have been synthesized and their metabolism processes in the body, but also can provide insight
anti-cancer activities have been evaluated.5,7,9,10 As a result, the Pt4+ into the understanding of the toxicity of the drugs at the
compounds have been proven to have advantages over the Pt2+ molecular level.
compounds because of their kinetic inertness and thus toxicity
In the present work, we synthesized a potential Pt4+ anti-
hepatoma drug cis,cis,trans-[Pt(NH3)2Cl2(O2CCH2CH2COOH)-
(OCONHC16H33)]. Multispectroscopic methods including
fluorescence, UV-vis absorption and circular dichroism (CD)
spectroscopy, coupled with a molecular docking technique were
employed to characterize the binding of the Pt4+ compound with
HSA and the effect of the Pt4+ compound on the molecular
Clinical College of Weifang Medical University, Weifang 261031, China.
E-mail: luzhongdoc1103@hotmail.com; Fax: +86-05368068895;
Tel: +86-05368068895
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5nj01103e
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conformation of HSA at physiological pH (pH 7.4). The study evaluated by the MTT assay. Cells were seeded on a 96 well plate
provides a quantitative understanding of the effect of the Pt4+ (2000 cells per well) in 200 mL RPMI or DMEM and incubated
compound on the structure of HSA, which could be useful for 24 h at 37 1C. The following day, solutions of the platinum
supports for the further design of a much more suitable Pt4+ compounds were freshly prepared in RPMI or DMEM media
compound with structural variants.
(Pt4+ compound), PBS (cisplatin), or obtained by FPLC isolation
and quantitated by GFAAS. Preparation of the samples for the
Pt4+ compound was achieved by ultrasonication of a suspension
of the solid platinum complex in cell culture medium for
30 min followed by filtration through a 0.2 mM syringe filter.
The cells were then treated with the platinum compounds,
separately at varying concentrations, and incubated for 72 h at
37 1C. The cells were then treated with 200 mL fresh medium
containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (0.8 mg mLꢀ1) and incubated for 3 h at 37 1C.
The medium was removed, 200 mL of DMSO was added to the
cells, and the absorbance of the purple formazan was recorded
at 570 nm using a BioTek Synergy HT multi-detection micro-
plate plate reader. Each experiment was performed in triplicate
for each cell line.
2. Materials and methods
2.1. Materials
HSA with a sharp molecular weight distribution at 66 400 g molꢀ1
was purchased from Amresco. Cisplatin was obtained from Hubei
Biocause Pharmaceutical Co., Ltd (Hubei, China; purity no less
than 99.7%). Phenylbutazone (99% purity) was purchased from
ACROS ORGANICS (New Jersey, USA); ibuprofen (99% purity) was
purchased from Shanghai Civi Chemical Technology Co., Ltd
(Shanghai, China); and digitoxin (99% purity) was purchased
from Hubei Huabei Biomedicine Co., Ltd (Wuhan, China). All
other chemicals were bought from Sinopharm Co., Ltd (Beijing,
China).The pH value of the mixture solutions for the tests was 7.4.
It should be noted that, since the isoelectric point of HSA occurs
at 4.9, under this condition it is in a negative form.
Fluorescence spectral analysis was carried out on a Hitachi
F-4500 fluorescence spectrophotometer using 1.0 cm quartz
cells. The excitation wavelength of HSA studied in this work was
280 nm. Synchronous fluorescence spectra were acquired by
the same spectrofluorometer. They were recorded and the
2.2. Methods
The asymmetrically functionalized Pt4+ compound 3 can be difference between excitation wavelength and emission wave-
obtained by the reaction of 2 with succinic anhydride using the length was kept constant (Dl = lem ꢀ lex). When the Dl was at
method from Dhar14 as described in Fig. 1. Compound 3 was 15 or 60 nm, the synchronous fluorescence spectrum gave the
placed into a 5 mL glass vial and the isocyanate C17H35NO2 characteristic information about Tyr residues or Trp residues.
