Synthesis, characterization, DNA and HSA binding studies of isomeric Pd (II) antitumor complexes using spectrophotometry techniques

Article abstract of DOI:10.1080/07391102.2017.1322536

Two new Palladium(II) isomeric complexes, [Pd (Gly)(Leu)](I) and [Pd (Gly)(Ile)](II), where Gly is glycine, and Leu and Ile are isomeric amino acids (leucine and isoleucine), have been synthesized and characterized by elemental analysis, molar conductivity measurements, FT-IR, ¹H NMR, and UV–Vis. The complexes have been tested for their In vitro cytotoxicity against cancer cell line K₅₆₂ and their binding properties to calf thymus DNA (CT-DNA) and human serum albumin (HSA) have also been investigated by multispectroscopic techniques. Interactions of these complexes with CT-DNA were monitored using gel electrophoresis. The energy transfer from HSA to these complexes and the binding distance between HSA and the complexes (r) were calculated. The results obtained from these studies indicated that at very low concentrations, both complexes effectively interact with CT-DNA and HSA. Fluorescence studies revealed that the complexes strongly quench DNA bound ethidium bromide as well as the intrinsic fluorescence of HSA through the static quenching procedures. Binding constant (K_b), apparent biomolecular quenching constant (k_q), and number of binding sites (n) for CT-DNA and HSA were calculated using Stern–Volmer equation. The calculated thermodynamic parameters indicated that the hydrogen binding and vander Waals forces might play a major role in the interaction of these complexes with HSA and DNA. Thus, we propose that the complexes exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA. The complexes follow the binding affinity order of I?>?II with DNA- and II?>?I with HSA-binding.

Full text of DOI:10.1080/07391102.2017.1322536

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Synthesis, Characterization, DNA and HSA Binding
Studies of Isomeric Pd (II) Antitumor Complexes
Using Spectrophotometry Techniques
Sareh Zareian-Jahromi & Hassan Mansouri-Torshizi
To cite this article: Sareh Zareian-Jahromi & Hassan Mansouri-Torshizi (2017): Synthesis,
Characterization, DNA and HSA Binding Studies of Isomeric Pd (II) Antitumor Complexes
Using Spectrophotometry Techniques, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2017.1322536
To link to this article: http://dx.doi.org/10.1080/07391102.2017.1322536
Accepted author version posted online: 23
Apr 2017.
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Date: 24 April 2017, At: 04:38

Publisher: Taylor & Francis
Journal: Journal of Biomolecular Structure and Dynamics
DOI: http://dx.doi.org/10.1080/07391102.2017.1322536
Synthesis, Characterization, DNA and HSA Binding Studies of Isomeric Pd (II) Antitumor
Complexes Using Spectrophotometry Techniques
a
a
Sareh Zareian-Jahromi and Hassan Mansouri-Torshizi*
a
Department of Chemistry, Faculty of science, University of Sistan and Baluchestan, Zahedan,
Iran,
*
Corresponding author. Tel./fax: +98-54-33431146, Fax: +98-54-33446565
E-mail: hmtorshizi@hamoon.usb.ac.ir
1

Synthesis, Characterization, DNA and HSA Binding Studies of Isomeric Pd (II) Antitumor
Complexes Using Spectrophotometry Techniques
a
a
Sareh Zareian-Jahromi and Hassan Mansouri-Torshizi*
a
Department of Chemistry, Faculty of science, University of Sistan and Baluchestan, Zahedan,
Iran,
*
Corresponding author. Tel./fax: +98-54-33431146, Fax: +98-54-33446565
E-mail: hmtorshizi@hamoon.usb.ac.ir
Abstract:
Two new Palladium(II) isomeric complexes, [Pd(Gly)(Leu)](I) and [Pd(Gly)(Ile)](II), where
Gly is glycine, and Leu and Ile are isomeric amino acids (leucine and isoleucine), have been
synthesized and characterized by elemental analysis, molar conductivity measurements , FT-IR,
1
H NMR and UV–Vis. The complexes have been tested for their In vitro cytotoxicity against
cancer cell line K562 and their binding properties to calf thymus DNA (CT-DNA) and human
serum albumin (HSA) have also been investigated by multispectroscopic techniques.
Interactions of these complexes with CT-DNA were monitored using gel electrophoresis. The
energy transfer from HSA to these complexes and the binding distance between HSA and the
complexes (r) were calculated. The results obtained from these studies indicated that at very low
concentrations, both complexes effectively interact with CT-DNA and HSA. Fluorescence
studies revealed that the complexes strongly quench DNA bound ethidium bromide as well as
the intrinsic fluorescence of HSA through the static quenching procedures. Binding constant
b q
(K ), apparent biomolecular quenching constant (k ) and number of binding sites (n) for CT-
DNA and HSA were calculated using Stern-Volmer equation. The calculated thermodynamic
parameters indicated that the hydrogen binding and vander Waals forces might play a major role
in the interaction of these complexes with HSA and DNA. Thus, we propose that the complexes
exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA.
The complexes follow the binding affinity order of I>II with DNA- and II>I with HSA-
binding.
2

Keywords: DNA-binding, Pd(II) complexes, Amino acids, HSA- binding, Cytotoxicity
Abbreviations:
Gly
SDS
Leu
glycine
Sodium Dodecyl Sulphate
leucine
Ile
isoleucine
CT-DNA
EB
calf thymus deoxyribonucleic acid
ethidium bromide
human serum albomine
apparent binding constant
Stern-Volmer constant
binding constant
HSA
K
app
K
sv
K
b
k
q
quenching constant
number of binding sites
enthalpy changes
n



H
S
entropy changes
G
free energy changes
1
. Introduction
Medicinal inorganic chemistry plays a key role in designing the metal-based anticancer drugs
which have a great interest as a potential therapeutic agent against cancer treatments (Hannon,
2
007; Kostova, 2006). Yet, as a chemical agent used during chemotherapy, these drugs may
cause serious side effects (Sparks & Scholz, 2009). While designing the novel therapeutic and
3

diagnostic metal- based anticancer drugs, the main goal is to produce new types of adducts on
DNA with reduced side-effects (Erdogan & Özalp-Yaman, 2014; Singh, Joseph, Kumar,
Bathini, & Lown, 1992). Recently, there has been a great interest in binding of metal complexes
with DNA, for it may provide important information about new cancer therapeutic agents and
potential probes of DNA structure and conformation (da Silveira et al., 2011; Gaber, El-
Ghamry, & Fathalla, 2015; Li et al., 2013). Likewise, due to their promising antitumor and
antibacterial activities, Pd(II) and Pt(II) complexes have attracted considerable attention in
recent years (El-Shahawi, Al-Jahdali, Bashammakh, Al-Sibaai, & Nassef, 2013; Ghani &
Mansour, 2012; Matesanz & Souza, 2007; Montagner, Gandin, Marzano, & Longato, 2011;
Özbek, Alyar, Alyar, Şahin, & Karacan, 2013; Özdemir, Akkaya, & Özbek, 2013). Nowadays,
2 2
Cisplatin (cis-[PtCl (NH3) ]) is an important anticancer drug and its activity is related to its
binding to DNA and protein (Icsel et al., 2015). This compound is used in the treatment of solid
tumors (Arnesano, Losacco, & Natile, 2013; Iglesias et al., 2015); however, its use in clinical
cancer settings has been limited by unwanted toxic effects, low water-solubility and acquired
resistance of cancer cells. Therefore, much effort has been put forward into the development of
new platinum, palladium and other metal complex-based anticancer drugs (Montana & Batalla,
2
009; Ott & Gust, 2007; Zalba & Garrido, 2013). Owing to higher liability of palladium versus
platinum analogs, amino acid ligands, which do not dissociate easily in aqueous solution, have
been used to synthesize palladium anticancer complexes (J. Zhang, Li, Wang, Zhang, & Li,
2
010). It has also been suggested that these palladium complexes may be useful for the
treatment of tumors of the gastrointestinal region where cisplatin fails (Puthraya, Srivastava,
Amonkar, Adwankar, & Chitnis, 1985). Recently, the metallic complexes with amino acids such
as ligands have received much attention because they proved to be useful antitumoral drugs
against sarcoma and leukaemia. In addition, the antibacterial agents applied against
Staphylococcus aureus, Escherichia coli and Candida albicans, etc., were used as nutritive
supplies for both human and animals (Stanila et al., 2009).
Generally, DNA is the primary intracellular target of anticancer drugs (Gumus et al., 2009). The
interaction between small molecules and DNA can often manipulate biodistribution, DNA
binding rate and mode. It can also be recognized by DNA-repair mechanisms, causing damage
to cancer cells and blocking the division of cancer cells, and even resulting in death of cancer
cells (Alagesan, Bhuvanesh, & Dharmaraj, 2014). Thus, it was thought worthwhile to study the
interaction of metal-based drugs with DNA for a better understanding of their pharmacological
properties and design new therapeutic agents (Katrahalli, Kalalbandi, & Jaldappagari, 2012).
4