(anhydrous DMF) solution (2 mL) was added. The suspension All the excitation and emission slits were set at 5/5 nm. The
was stirred overnight. The next day, the suspension had experiments were carried out at 298 K.
become a clear green solution. The solution was then filtered
CD spectra were recorded over a wavelength range of
and the solvent was removed under reduced pressure at 65 1C. 190–250 nm at 0.2 nm intervals at 298 K in a thermostated cell
Ethyl ether (2 mL) was added to the oily residue, and the holder on a MOS-450 CD spectrometer. The scanning speed was
mixture was ultrasonicated for 1 min and centrifuged. The set at 200 nm minꢀ1 and the cell length was 1 cm. Results are
solid was further washed with DCM (4 mL) and diethyl ether expressed as ellipticity (mdeg), which was obtained in mdeg
(2 mL). The washed solid was then left under vacuum overnight. directly from the instrument. Three scans were made and
The human ovarian carcinoma A2780, cisplatin resistant averaged for each CD spectrum.
A2780/CP70 cell lines and the human lung carcinoma cell line
Fluorescence lifetimes have been measured using a time-
A549 were kindly provided by Dr Sufang Zhang. Cytotoxicity correlated-single-photon counting (TCSPC) spectrophotometer
against different cell lines (A549, A2780, and A2780/CP70) was (Horiba Jobin Yovin) with full width at half maximum (FWHM)
Fig. 1 Route of Pt4+ compound synthesis.
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ca. 300 ps, repetition rate 1 MHz and the resolution was 28 ps (Fig. S2, ESI†), which shows that the Pt4+ compound takes more
per channel. The excitation and emission wavelengths have than 95% in the final product.
been chosen to be 280 and 340 nm, respectively and MCP-PMT
3.2. Cytotoxicity and uptake of the Pt4+ compound
as a detector. The emission from the samples was collected at a
right angle to the direction of the excitation beam maintaining
The cytotoxicity of the Pt4+ compound and cisplatin was examined
magic-angle polarization (54.71). The data have been fitted to
using the on-target CCRF-CEM leukaemia cell line and the off-
multiexponential functions after deconvolution of the instru-
target ovarian carcinoma cell line A2780 and its cisplatin-resistant
ment response function by an iterative reconvolution technique
derivative A2780/cp70. Cancer cells were treated with cisplatin or
using IBH DAS 6.2 data analysis software in which reduced w2
Pt4+ compound for 48 h and cell viability was evaluated. IC50
values, which represent the concentration required to inhibit
serves as a parameter for goodness of fit.
Measurements of FT-IR were carried out on a Nicolet Avatar
330 FT-IR spectrometer equipped with a germanium attenuated
growth by 50%, are given in Fig. 2A. The IC50 for cisplatin at
48 h is 0.92 ꢁ 0.04 mM, and that for the Pt4+ compound is 0.33 ꢁ
total reflection (ATR) accessory, adeuterated triglycine sulfate
0.03 mM. In addition, we further evaluated the cytoxicity of the
(DTGS) detector, and a KBr beam splitter. All the FT-IR spectra
Pt4+ compound in normal human cells. The IC50 value of the Pt4+
were taken via the ATR method with a resolution of 4 cmꢀ1 and
compound in the MRC-5 (normal lung tissue) cell line is 0.74 ꢁ
128 scans at room temperature. The background (containing all
0.05 mM, which is two times that in the A2780 ovarian cancer cell
system components except protein) was collected under the
same conditions and subtracted from the spectra of sample
solution to obtain the FT-IR spectra of protein.
lines (IC50 = 0.33 ꢁ 0.03 mM).
The extent of cellular uptake was investigated by treating
A2780 ovarian cancer cells with 5 mM of cisplatin or Pt4+
The PDB entry of the HSA crystal structure employed in the
compound for 5 h. The whole cell concentration of platinum
docking study was 1H9Z. The docking study was conducted by a
was then evaluated by inductively coupled plasma mass spectro-
Surflex Dock program in Sybyl 8.1 package. The protocol was
metry. Notably, from cisplatin to Pt4+ compound, the 34.57-fold
generated by residues and ligand mode. The threshold was set
increase in cellular uptake is mirrored by increase in cytotoxicity,
at 0.50, while the bloat was 0. The parameters in the docking
suggesting that the increase in cytotoxicity can be attributed in
work were set as follows: additional starting conformation per
large part to the increase in uptake. As shown in Fig. 2A, the Pt4+
molecule: 20; angstroms to expand search grid: 6; max con-
compound also displays a lower resistance factor in ovarian
formation per fragment: 20; max number of rotatable bonds per
cancer cell lines than cisplatin, and the treatment with cisplatin
molecule: 100. The structure of the Pt4+ compound was drawn by
or Pt4+ compound at equimolar concentrations led to drastically
Sybyl 8.1 package. After being charged using the Gasteiger and
different degrees of cell survival.
Marsili method, the molecule was optimized in energy and
geometry using Tripos Force Field.
3.3. Quenching mechanism, binding number and affinity
determination of the protein-compound system
Analysis of Trp fluorescence quenching for the determination
of interactions between the Pt4+ compound with HSA were
3. Results and discussion
performed through titration of the Pt4+ compound against
protein. Fig. 3A shows that HSA has a strong fluorescence
emission peak at B340 nm. Thus, following quenching of the
HSA fluorescence on addition of the compound can conclude
3.1. Synthesis of the Pt4+ compound
The amphiphilic Pt4+ compound construct was prepared using
the synthetic approach shown in Fig. 1. Hydrogen peroxide
oxidation of cisplatin (1) in water affords 2. The asymmetrically
functionalized Pt4+ compound 3 can be obtained by the reaction
of 2 with succinic anhydride. The fortuitous acetone solubility of
the disuccinate compound and the insolubility of 3 provide an
effective means of removing the undesired side product.14 The
ability of isocyanate reagents to undergo nucleophilic attack by
the platinum-bound hydroxide ligand of 3 was exploited to form
a Pt4+ carbamate species. Compound Pt4+ bear a hydrophobic
unbranched aliphatic chain with a length of C16.
The Pt4+ compound was fully characterized by multinuclear
(1H, 13C, and 195Pt) NMR spectroscopy and electrospray ioniza-
tion mass spectrometry (ESI-MS) (Fig. S1, ESI†). The Pt4+ com-
pound displays a 195Pt NMR signal around d = 1240 ppm,
confirming the 4+ oxidation state of the platinum. In the ESI
mass spectra, well defined isotopic distribution patterns provided
further confirmation of chemical composition. Chemical purity
was established with combustion analysis and analytical HPLC
Fig. 2 (A) The in vitro cytotoxicity using growth inhibition assays of the
compounds after 48 h of exposure. (B) Whole cell uptake of the com-
pounds. Error bars are the standard deviations of three runs.
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Fig. 3 (A) Effect of the Pt4+ compound on the fluorescence intensity of HSA. (B) Stern–Volmer and (D) Hill plots for HSA interacting with the Pt4+
compound at 283 K, 298 K, and 310 K. Conditions: T = 298 K, pH 7.4, salt concentration 0.9%; c(HSA) = 1.0 ꢂ 10ꢀ6 M; c(Pt4+): 0, 1.0, 3.0, 5.0, 8.0, 11.0,
and 15.0 ꢂ 10ꢀ5 M. (C) The time-resolved fluorescence decay for HSA interacting with the Pt4+ compound at 298 K, pH = 7.4, salt concentration 0.9%.
c(HSA) = 1.0 ꢂ 10ꢀ6 M; c(Pt4+) 1–3: 0, 5.0 and 15.0 ꢂ 10ꢀ5 M. Error bars are the standard deviations of three runs.