Serum albumin has long been the center of attention of the pharmaceutical industry mostly
because of its ability to bind with various drug molecules and to alter the overall distribution,
metabolism, and efficacy of many drugs which work based on their affinity to serum albumin
(
Jayanthi, Anusuya, Bhuvanesh, Khalil, & Dharmaraj, 2015; Katrahalli, Jaldappagari, &
Kalanur, 2010). Among the investigated proteins, human serum albumin (HSA) is the most
abundant carrier protein in blood stream which has high affinity with a wide variety of ligands
such as metabolites and drugs. The most important physiological functions of this protein are
the storage and transport of great range of endogenous and exogenous compounds such as fatty
acids, hormones, bilirubin, and drugs and the maintenance of osmotic pressure and pH of the
blood plasma (Huang, Kim, & Dass, 2004; Wang et al., 2008). The crystallographic analysis of
HSA revealed that this protein contains a single chain of 585 residues with a molecular mass of
6
6.438 kDa. It constitutes three homologous alpha-helical domains I(1–195), II(196–383) and
III(384–585) that are packed in two separate sub-domains A and B. HSA contains a single Trp
residue at position 214. The principal region of ligand binding on serum albumin is located
between the sub-domains IIA and IIIA. The HSA/metal binding occurs mainly near Trp residue
in sub-domain IIA, which has also been identified as a primary binding site for many drugs
(
Ascenzi & Fasano, 2010; Yousefi et al., 2015).
Several palladium (II) complexes of amino acid chelating ligands have been reported which
interact with DNA and the proteins like HSA and BSA (Divsalar, Saboury, Yousefi, Moosavi-
Movahedi, & Mansoori-Torshizi, 2007; Eslami Moghadam et al., 2016; J. Zhang et al., 2010).
Their results suggested that the presence of different amino acids in these complexes can i)
modulate their solubility ii) improve the antiproliferative properties iii) decrease structural
inducing harmful efforts on the carrier proteins and iv) increase drug concentration in cancer
cells. Keeping these criteria in mind, two new, water soluble, neutral and isomeric palladium (II)
complexes [Pd (Gly) (Leu)](I)and [Pd (Gly) (Ile)](II), were synthesized and characterized.
Their cytotoxicity against human tumor cell line K562 was investigated by MTT assay. The
interaction of these compounds with DNA or HSA was studied by UV–Vis, fluorescence, FT-IR
spectroscopy and gel electrophoresis. On the basis of the spectroscopic data, the binding
q b
parameters (Kapp, L1/2, Ksv, k , K and n), thermodynamic parameters (∆Hº, ∆Sº and ∆Gº) and
binding modes in the interaction of the Pd (II) complexes to CT-DNA and HSA were calculated.
The obtained results may have important applications in drug delivery and drug design
procedures.
2
. Experimental
5

2
.1. Materials and methods
All chemicals including Palladium (II) chloride, glycine, leucine, isoleucine, sodium chloride,
sodium bicarbonate and Tris- HCl buffer ( tris-( hydroxymethyl)methylamine) were purchased
from Merck Chemical Co. and were used as received. Highly polymerized calf thymus
deoxyribonucleic acid sodium salt Type 1 (CT-DNA) and Human serum albumin (HSA, free
fatty acid fraction V, purity > 97%) were also obtained from Sigma–Aldrich.
FT-IR (Fourier transform Infrared) spectra of amino acids as ligands and their corresponding Pd
(II) complexes were recorded on a JASCO-460 plus FT-IR spectrometer in the range of 4000 to
-1
4
00 cm in KBr pellets. The contents of carbon, hydrogen and nitrogen were determined by
1
Herause CHNO- RAPID elemental analyzer. H NMR (proton nuclear magnetic resonance)
spectra of the compounds were recorded on a Brucker DRX- 250 Avance spectrometer at
2
6
50MHz in DMSO-d (deuterated dimethyl sulfoxide) using tetramethylsilane (TMS) as the
internal reference. UV-Vis (Ultraviolet- Visible) spectra of the metal complexes and ligands
were further measured on a Rayligh- 1601 spectrophotometer. Melting points were measured on
a Unimelt capillary melting point apparatus and were reported uncorrected. Conductivity
measurements of the above Pd (II) complexes in deionized water were carried out on a
Systronics conductivity bridge 305, using a conductivity cell of cell constant 1.0. Fluorescence
spectra were recorded using a Cary Eclips spectrometer. Finally, the agarose gel electrophoresis
was performed using gel documentation systems.
2
.2. Synthesis of palladium (II) complexes
.2.1. Preparation of glycinatoleucinatopalladium(II), [Pd (Gly) (Leu)[ (I):
2
PdCl
water. The reaction mixture was stirred for 2h at 60-70°C to yield Na
solution was cooled and filtered. In the other beaker containing about 10mL of water, glycine
75mg, 1mmol) and NaHCO (84mg, 1mmol) were mixed and stirred for 15 minutes to get
sodium salt of glycine and filtered. This solution was dropletly added to the stirred solution of
Na [PdCl [and was heated for about 2h at 50°C to get the yellow solution containing Na[Pd
Gly) (Cl
NaHCO
2
(177mg, 1mmol) and NaCl (117mg, 2mmol), were added to 20mL of double distilled
2
[PdCl [. This brown
4
(
3
2
4
(
2
)] and finally, it was filtered. In another beaker, leucine (131mg, 1mmol) and
3
(84mg, 1mmol) were dissolved in 20mL water and were stirred at 40°C for 10min to
convert the leucine into its sodium salt. Then, this solution was slowly added to the above
solution containing Na[Pd (Gly) (Cl )]. The reaction mixture was stirred at 40°C for 2h and was
2
concentrated to 20mL to give cream colored precipitate of the desired complex. It was then
6