whether the Pt4+ compound interacts with the protein at the site
Fluorescence quenching can be either dynamic or static in
close to Trp-214. There is a continuous decrease in the emission nature. To know about the quenching mechanism of HSA by
spectral intensity of HSA without significant change in the wave- the Pt4+ compound, we analyzed the data by the Stern–Volmer
length of maximal fluorescence emission (lmax). The decrease in plots (Fig. 3B). Kq is 2.24 ꢂ 1012
M
ꢀ1 sꢀ1, which lies in the order
fluorescence intensity upon addition of the compound was ana- of 1012, as shown in Table 1. However, the Kq for the HSA–Pt4+
lyzed according to the Stern–Volmer equation:12 F0/F = 1 + Kqt0[Q] = compound system is 100 times higher than the maximum
1 + KSV[Q], where F0 and F are the fluorescence intensities in the scatter collision quenching constant of various quenchers with
absence and presence of the quencher, respectively. Kq and KSV are polymers (2 ꢂ 1010
M
ꢀ1 sꢀ1). This shows that quenching is not
the quenching rate constants of the bimolecular and the Stern– initiated by dynamic diffusion but occurs by the formation of a
Volmer dynamic quenching constant, respectively. [Q] is the strong complex between HSA and the Pt4+ compound. Further-
concentration of the quencher and t0 is the average lifetime of more, the temperature dependence of KSV is studied to distin-
HSA without a quencher.
guish the type of quenching by the differential response toward
Table 1 Stern–Volmer quenching constants, binding parameters, and thermodynamic parameters of the HSA–Pt4+ drug system at different
temperatures
Stern–Volmer quenching constants
Binding parameters
Thermodynamic parameters
T (K)
Kq (Mꢀ1 sꢀ1
)
KSV (Mꢀ1
)
R
KA (Mꢀ1
)
n
R
DG1 (J molꢀ1
)
DS1 (J molꢀ1 Kꢀ1
)
DH1 (J molꢀ1
ꢀ1.07 ꢂ 104
)
283
298
310
2.93 ꢂ 1012
2.24 ꢂ 1012
1.78 ꢂ 1012
2.93 ꢂ 104
2.24 ꢂ 104
1.78 ꢂ 104
0.9968
0.9976
0.9973
5.07 ꢂ 103
4.51 ꢂ 103
3.37 ꢂ 103
1.11
1.05
1.05
0.9998
0.9991
0.9992
ꢀ2.01 ꢂ 104
ꢀ2.08 ꢂ 104
ꢀ2.09 ꢂ 104
33.5
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temperature and viscosity. In static quenching, KSV decreases
with an increase in temperature due to the formation of
complex with protein, which undergoes dissociation on
increasing temperature. However, for dynamic quenching, KSV
increases with temperature as in this case higher temperature
results in faster diffusion of the quencher and hence larger
extent of collisional quenching.15 We found that upon increas-
ing temperature, the KSV value decreases, which is good indica-
tion of static quenching.
Time-resolved fluorescence spectroscopy is a tool that
can probe the interaction between ligands and proteins. The
fluorescence lifetime can be used to directly distinguish
between dynamic and static quenching. For static quenching,
the lifetime does not depend on the quencher concentration
(i.e., t0 = t). Time resolved fluorescence spectra of the free HSA
and HSA–Pt4+ complexes are shown in Fig. 3C using the
fluorescence decay parameters. The fluorescence decay of the
Fig. 4 van’t Hoff plot for the HSA–Pt4+ compound system. Error bars are
the standard deviations of three runs.
unquenched protein is almost the same with the quenched while the free energy change (DG) is then calculated from the
protein. The average fluorescence lifetime of Trp-214 stayed following relationship: DG = DH ꢀ TDS = ꢀRT ln K. The corres-
still with increasing Pt4+ compound. These results suggested ponding results were presented in Table 1, while the values of DH
that the HSA–Pt4+ compound quenching mechanism was static. and DS of the binding site were obtained from the slopes and
When small molecules bind independently to a set of ordinates at the origin of fitted lines. The values of DH and DS are
equivalent sites on a macromolecule, the binding number found to be ꢀ1.07 ꢂ 104 J molꢀ1 and 33.5 J molꢀ1
K
ꢀ1, which
between them has also been studied through the following indicated that the electrostatic force played the major role in the
double-logarithmic equation:16,17
process of forming the Pt(IV) complex.19,20 This was also con-
firmed by the salt effect experiments (Fig. S3, ESI†). The negative
sign for DG means that the binding process is spontaneously
driven by enthalpy and entropy together.
F0 ꢀ F
lg
¼ lg KA þ n lg½Qꢃ
F
where F0 and F stands for the fluorescence intensity in the
absence and the presence of the quencher at various concen-
trations of [Q] respectively, KA refers to the binding constant,
and n is the binding number. According to the equation, the
binding number can be obtained from the slope of the plot of lg
(F0/F ꢀ 1) vs. lg[Q] (Fig. 3D). Table 1 demonstrates that the value
of n is close to 1 and KA is in the order of 103, which suggests
that the Pt4+ and HSA have one binding site and exhibits a weak
binding force during their interaction process. In general, the
interaction forces between ligands and proteins may include
electrostatic interactions, multiple hydrogen bonds, van der
Waals interactions, hydrophobic and steric contacts within the
antibody-binding site, etc. To elucidate the interaction between
the Pt4+ compound and HSA, the temperature-dependent thermo-
dynamic parameters were calculated from the van’t Hoff plots. It
is supposed that the enthalpy change (DH) does not vary signifi-
cantly in the temperature range studied and can be considered
as a constant, then both the enthalpy change (DH) and entropy
change (DS) can be evaluated from the van’t Hoff equation:18
3.4. Conformational changes of HSA induced by the Pt4+
compound
Synchronous fluorescence spectra can give information about
the molecular environment in the vicinity of chromosphere
molecules in low concentrations under physiological conditions.
It is a sensitive technique for detecting microenvironmental
changes in these chromophores, with several advantages, such
as spectral simplification, spectral bandwidth reduction and
avoiding different perturbing effects. When the D values (Dl)
between excitation and emission wavelengths are stabilized at
15 or 60 nm, synchronous fluorescence gives the characteristic
information about Tyr or Trp residues.21 The effect of the Pt4+
compound on HSA synchronous fluorescence spectra is shown
in Fig. 5, from which it is apparent that the maximum emission
wavelength had a slight red shift of 5 nm in the investigated
concentration range when Dl = 60 nm, which suggested a more
polar (or less hydrophobic) environment of Trp residues; while
when Dl = 15 nm the maximum emission wavelength moved
only 2 nm at the investigated concentration range, which indi-
cated that there was less change in the microenvironment of
Tyr residues. FT-IR spectroscopy has emerged as an established
DH DS
RT
ln K ¼ ꢀ
þ
R
where K is analogous to the effective quenching constants technique to investigate the structural characteristics of pro-
at the corresponding temperatures and R is the gas constant teins. Among the amide bands of the protein, the amide I band
(8.314 J molꢀ1
K
ꢀ1). The temperatures used in our experiment (1700–1600 cmꢀ1, mainly CQO stretch) and amide II band
were 283, 298 and 310 K. From Fig. 4 we can see that the linear (1600–1500 cmꢀ1, C–N stretch coupled with N–H bending mode)
relationship between ln K and 1/T is good. The enthalpy change both have a relationship with the secondary structure of protein,
(DH) is obtained from the slope of the van’t Hoff relationship, which range from gross aspects to subtle rearrangements
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Fig. 5 Synchronous fluorescence spectra of the Pt4+ compound with
HSA with Dl = 15 nm or Dl = 60 nm at 298 K. Concentrations are
consistent with the steady-state fluorescence study.