filtered and washed with water, acetone and was dried at 50°C in an oven overnight. Its yield
was 217.7mg (70%). It decomposes at 210- 212ºC. Elemental analysis was also calculated for
C
8
H
16
O
4
N
2
Pd (311): C, 30.86; H, 5.14; N, 9.00. Found: C, 30.87; H, 5.13; N, 9.10.
2
.2.2. Preparation of glycinatoisoleucinatopalladium(II), ]Pd (Gly) (Ile)](II):
This neutral complex was synthesized and isolated following the procedure as given for ]Pd
Gly) (Leu)[ complex except that instead of leucine, isoleucine (131mg, 1mmol) was used. Its
yield was 192.8mg (62%). It decomposes at 203-206ºC. Elemental analysis was also calculated
for C Pd (311): C, 30.86; H, 5.14; N, 9.00. Found: C, 30.85; H, 5.15; N,9.12.
(
8
16 4 2
H O N
2
.3. Cytotoxic Studies
.3.1. Cell Culture
2
The chronic myelogenous leukemia cell line, K562, was obtained from the Cell Bank of Pastor
Institute of Biochemistry and Cell Biology (Iran). The cells were grown in the RPMI medium.
This medium was supplemented with L-glutamine (2mM), Streptomycin and penicillin (5
2
µg/mL) and 10% heat-inactivated fetal calf serum at 37ºC under a 5% CO /95% air atmosphere
(
Divsalar, Razmi, Saboury, Mansouri-Torshizi, & Ahmad, 2015).
2
.3.2. Cell Proliferation Assay
The above Pd (II) complexes inhibit the growth of chronic myelogenous leukemia cell line,
562. This growth inhibition was measured by means of MTT (3- (4, 5-dimethylthiazol)- 2,5-
K
diphenyltetrazolium bromide) assay. The cleavage and conversion of the soluble yellowish MTT
to the insoluble purple formazan by active mitochondrial dehydrogenase of living cells has been
used to develop an assay system which was an alternative to other assays for measurement of
4
cell proliferation. Harvested cells were seeded into 96-well plate (1×10 cell/mL) with varying
concentrations of the sterilized Pd(II) complexes (0-250µM) and were incubated for 24h. Four
hours to the end of incubations, 25µL of MTT solution (5mg/mL in PBS) was added to each
well containing fresh and cultured medium. At the end, the insoluble formazan produced was
dissolved in a solution containing 10% SDS and 50% DMF (left for 2h at 37ºC in dark
conditions) and the optical density (OD) was read against a reagent blank with multi-well
scanning spectrophotometer (ELISA reader, Model Expert 96, AsysHitchech, Austria) at the
wavelength of 570nm. The OD value of the study groups was divided by the OD value of the
untreated control and was presented as the percentage of control (as 100% ) (Divsalar et al.,
7

2
015). The 50% cytotoxic concentration (Cc50) of each compound was determined from the plot
of % cell growth versus [complex] for each sample.
2
.4. DNA/HSA Binding studies
2
.4.1. Electronic absorption spectroscopy
All the experiments involving interaction of the pd(II) complexes with DNA or HSA were
carried out in a Tris-HCl buffer (5mM Tris- HCl, 50mM NaCl and pH 7.2).The stock solutions
-4
of DNA (4mg/mL), HSA (2mg/mL), and each of the synthesized Pd(II) complex (10 M) were
freshly prepared in the Tris buffer. The concentrations of DNA and HSA were determined by
-1
-1
using the extinction coefficients of DNA, 6600M cm at 260nm (Jayanthi et al., 2015) and
-1
-1
HSA, 36500M cm at 280nm (Yousefi et al., 2015). A solution of CT-DNA gave a ratio of
UV- Vis absorption at 260nm and 280nm of about 1.7–1.9, indicating that the CT-DNA was
sufficiently free of protein (Annaraj, Balakrishnan, & Neelakantan, 2016; Raman & Raja, 2007).
Two experiments were performed to investigate the interactions of complexes I and II, with
DNA and HSA using an electronic absorption spectroscopy in the UV-Vis region.
First: a fixed amount of DNA (25µL stock solution +1.975mL Tris-HCl buffer, i.e. 0.18mM)
and/or HSA (100µL stock solution +1.9mL Tris-HCl buffer, i.e. 0.013mM) was titrated
separately by each metal complex (0.000- 0.042mM for DNA and 0.000- 0.040mM for HSA) at
3
00K. After each addition of the complex fraction, the solutions (DNA-complex / HSA-
complex) were incubated for 5 min, and then the absorption spectra were recorded as 200-
50nm for DNA and 230-350nm for HSA. The titration processes were continued until no
3
change was observed in the absorption spectra. The absorption data were analyzed using
Eq.(1)(Abu, Ghithan, Darwish, & Abu-Hadid, 2012).
ꢃ
ꢅꢆ
(1)
ꢀ
ꢁꢀꢂ
ꢀ ꢁꢀ
ꢉꢀ ꢁꢀ ꢊꢋꢌꢍꢎꢏꢐꢑꢒꢓꢌꢔꢐꢍꢕꢖ
ꢇꢈꢈ ꢄ ꢂ
ꢄ
ꢂ
where A
of metal complexes, A
sites on DNA or HSA were occupied by the desired metal complex), and A is the recorded
0
is the initial absorption of DNA or HSA at 260 or 280nm respectively in the absence
∞
is the final absorption of the ligated DNA or HSA (i.e. when all binding
absorption at various concentrations of metal complexes. The double reciprocal of 1/A - A
0
vs.
1
/[metal complex] is linear and the apparent binding constant (Kapp) can be calculated from the
ratio of the intercept to the slope.
8

In the above experiments, the selected spectra were overlaid to see if there is any isosbestic
points and or hypo-, hyper-, hypso- and batho-chromic shifts.
Second: In this experiment, the conformational changes of the native CT-DNA and HSA
induced by the complexes I or II were measured as a function of spectral changes (Mansouri-
Torshizi, Eslami-Moghadam, Divsalar, & Saboury, 2011). Here, a constant parameter, L1/2, i.e.
the concentration of each metal complex at midpoint transition of CT-DNA or HSA from native
to unfolded was determined (Mansouri-Torshizi, Mahboube, Divsalar, & Saboury, 2008).The
sample cell was filed with 1.8mL of 0.17mM DNA solution (1.775mL Tris-buffer + 0.025mL
stock solution of DNA) or 1.8mL of 0.014mM HSA solution (1.7mL Tris-buffer + 0.1mL stock
solution of HSA). However, the reference cell was filled with 1.8mL of Tris-buffer only. Then,
both cells were kept at the constant temperature of 300K or 310K, and 10µL stock solution of
either Pd(II) complex was added to both cells. After a 5-min incubation, the absorption was
recorded at 260nm for DNA, or 280nm (denoted A
) and 640nm (denoted A640) for HSA
m
(
Aminzadeh, Mansouri-Torshizi, & Modarresi-Alam, 2016; Islami-Moghaddam, Mansouri-
Torshizi, Divsalar, & Saboury, 2009). Addition of Pd (II) complex to both cells was continued
until no further change in the absorption readings was observed. As a result, the data obtained
from this experiment are: A
volume of solution in the sample cell after each addition of Pd(II) complex, V
i.e. 1.8mL, and [L] is total concentration of Pd(II) complex in each time of addition). On
plotting a (A -A640) against [L] , two types of sigmoidal plots (ascending or descending) may
m
, A640, [L]
t
and a=V
2
/V
1
(where, a is dilution coefficient, V
2
is total
1
is initial volume,
t
m
t
result. From these plots, the concentration of each metal complex at midpoint of transition, L1/2
could be deduced for both temperatures, 300K and 310K, separately.
,
2
.4.2. Fluorescence spectra
Fluorescence titration is one of the helpful method used for finding the interactions of metal
complexes with the biomacromolecules such as DNA, HSA, BSA,…etc. The relative binding
propensity of the Pd(II) complexes to DNA or HSA was studied by the fluorescence titration
method. In these experiments, the excitation wavelengths of 471nm and 295nm were selected
for DNA and HSA, respectively. The emission spectra were recorded from 525 to 725nm and
from 300 to 500nm for DNA and HSA binding, respectively. Measurements were made using a
1
cm path length fluorescence cuvette. The band slits for both excitation and emission were 5nm.
The maximum fluorescence intensity was used in order to calculate the thermodynamic
parameters. In the interaction studies between the above-mentioned Pd(II) complexes and DNA
or HSA, the following procedures were carried out:
9