Fig. 6 Upper: FT-IR spectra of free HSA (solid curve, 1.0 ꢂ 10ꢀ4 M), the
HSA–Pt4+ compound complex (the molar ratio of HSA/Pt4+ compound
was maintained at 1 : 3 for the dashed curve). Lower: CD spectra of HSA in
the absence and presence of Pt4+ compound. The concentration of HSA
was 2.0 ꢂ 10ꢀ7 M, and the concentrations of the Pt4+ compound were 0 (a),
2.0 (b) and 5.0 (c) ꢂ10ꢀ7 M. T = 298 K, pH = 7.4, salt concentration 0.9%.
associated with ligand binding. In the infrared spectra, the
amide I region occur approximately at 1600–1700 cmꢀ1, while
the amide II region occurs nearly at 1548 cmꢀ1. The former
region appears due to the CQO stretching vibration, which has
significantly higher signal intensity; the later one occurs due to
C–N stretch coupled with N–H bending. The conformational
sensitivity of amide bands is primarily due to the hydrogen
bonding and the coupling between transition dipoles. Fig. 6(upper)
compares the FT-IR spectra of HSA and the difference spectra of
HSA–Pt4+ complex in phosphate buffer solution at 298 K. The
difference spectra of the HSA–Pt4+ complex were obtained by
subtracting the spectra of the Pt4+ compound from the spectra of
the HSA–Pt4+ complex. The shift in the peak position of the
amide I band of HSA from 1651 to 1649 cmꢀ1 after addition of
the Pt4+ compound indicates the change around the amido bond
(CQO) of HSA upon interaction with the Pt4+ compound.
Analysis of the secondary structure of HSA and Pt4+ complex
was carried out on the basis of the CD technique. The CD spectra
of HSA in the absence and presence of the Pt4+ compound were
shown in Fig. 6. As Fig. 6(lower) showed, HSA exhibited two
negative ellipticities at 208 and 220 nm in the UV region, which
were characteristic of the typical a-helix structure of protein.22
The binding of the Pt4+ compound to HSA caused a decrease in
band intensity at all wavelengths of the far-UV CD without any
significant shift of the peaks, indicating the decrease of the
unfolding even more. However, the CD spectra of HSA in the
presence and absence of the Pt4+ compound are similar in shape,
indicating that the structure of HSA is also predominantly
a-helical. From the above results, it is apparent that the effect
of the Pt4+ compound on HSA causes a conformational change of
the protein, with the loss of a-helical stability.23,24 The calculat-
ing results exhibited a reduction of a-helix structures from 58.4%
of native protein to 52.6% at molar ratio Pt4+ compound/HSA =
10: 1, while the fractions of random coil increased from 20.6% to
25.3%, indicating the adaptive structural alterations (Table 2).25
The three-dimensional fluorescence spectra are used to
further study the change in the configuration of proteins
induced by small molecules. The three-dimensional fluorescence
Table
2
Secondary structural content of HSA in the absence and
presence of the Pt4+ drug
Content (%)
Sample
a-Helix
b-Sheet
b-Turn
Random coil
Pure HSA
58.4
55.6
54.9
52.6
10.3
10.9
11.5
13.0
10.7
10.5
10.0
9.1
20.6
23.0
23.6
25.3
HSA : Pt4+ = 1 : 5
HSA : Pt4+ = 1 : 8
a-helical content in protein, which means the peptide strand HSA : Pt4+ = 1 : 10
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spectra and contour ones are shown in Fig. 7. Four peaks can be The above phenomena and analysis of the fluorescence character-
seen in Fig. 7. The peak where lex = lem is the first-ordered istic of the peaks revealed that the binding site is near Trp and Tyr
Rayleigh scattering (RLS) peak. Peak a (lex = 280 nm, lem
=
residues and induced the fluorescence quenching of intrinsic
334 nm) relates to the fluorescence behavior of Trp and Tyr fluorescence of HSA.29,30 The binding of the Pt4+ compound
residues; peak b (lex = 230 nm, lem = 335 nm) mainly reveals the induced slight unfolding of the polypeptides of protein.