(i) DNA interaction studies:
The fluorescence intensity of the solutions of calf thymus DNA was checked and their emission
intensities were found to be very small and negligible. Thus, ethidium bromide (EB) was used to
probe the course of the interaction. In this experiment, in a quartz cell containing 1ml Tris-HCl
buffer of pH=7, 25µL of DNA (stock solution) and 68µL aqueous EB (0.023mg/mL) were
added and the volume was adjusted to 2mL by the same buffer. In these solution mixtures, the
fluorescence of EB enhanced about 50 fold at ~ 600nm as it intercalates between the base pairs
of DNA double helix (Saeidifar et al., 2014; Shahraki et al., 2014). Next, the effects of each
palladium (II) complex on the emission intensity of interacted DNA-EB were studied by adding
increasing concentrations of each metal complex (10-100µL of the stock solution).After each
addition step and equilibration for 5min, an emission spectrum was recorded between 525 to
725nm. At the end of the experiment, all spectra were overlaid to observe the changes in their
emission intensities. This experiment was performed separately for complexes I and II at three
different temperatures of 293, 300, and 310K. To analyze the interaction of DNA and
synthesized compounds, the quenching constant, Ksv, was determined by using Stern–Volmer
equation (2) (Khosravi & Mansouri-Torshizi, 2017; Niroomand, Khorasani-Motlagh,
Noroozifar, & Moodi, 2012).
ꢗꢂ
ꢃꢘꢅꢙ ꢋꢜꢖꢃꢘꢅꢝ ꢟ ꢋꢜꢖ
(2)
ꢚꢛ
ꢞ ꢠ
ꢗ
0
where, F and F are the fluorescence intensities of DNA-EB in the absence and presence of
various concentrations of quencher, i.e. metal complex, [Q] is the total concentration of each
Pd(II) complexes, τ is the average fluorescence lifetime of fluorophore (in this case, EB which
is 10 ) in the absence of quencher and k is the apparent biomolecular quenching constant
which equals to K /τ . Therefore, the slope of the plot F /F versus [Q] gives the value of K
SV
0
-12
q
SV
0
0
b
for each sample. Also, the binding constant (K ) and the number of binding sites (n) on DNA
were calculated according to Equation (3) (Icsel et al., 2015).
ꢗꢂꢁꢗ
ꢡꢢꢣꢑꢉ
ꢊꢃꢡꢢꢣꢙ ꢅꢥꢡꢢꢣꢋꢜꢖ
(3)
ꢤ
ꢗ
Here, from the plot of log (F
Knowing K
equation (Khorasani-Motlagh, Noroozifar, & Mirkazehi-Rigi, 2010).
0
-F/F) versus log [Q], the values of n and K
b
could be calculated.
b
, thermodynamic parameters can be evaluated using equation (4) and Van’t Hoff
1
0

ꢨꢩꢪ
ꢫꢬ
ꢨꢚ
ꢪ
ꢡꢦꢙ ꢃꢧ
ꢅ ꢫ
(4)
ꢤ
b
where K is the binding constant at the corresponding temperature, R represents the gas constant,
T is the absolute temperature (K), and ΔHº and ΔSº are enthalpy and entropy changes,
respectively. These two later parameters were obtained separately at 293, 300 and 310K by
b
plotting lnK versus 1/T. In this plot, ΔHº and ΔSº were determined from the slope (-ΔHº/R) and
the intercept on y axis (ΔSº/R), respectively.
Finally, the free energy changes (ΔGº) in the interaction between each Pd(II) complex and
DNA-EB can be calculated according to Equation (5) (Khorasani-Motlagh et al., 2010).
ꢨꢭ ꢃꢨꢮ ꢧꢯꢨꢰ ꢃꢧꢱꢯꢡꢦꢙꢤ
(5)
(ii) HSA interaction studies:
The procedure followed to study the interaction between HSA and each metal complex was the
same as the above procedure for DNA interaction. The only difference was that there is no need
of using fluorophore (EB) as in the case of DNA interaction studies. That is because HSA is
-8
0
intrinsically fluorescence active (τ = 10 ). In fact, the autofluorescence activity of HSA comes
from the tryptophan, tyrosine and phenylalanine residues. The intrinsic fluorescence of HSA is
almost contributed to tryptophan alone, because phenylalanine has a very low quantum yield
and the fluorescence of tyrosine is almost totally quenched if it is ionized or located near an
amino group, a carboxyl group or a tryptophan (Abu et al., 2012). Moreover, the fluorescence
activity of HSA is very sensitive to its microenvironment, and variety of molecular interactions
can diminish the fluorescence intensity of this protein.
2
.4.3. CT-DNA gel electrophoresis
The interactions between [Pd(Gly)(Leu)](I) and [Pd(Gly)(Ile)](II) complexes with CT-DNA
were monitored using agarose gel electrophoresis on a horizontal gel apparatus. The gel was
stained with EB and was photographed under UV light. In this experiment, first, a reaction
mixture containing 10µL of CT-DNA (stock solution) was placed in a clean and sterile Ependorf
tube and in the other 4 tubes, same amounts of CT-DNA and diverse concentrations of each
Pd(II) complexes I or II (5, 10, 15 and 20µL from stock solution) were added, so, the volumes
of all tubes were then increased to 40µL by Tris-HCl buffer. All tubes were mixed slowly and
incubated for 1h at 310K. The reactions in the tubes were terminated by the addition of 5µL
1
1

loading buffer containing 0.25% blue bromophenol and of these mixtures, only 4µLwas finally
loaded into the gel (Mohamed, Shoukry, & Ali, 2012). The product resulting from the
interactions of the complexes I and II with CT-DNA electrophoresis on the agarose gel was
prepared in 1% (w/v) of TAE buffer ( 40mMTris-acetate, 1mM EDTA)(pH=8.28) at constant
voltage of 50V for 2h, until the blue bromophenol had travelled through 75% of the gel. After
electrophoresis, the gel was stained for 30min by soaking in an aqueous ethidium bromide
(0.5µg/mL). The gel was then destained for 10min by being kept in sterile distilled water. The
interacted CT-DNA bands were visualized by viewing the fluorescence of intercalated EB under
UV light, and then, were photographed. The binding ability was observed and assessed for the
intensity and mobility of the bands (Shoukry & Mohamed, 2012).
2
.4.4. Calculations of energy transfer
The efficiency of energy transfer (E) in biochemistry can be used to evaluate the distance
between the metal complex and the fluorophores in the protein .According to Foster Resonance
Energy Transfer (FRET), the efficiency of energy transfer (E) could be calculated by Eq.(6)
(
Matei, Ionescu, & Hillebrand, 2011):
ꢗ
ꢳ
ꢳ
ꢳ
ꢲꢃꢱ ꢵꢉꢱ ꢅꢑꢴ ꢊꢃꢘꢧ ꢑꢑꢑꢑꢑꢑꢑꢑꢑꢑꢑꢑꢑꢑꢑꢑ
(6)
ꢠ
ꢠ
ꢠ
ꢗ
ꢂ
where F and F
0
are the fluorescence intensities of HSA in the presence and absence of metal
complex, r is the distance between acceptor (bound metal complex) and donor (tryptophan
residue) and R
0
is the critical distance when the transfer efficiency is 50%. The value of R
0
was
calculated using the following equation (Matei et al., 2011):
ꢳ
ꢁꢺꢻ
ꢺ
ꢁꢽ
ꢱ ꢃꢶꢷꢶꢸꢘꢹ ꢙ ꢼ ꢾꢿꢑꢑ
(7)
ꢠ
2
In the above equation, K is the spatial orientation factor describing the relative orientation in
space of the transition dipoles of the donor and acceptor, N is the refractive index of the
medium, ɸ is the fluorescence quantum yield of the donor and J is the overlap integral of the
fluorescence emission spectrum of the donor with the absorption spectrum of the acceptor which
could be calculated by Eq. (8) (Matei et al., 2011):
ꢽ
ꢿ
ꢃꢑꢉꣀꣁꢉꣂꢊꣃꢉꣂꢊꣂ ꢨꣂꢊꢵꢉꣀꣁꢉꣂꢊꢨꣂꢊꢑ
(8)
1
2

where F(λ) is the corrected fluorescence intensity of the donor at wavelength λ and ԑλ is the UV
molar absorption coefficient of the acceptor at the wavelength λ. Under the experimental
2
condition, the value of K = 2/3, N = 1.336 and ɸ= 0.188(Matei et al., 2011).
2
.4.5. FT-IR Spectroscopy
To further understand the structural alterations of HSA induced by binding of the isomeric
Pd(II) complexes, FT-IR spectra of HSA alone, and complexes I- or II–HSA systems in the
-1
region of 1800-1400cm in KBr pellets were recorded at room temperature. Spectra of buffer
solution were collected at the same condition. Then, the absorbance of buffer solution was
subtracted from the spectra of sample solution. Finally, the difference spectra of HSA were
obtained by subtracting the spectrum of each metal complexes I- or II-free form from that of I-
or II–HSA bound forms with the same concentrations (Yue, Chen, Qin, & Yao, 2009).
3
. Results and discussion
Two new, neutral and sufficiently water soluble complexes, [Pd(Gly)(Leu)](I) and
Pd(Gly)(Ile)](II), (where Gly is glycine, Leu is leucine, and Ile is isoleucine), were prepared by
interaction of Na[Pd(Gly)(Cl )] with an appropriate amount of leucine and isolucine sodium
[
2
salts. As shown in Fig.1, since leucine and isoleucine are the only isomeric pair of amino acids
among the twenty essential amino acids present in the structure of proteins, resulting Pd(II)
complexes can show isomeric from this source. This is the delicacy of the present research work
which has been looked at to see if there is much difference in their behaviors. The complexes
were isolated as yellow solids and our attempts to make single crystal suitable for X-ray
crystallography were unsuccessful.
-4
2
-
Molar conductance of 10 M solution of complexes I and II in water were 8 and 9cm .ohm
1
-1
.
mol , respectively, indicating that these Pd (II) complexes behave as non-electrolytes
(
Angelici, 1969).
Electronic absorption spectra of both complexes show two bands at 206nm and 320nm for
complex I and 202nm and 318nm for complex II assigned respectively to π→ π* and n→
π*transition of C=O groups of amino acids(Stanila et al., 2009). However, the absorption peak
of both complexes at 206nm and 202nm showed a red shift of ~14nm from more polar water to
less polar methanol , indicating that this peak is not only due to π→ π* transition of amino acid
ligands but also may be due to metal-ligand charge transfer overlaying each other (Cotton,
Wilkinson, Bochmann, & Murillo, 1999).
1
3

Information about the metal ions coordination was obtained by comparing the FT-IR
frequencies of free amino acids with their corresponding Pd(II) complexes. In the spectrum of
the leucine, isoleucine and glycine the ν (N–H) stretching vibration appears at 3055, 2958 and
-1
-1
2
900cm respectively and are shifted in their corresponding Pd (II) complexes to 3286cm in I
-1
and 3244cm in II proving the involvement of the -NH
2
groups in the complex formation
-1
(
Stanila et al., 2009). The absorption band at 1608cm was attributed to the ν (C=O) stretching
-1
-1
vibration in the spectra of the free amino acids. This band appearing at 1635cm and 1654cm
in the spectra of I and II shows that -COO group is coordinated to the metal center (Stanila et
al., 2009).
1
The H NMR spectra of I and II were recorded in DMSO-d
6
using TMS as an internal reference.
1
The proposed structures and NMR numbering schemes are given in Fig. 1. In the H NMR
spectrum of I, three multiple signals appeared at 0.86, 1.51, and 3.08ppm are assigned to
H
a
(6H), H
leucine (4H, Hffʹ) resonated as four broad signals with low intensity in the range of 4.03 to
.25ppm. The large upfield shift of 0.62 and 0.55ppm of H and H protons as compared with H
and H protons of free amino acids supports the binding of N of the -NH groups and O of the -
b c d e 2
; H (3H) and H ; H (3H), respectively. The protons of NH groups of glycine and
5
d
e
d
e
2
COO groups of glycine and leucine to Palladium(II) (Puthraya et al., 1985).
1
The H NMR spectrum of II exhibits five signals at 0.87, 1.01, 1.42, 1.78, and 3.07ppm which
are assigned to H
of NH groups of glycine and isoleucine (4H, Hffʹ) resonate as four broad signals with low
intensity in the range of 3.67 to 4.91ppm. The large upfield shift of H (0.92ppm) and H
0.55ppm) protons supports the binding of N of the -NH group and O of the -COO group of
these amino acids to Pd(II) (Puthraya et al., 1985).
a b g c d e
( 3H ), H (2H), H (3H), H (1H) and H ; H (3H), respectively. The protons
2
d
e
(
2
On the basis of the above characteristic data of elemental analysis, conductivity measurements,
1
UV-Vis, FT-IR and H NMR spectra, the proposed structural formula of the isomeric Pd (II)
complexes are given in Fig.1.
Figure.1.
In addition to the above characterization data, the results of following experiments are discussed
here.
i) Cytotoxic studies. ii) Apparent binding constant (Kapp), in the interaction of each Pd(II)
complex with CT- DNA or HSA and ability of each metal complex to unfolding CT- DNA or
HSA ( L1/2) by using UV- Vis technique. iii) Ethidium bromide displacement, Stern Volmer
1
4

b
quenching constant (Ksv), binding constant (K ), number of binding sites (n), thermodynamic
parameters (∆Hº, ∆Sº and ∆Gº) and binding mode by using fluorescence technique. iv) Gel
electrophoresis. V) Energy transfer by using UV-Vis and fluorescence techniques and VI)
alteration of HSA secondary structure by using FT-IR technique.
3
.1. Cytotoxicity tests of the Pd (II) amino acid complexes
The Invitro effect of the prepared complexes on human leukemia cell lines was evaluated by
MTT assay. In these studies, different concentrations of the isomeric Pd (II) complexes I or II
from 0 to 100μM of stock solutions were used in the culture medium of the cell line (Fig.2). The
5
0% cytotoxic concentration (Cc50) of complexes I and II after 24h incubation time are 40μM
and 50μM, respectively. As shown in Fig.2, cells growing after 24h was significantly reduced in
the presence of various concentrations of the Pd (II) complexes and the Pd (II) complexes
produce a dose-response suppression on growing of K562 leukemia cell line. Furthermore, the
Cc50 value of cis-diamminedichloroplatinum (II), the well-known anti-cancer drug was
measured under the above same experimental conditions. This value (154μM) is much higher
than the Cc50 values of the above two Pd (II) complexes. It can be noticed that the presence of
amino acids in the structure of these synthesized complexes as well as their higher water
solubility (~ 60mg/mL) as compared to cisplatin (~2.53mg/mL)(Yi & Bae, 2011) might
influence the anti-tumor activities of these complexes on K562 cell line.
Figure.2.
3
.2. The UV-Vis absorption studies in the interaction between isomeric Pd (II) complexes with
CT-DNA and HSA
Application of electronic absorption spectroscopy is one of the most useful techniques for
probing the interactions of metal complexes with DNA and HSA, as any interaction with the
macromolecule will cause electronic perturbations in them (Abdi, Hadadzadeh, Salimi,
Simpson, & Khalaji, 2012). A fixed amount of CT-DNA (0.18mM) or HSA (0.013mM) was
titrated by increasing the concentrations of the complexes I and II (0.000-0.042mM for DNA
and 0.000-0.040mM for HSA) at 300K. The decrease in the absorption maxima of DNA
(260nm) and HSA (280nm) without any obvious change in these peak positions are shown in
Fig.3. These observations indicate the interaction between both Pd (II) complexes with CT-
DNA and HSA molecules. There was no change in the absorption spectra if more metal
complexes were injected to the systems, suggesting saturation of CT-DNA and HSA binding
1
5