fluorescence spectral behavior of the polypeptide backbone
3.5. The Pt4+ compound was located at site I of HSA
structure, and its fluorescence intensity is correlated with the
secondary structure of protein.26,27 The production of RLS
is correlated with the formation of certain aggregates and pri-
marily the particle dimension of the formed aggregate in solution
dominates the RLS intensity. Bearing these points in mind, it is
inferred from the results that the size of the HSA–Pt4+ compound
complexes may be not changed obviously comparing that of HSA,
for the peak intensity are all around 2000. Analyzing from the
intensity changes of peak a and peak b, they decreased obviously
in the presence of the Pt4+ compound, the fluorescence intensity
ratios of peak a and peak b are 1.70 : 1 and 1.27 : 1, respectively.
To study the distinct binding site of the Pt4+ compound on HSA,
site-specific ligands were used: warfarin and furosemide for site
I and ibuprofen for site II.28 Fig. 8A shows that the fluorescence
intensity significantly decreased after the addition of warfarin
and furosemide but not after adding ibuprofen, suggesting that
the Pt4+ compound binds to site I of HSA. It could be presumed
that the Pt4+ compound is situated in the binding cavity of the
Fig. 8 (A) Molecular probing experiment showing site I is the major
binding site. Concentration of the probe molecules is fixed at 1.0 ꢂ
10ꢀ6 M, pH = 7.4, salt concentration 0.9%. Molecular docking results of
the HSA–Pt4+ compound interacting: (B) location of the compound in HSA
showing that the main binding site is site I; (C) amino acid residues around
4 Å of the Pt4+ compound molecule.
Fig. 7 The three-dimensional fluorescence spectra of the HSA–Pt4+
compound system. Conditions: T = 300 K, pH 7.4, salt concentration
0.9%.; c(HSA) = 1.0 ꢂ 10ꢀ6 M; c(Pt(VI)): 0 (A) and 14.0 (B) ꢂ10ꢀ6 M.
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Paper
NJC
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site I and negative carbonyl groups may slightly interact with
positively charged residues such as Lys-195, Lys-199, Arg-218,
Arg-222, His-242, and Arg-257 which are located at the entrance
of the site I cavity.
The molecular docking has been employed to further under-
stand precisely the binding site of the Pt4+ compound and HSA.
HSA comprises of three homologous domains (I–III): I (residues
1–195), II (196–383), III (384–585), each domain comprises of
subdomains that posses common structural motifs. The principal
regions of ligand binding to HSA are located in hydrophobic
cavities of subdomains IIA and IIIA, which are consistent with
Sudlow sites I and II, respectively. In the present study, Autodock
program is applied to calculate the possible conformation of the
Pt4+ compound that binds to the protein. Fig. 8C shows that the
Pt4+ compound more favorably fits in the cavity of subdomains IIA,
which corresponds to site I. His-288, Glu-153, Arg-257, Gln-196,
Glu-292 and Arg-222 of site I were around the 4 Å of the
compound, of which Arg-257, Arg-222 and His-288 may be involved
in the electrostatic interaction.
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In this paper, a potential anti-hepatoma drug cis,cis,trans-
[Pt(NH3)2Cl2(O2CCH2CH2COOH)-(OCONHC16H33)] was synthe-
sized and it showed excellent anti-liver cancer activity. The
interaction of human serum albumin and the compound was
investigated employing different spectroscopic and molecular
docking techniques. The results revealed that the secondary
structure of protein was affected upon interaction with the
compound. Fluorescence results indicated the presence of the
static quenching mechanism in the binding of the Pt4+ com-
pound to the protein. Based on spectral data we have concluded
that the Pt4+ compound bound to site I of protein, which is
located in the hydrophobic pocket of subdomain IIA.
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Acknowledgements
This work was supported by grants from Medical and Techno-
logic Development Project of Shandong province (2011QZ028).
The authors thank Dr Yamin Chang for his technical support in
Pt4+ compound synthesis and Dr James. H. Haywood of Michigan
State University for editing the manuscript.
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