sites; thus, no further interaction occurred. However, in the titration of CT-DNA and HSA with
different amounts of complex I, two isosbestic points were observed respectively at 240 and
3
00nm for DNA and at 260 and 295nm for HSA systems (see Fig. 3, a and c). The presence of
these isosbestic points suggest that: (i) There is an equilibrium between DNA and or HSA
bound and free forms of metal complex I and these two forms have same absorption at the
isosbestic points (Huheey, Keiter, & Keiter, 1983) (ii) A good indicator of there being only two
absorbing species in solutions, and the final spectrum corresponding to that of interacted CT-
DNA-Pd or HSA-Pd complex, and (iii) Two isosbestic points indicate that the reaction proceeds
without any side reactions (such as hydrolysis). Utilizing Eq. (1), the overall binding constant of
0
complexes I and II to CT-DNA and HSA were estimated. In the plot of 1/ (A - A ) vs. 1/[metal
complex], the apparent binding constant (Kapp) can be obtained from the ratio of y intercept to
slope (see Fig.3 insert). The values are given in Table 1.
Table 1:
These Kapp in the interaction of these complexes with CT-DNA are lower than those reported for
classical intercalator such as Ethidium bromide and [Ru(phen)DPPZ] whose binding constant
6
7
has been found to be in the order of 10 -10 M(Xu, Chen, Xi, Liu, & Zeng, 2008). These results
suggest the binding modes of the complexes to CT-DNA should be non-intercalation and differ
from that of EB and [Ru(phen)DPPZ]. Moreover, binding with DNA via intercalation usually
results in hypochromism and bathochromism of the absorption. This is due to a consequence of
strong π-π* staking interaction between planar aromatic moiety of the complexes and DNA base
pairs (Chaveerach, Meenongwa, Trongpanich, Soikum, & Chaveerach, 2010). These results are
in agreement with the proposed structure of the complexes I and II having no planar aromatic
moieties to act as intercalator.
Figure.3.
3
.3. Determination of concentrations of metal complexes in midpoint transition, L1/2.
The profiles of the conformational changes in the structure of native CT-DNA or HSA which
were induced by complexes I and II are shown in Fig. 4. These conformational changes
occurred in such a manner that the absorbing groups of DNA (purine and pyrimidine bases) and
HSA (mainly tryptophan residues) get hidden from UV light, resulting in a decrease in the
intensity of the absorption peak with an increase in the concentration of Pd(II) complexes at the
same wavelength. Thus, the native and interacted CT-DNA or the unloaded and loaded HSA by
1
6

any of synthesized Pd(II) complexes have different conformations, so the transition between
these two conformations is discussed under the above title. The concentration of both Pd(II)
complexes in the midpoint transition, L1/2, was needed for transition of the native to the
interacted CT-DNA or the unloaded to loaded HSA at 300 and 310K is quite low (see Fig. 4 and
Table 1). This is one of the most important observations made in this research work, since a
lower side effect may be observed in the case of compounds having activity at lower
concentration (Edwards & Aronson, 2000). Looking at L1/2 values given in Table 1, the
following points could be deduced: (i) L1/2 values of CT-DNA are lower than those of HSA
indicating that the complexes show more interaction affinities towards DNA than with HSA.
This is expected because there may be more available binding sites on DNA as compared with
those of HSA. (ii) L1/2 values of CT-DNA and HSA at 300K are lower than those of 310K. The
results indicated that the reaction between the Pd(II) complexes with CT-DNA and HSA may be
exothermic and (iii) In the interaction between complex I with CT-DNA, L1/2 values at
temperatures of 300 and 310K, are lower than those for complex II suggesting that the
interaction affinity of the complex I is more than that of complex II. However, in the interaction
between the complex II with HSA, L1/2 values in both temperatures of 300 and 310K, are lower
than those for the complex I suggesting an interaction affinity of the complex II with HSA is
more than that of the complex I. Similar results have also been reported for the complex [Pd
(
Hex-dtc) (phen)]NO
3
(Mansouri-Torshizi et al., 2011).
Figure.4.
3
.4. Fluorescence experiments with calf thymus DNA
In order to gain further support for the interaction mode between the metal complexes and DNA,
we have also performed a fluorescence titration experiment(Abdi et al., 2012). It is well known
that small molecules can bind to DNA through four major modes of interactions: covalent-,
intercalation- , surface-, and groove-binding (Khorasani-Motlagh et al., 2010). In the case of
covalent binding, there should be a labile ligand (generally monodentate) coordinated to the
central metal ion of the complex to be substituted by a nitrogen base of DNA such as guanine
N7 which is a well-known binding type of cisplatin (Bobek et al., 2006). This type of binding
may not be possible in our cases, since both ligands around Pd (II) center are coordinated as
bidentate chelate and cannot be easily replaced (see Fig.1). Intercalation of a ligand between
DNA base pairs causes DNA to be lengthened and bent, while highly charged cations, such as
cobalt(III) and polyamines induce condensation through charge neutralization of backbone
1
7

phosphate groups (Khorasani-Motlagh et al., 2010). Considering the structures of two new
isomeric metal complexes )see Fig.1), intercalation of these compounds between base pairs of
DNA may not be in favor, since there are no aromatic and planar moieties present in the
structures of the Pd(II) complexes. The surface binding which is mainly consisted of
electrostatic attraction and hydrogen bonding is proposed as the primary factors for the sequence
specific groove binding from the theoretical analysis. As shown in Fig.1, both complexes
reported here are neutral, so the possibility of electrostatic binding between these complexes and
CT-DNA is nil. Therefore, we expect groove binding through other forces such as H-binding
and Van der Waals forces being the major forces acting between complexes I or II with CT-
DNA. This type of interaction is further supported by the following experiments.
3
.4.1. Competitive DNA-binding studies with EB
To further investigate the interaction between the synthesized metal complexes and DNA, a
competitive binding assay was carried out between Ethidium bromide and each Pd (II) complex
with DNA. EB is a known DNA intercalator which inserts itself between the base pairs in the
double helix and forms soluble complexes with nucleic acids. The fluorescence intensity of EB
is very weak in aqueous solution, but its intensity increases ~50-fold in the presence of DNA
(
Saeidifar et al., 2014; Shahraki et al., 2014). When a metal complex which could bind to DNA
more strongly than EB was added to a solution containing the EB–DNA system, the
fluorescence quenching of EB–DNA is observed. The competitive binding studies involve an
addition of Pd(II) complexes to DNA incubated with EB ([DNA]/[EB] = 30) and then
measuring the emission intensities of the EB– DNA system. As shown in Fig. 6, the emission
intensity of DNA–EB at 600nm decreased with addition of different amounts of each Pd (II)
complex to be interacted DNA-EB. The observed quenching of the DNA–EB fluorescence
intensity suggests that these complexes can displace EB from the DNA–EB system (Anjomshoa,
Fatemi, Torkzadeh-Mahani, & Hadadzadeh, 2014). Also, the experiment revealed that addition
of the complexes I or II to DNA bound EB does not cause releasing EB molecules, while the
emission intensity decreases steadily. In case of EB release, a weak intensity spectrum due to
free EB near the base line should appear which has not been observed in our cases (see Fig. 5).
This implies that the two probes, the complexes and EB, probably bind independently with
DNA and at the same time, binding of each complexes I or II affects the binding of the EB by
slight slip out of EB from hydrophobic to hydrophilic regions diminuendo in emission
intensities. Thus, this experiment supports the above groove binding expectation(Khorasani-
Motlagh et al., 2010).
1
8

Figure. 5.
3
.4.2. Stern-Volmer quenching constants
Titration of CT-DNA-EB with various amounts of complexes I or II was quantified by
measuring the fluorescence quenching of the CT-DNA-EB solution. In fact, two quenching
processes are known: static and dynamic. Dynamic quenching or collisional quenching requires
the contact between the excited lumophore and the quenching species, the quencher. The rate of
quenching is diffusion controlled and depends on temperature and viscosity of the solution. The
quencher concentration must be high enough, so the probability of collision between the analyte
and quencher is significant during the lifetime of the excited species (Kashanian, Khodaei, &
Pakravan, 2010). The other form of quenching is static quenching in which the quencher and the
fluorophore in the ground state form a stable complex. Dynamic and static quenching can also
be discerned by their differing dependence on temperature. Dynamic quenching depends upon
diffusion. Since higher temperatures lead to larger diffusion coefficients, the Ksv can be
increased by raising the temperature. In contrast, this increased temperature is likely the result of
decreased complex stability, and, thus, lower static quenching constant is resulted (Baguley &
Le Bret, 1984). In order to clarify the quenching mechanism of CT-DNA-EB by complexes I
and II, the Stern-Volmer (Equation (2)) is used to analyze the fluorescence quenching data. This
equation has been explained in the experimental section. The Ksv values for the above Pd(II)
0
complexes are obtained from the slope of the plot F /F versus [Q] at three temperatures.
According to the classified Stern-Volmer equation, the quenching plots (Fig.6) illustrate that the
quenching of EB bound to DNA by the complexes are in good agreement with the linear Stern-
Volmer equation (Derakhshankhah et al., 2012). Finally, the Ksv and k
were obtained. The results are shown in Figure 6 and Table 2.
q
at different temperatures
Table 2:
In Table.2, the results show that the values of Ksv and k
q
decrease with increasing of the
10
-1 -1
q
temperature and the values of k is greater than 2.0×10 Lmol S , which indicates that the
probable quenching mechanism of DNA-EB solution is a static quenching (Hussein, 2011;
Shaikh, Seetharamappa, Kandagal, & Manjunatha, 2007). Similar results have also been
reported by A. Heydari, et. al. for [Pd(acac)(Ala)] complex(Heydari & Mansouri-Torshizi,
2
016).
Figure.6.
1
9

3
b
.4.3. Binding constant (K ) and binding stoichiometry (n)
Fluorescence titration data were used to determine the binding constant (K ) and the number of
b
binding sites (or the binding stoichiometry) (n) for the complex formation of CT-DNA-EB with
complexes I or II (Kashanian et al., 2010). Based on Equation (2), the values of K
determined from the intercept and slop of (log[(F -F)/F] versus log [Q], respectively (Fig.7).
These corresponding calculated results are listed in Table 2. Data on Table 2 show that the
values of K decrease with increasing of temperature, indicating that the stability of DNA-EB
b
and n were
0
b
complex reduces as the temperature rises. The number of binding sites n is approximately equal
to unity, indicating that there is one independent class of binding sites on DNA for each Pd(II)
complex. Similar observations were made for the reported metal complex (Ahmadi, Alizadeh,
Shahabadi, & Rahimi-Nasrabadi, 2011; Mukherjee et al., 2013). However, the values of n and
K
b
which decrease with rises in temperature demonstrate that the complex formation between
CT-DNA-EB and Pd(II) complexes can be regarded to be an exothermic procedure. Finally, the
values of K had a trend similar to the Kapp values (Table1) for title complexes and values of K
represent lower binding affinity of complex II to DNA than complex I. This is a noticeable
point which might be due to the difference between leucine side chain (-CH -CH(CH ) and
) which also plays an important role in the above
b
b
2
3 2
)
3 2 3
isoleucine side chain (-CH(CH )-CH -CH
interactions.
Figure. 7.
3
.4.4. Thermodynamic studies
To have a better understanding of thermodynamic of the complication reaction between
isomeric complexes I or II and DNA-EB, contributions of enthalpy and entropy should be
determined in the reaction. Thermodynamic parameters describing the binding reactions can be
divided into three contributions. The first contributions are due to the hydrogen bonding and the
hydrophobic interactions between the Pd(II) complexes and CT-DNA-EB-binding sites. The
second contribution arises from the conformational changes in either the nucleic acid or the
complexes I or II upon binding. The third contributions are provided by the coupled processes
such as ion release, proton transfer, or changes in the hydration water. Evaluation of the
formation constant for the each Pd(II) compound- DNA-EB complex at three different
temperatures (293, 300 and 310K) allows thermodynamic parameters of I- or II–DNA
formation to be determined via Van’t Hoff equation (Eq. 4). By plotting lnK
b
versus 1/T (Fig.
8
), ∆Hº and ∆Sº were determined (Kashanian et al., 2010). Knowing these two values, ∆Gº was
2
0

calculated from the following standard equation (Eq. 5).Thus, according to Eqs. (4) and (5), the
obtained values of ΔHº, ΔSº and ΔGº and the results are shown in Table 3.
Table 3:
Generally, the intercalation interactions are driven by a large favorable enthalpy reduction and
an unfavorable entropy decrease, but the groove binding reactions are mostly driven
entropically. The electrostatic interactions exhibit small or zero enthalpy and positive entropy
changes, but hydrogen bonding and Van der Waals interactions are usually along with negative
∆Hº and ∆Sº. However, the hydrophobic interactions are characterized by positive entropy and
enthalpy changes (Niroomand et al., 2012). Considering the thermodynamic parameters given in
Table 3, the binding forces are spontaneous because the Gibbs free energy change value is
negative. It is also demonstrated that both enthalpy and entropy changes are negative,
suggesting that the Pd(II) complexes were hold in DNA grooves via hydrogen and Van der
Waals binding. It could also be explained that, owing to the binding of complexes to DNA,
some conformational changes may induce the results to the partial destacking of some base
pairs. It was showed by the decrease in fluorescence emission intensity of DNA-EB upon
addition of the Pd (II) complexes. These results are quite in agreement with the proposed
structures of the Pd (II) complexes (Fig.1). As shown in Figure 1, the carbonyl and -NH
of amino acids coordinated to Pd (II) center are susceptible for H-binding and –CH -CH(CH
group of leucine and –CH(CH )-CH -CH group of isoleucine can interact via Van der Waals
2
groups
2
3 2
)
3
2
3
forces in groove binding. Moreover, the negative values of ∆Hº indicate that the above
interaction proceeds exothermically.
Figure. 8.
3
.5. Fluorescence experiments with Human Serum Albumin (HSA)
In order to determine the quenching mechanism of the interaction between HSA and the
synthesized isomeric Pd(II) complexes, the fluorescence experiments were carried out at three
different temperatures (293, 300 and 310K). The fluorescence emission spectra of HSA in the
absence and presence of different concentrations of the Pd (II) complexes at 293K are shown in
Figure 9.
Figure. 9.
2
1

As shown in this figure, the HSA fluorescence intensities were gradually decreased by
increasing the concentration of each Pd(II) complex, indicating that the Trp residue was
transferred into a more hydrophobic environment during the interaction. The similar emission
profiles were observed as the experiments were repeated at 300 and 310K (Yousefi et al., 2015).
In later parts of these studies, it was observed that the number of binding sites for each metal
complex per HSA molecule equals unity, i.e., there is only one binding site for each Pd(II)
complex on HSA and once it gets occupied, no further change was observed in the emission
spectrum, suggesting that the interaction between each neutral Pd(II) complex and HSA
includes the occupation of one binding site. Probably, there are no more interactions with other
parts of HSA molecule causing structural damage to bring about possible side effects in the
physiological systems (Mansoori-Torshizi, Islami-Moghaddam, & Saboury, 2003). This is in
fact the normal function of the protein HSA in the blood stream that should transfer the drug
agents without damaging itself.
For better quantitative understanding of the magnitude of the complexes to quench the emission
0
intensity of HSA (see Fig. 9), Stern-Volmer Eq.(2) can be used. A plot of F /F against the
concentration of complexes I and or II results in liner plots (Fig. 10) and the Ksv values are
obtained from the slopes. The observed Ksv values indicated good HSA binding affinities of the
two complexes, but the complex II exhibited a higher protein binding ability (Table 4).
Table 4:
1
2
-1 -1
12 -1 -1
q
The k values for complexes I and II are 0.32×10 M S and 0.42×10 M S at 300K,
10
-1 -
respectively. From the other side, k
q
of various quenchers with biopolymers is 2.0×10 Lmol S
1
(
Lakowicz & Weber, 1973). Thus, the calculated k in the protein quenching procedure is
q
greater than the maximum scatter collision-quenching constant of biopolymers. This indicated
that a static quenching mechanism is operating (Anbazhagan & Renganathan, 2008; Banerjee,
Ghosh, Sarkar, & Bhattacharya, 2011). Moreover, the curves F
support the static quenching, too.
0
/F versus [Q] which are linear
Figure. 10.
The binding constant, K
n, on HSA available for the complexes I and II were calculated using Eq. (3) (Table 4). The K
values were determined from the slope of plot log (F -F/F) versus log [Q] (Fig. 11). The results
are consistent with greater binding affinity of complex II as compared to that of complex I.
b
, of the quenchers (I and II) with HSA and the number of binding sites,
b
0
2
2

Similar results were obtained for the reported metal complexes (Jin et al., 2015; L.-W. Zhang,
Wang, & Zhang, 2007). The same observations were made in the UV-Vis absorption
experiments (Kapp value of II is greater than that of I). The values of n for both complexes at the
experimental temperatures approximately equal to 1, indicating that there is just a single binding
site on HSA for each Pd (II) complex. As the complexes are natural, they most likely bind to the
hydrophobic pocket in the sub-domain IIA of HSA. The above observations have been made by
A. Heydariet. al.(Heydari & Mansouri-Torshizi, 2016).
Figure. 11.
By plotting the binding constants, K
b
, from Table 4, versus temperatures and using Eqs. (4) and
(5), the thermodynamic parameters were obtained (Fig. 12). The calculated enthalpy, free
energy, and entropy changes are summarized in Table 5. In this study, the negative values of
ΔGº indicated that the interaction of I and II with HSA are spontaneous. On the other hand, the
negative values of ΔHº and ΔSº suggested that hydrogen bond and Van der Waals interactions
are involved in the binding of these two Pd(II) complexes with HSA. Therefore, these two
forces may play a main role in holding Pd(II) complexes in the hydrophobic pocket located in
the sub- domain IIA of HSA molecule (Bi et al., 2005).
Table 5:
Figure. 12.
3
.6. Interaction of CT-DNA with complexes I and II by DNA mobility shift experiments in
agarose gel electrophoresis
Gel electrophoresis is a technique that works on the migration of DNA in the agarose gel under
the influence of electric potential as a function of their mass, charge and shape (Budagumpi,
Kulkarni, Kurdekar, Sathisha, & Revankar, 2010). Here, several gel electrophoresis studies were
done to unravel the effects of the new synthesized compounds I and II on CT-DNA. In these
experiments, changes in electrophoretic mobility of CT-DNA in absence and presence of metal
complexes are usually taken as the evidence of metal complex- DNA interaction. Retardation of
the electrophoretic mobility of a DNA band could be attributed to the binding of certain
molecules of the compound able to increase its molecular weight, while precession of the
electrophoretic mobility could be attributed to the degradation ( or damage) of the initial DNA
substrate, mirrored to a decrease in its molecular weight. DNA molecules with molecular
2
3

weights lower than those of the initial DNA molecule derived from DNA breakage effect
migrate faster, while DNA molecules with higher molecular weight delay more than the
mobility of the initial DNA (Protogeraki et al., 2014). In other words, the larger the retardation
of DNA, the greater the DNA unwinding (or interaction) produced by the given compound(Fox,
1
997). However, depending on the type of DNA, the interaction of metal complex with DNA
may decrease or increase the mobility. For instance, binding of cisplatin to plasmid DNA results
in a decrease in mobility of supercoildplasmid DNA (Form I) and an increase in mobility of the
open circular plasmid DNA (Form II). The ability of complexes I and II to interact with double
stranded CT-DNA were investigated using an electrophoretic analysis through the examination
of the effect of different concentrations of new Pd(II) complexes I and II (1.25, 2.50, 3.75 and
5
.00mM) on the DNA. As shown in Fig. 13, with an increase in the complex concentration, the
intensity of CT-DNA band decreased as compared to the band of free CT-DNA. This decrease
in the intensity of the bands is believed to be due to the quenching fluorescence of CT-DNA-
intercalated Ethidium bromide by increasing concentrations of Pd(II) complexes. These
observations are in agreement with the observed static quenching of DNA-EB by these two
complexes upon interaction via the groove binding. It has been reported that such a groove
binding may bring about conformational changes resulting in partial slip of intercalated EB from
hydrophobic to hydrophilic moieties and a decrease in fluorescence intensity (Khorasani-
Motlagh et al., 2010). Moreover, a small up-shift in the mobility of the bands was recorded for
both complexes I and II after binding to CT-DNA as compared to free CT-DNA. This delay in
the electrophoretic mobility of the bands was barely visible at the bottom of the bands, leading
to the formation of V letter by connecting bottom tip portions of all bands. The delay in the
electrophoretic mobility of the bands may be explained by the bases of binding of the Pd (II)
complexes to CT-DNA increases its molecular mass. It could also be attributed to changes in
conformation of CT-DNA due to its binding with metal complexes (Fox, 1997).
Figure. 13.
The above observations converge towards the conclusion that both complexes I and II bind to
CT-DNA and this result is in accordance and reinforces the view expressed by the spectroscopic
experiments, which refer to the potential modes of binding with CT-DNA.
3
.7. Energy transfer between complexes I and II with HSA
The energy transfer efficiency and distance between the synthesized Pd (II) complexes and HSA
can be estimated using Forster’s Resonance Energy Transfer (FRET) theory. FRET is a distance
2
4

dependent interaction in which excitation energy is transferred nonradiatively from a donor
Trp214 residue of HSA) to an acceptor molecule (here complexes I or II), with the prerequisite
(
that a non-zero overlap exists between the emission spectrum of the donor and the absorption
spectrum of the acceptor, which is fulfilled for the HSA–I /-II pair (Fig. 14) (Matei et al., 2011).
The rate of energy transfer depends on: (i) the extent of overlapping between fluorescence
emission spectrum of donor and the absorption spectrum of acceptor, (ii) the relative orientation
of the donor and acceptor dipoles, and (iii) the distance between the donor and the acceptor
(
0
Tabassum, Al-Asbahy, Afzal, & Arjmand, 2012). From Eqs. (6)– (8), the values of r, R , J and
E were calculated (Table 6). The average distance of 2–8nm between a donor and acceptor pair
indicates that the energy transfer from HSA to metal complexes occurs with high probability. As
can be inferred from results, the values of r suggest that the energy transfer from HSA to the
synthesized Pd (II) complexes can occur with high probability.
Table 6:
Figure. 14.
3
.8. Changes of the protein’s secondary structure induced by metal complexes binding
FT-IR spectroscopy is a helpful technique for the study of hydrogen bonding and has been
identified as one of the few techniques that are established in the determination of protein
secondary structure at different physiological systems. The information on the secondary
structure of proteins arises from the amide bands which result from the vibrations of the peptide
groups of proteins. When small molecules bind to a globular protein like HSA, changes in the
hydrogen bonding which is involved in the peptide linkages would occur, resulting in changes in
the vibrational frequency of the different amide modes. The modes most widely used in the
protein structural studies are amide I, amide II, and amide III. Amide I band ranges from 1600 to
-1
1
700cm and arises principally from the C=O stretching. Amide II band is primarily an N-H
-1
bending with a contribution from C-N stretching vibrations, and ranges from 1480 to 1600 cm .
-1
Amide III band ranges from 1220 to 1330cm because the C-N stretching mode coupled to the
in-plane N-H bending mode (Abu et al., 2012; Neault & Tajmir-Riahi, 1998). Fig. 15 shows the
FT-IR spectrum of free HSA and Pd(II) complex-bound form of HSA with its difference
spectrum of these two. The spectrum in Fig. 15(a) was obtained by subtracting the FT- IR
absorption of the Tris–HCl buffer from the spectrum of the protein solution. The spectrum in
Fig. 15(b) was obtained by the FT- IR absorption of Pd (II) complex was bounded to HSA and
2
5

the spectrum in Fig. 15(c) was obtained by subtracting the FT- IR absorption of the Pd(II)
complex-free form from that of the Pd(II) complex-bound form. As shown in Fig. 15, the peak
−
1
-
position of amide I bands has changed from 1657.12 to 1659.78cm and 1657.12 to 1664.76cm
1
for complexes I and II, respectively, which indicated that the secondary structure of the protein
has been changed after Pd (II) complexes were interacted. These results also indicated that these
synthesized Pd (II) complexes interacted with the C=O and C–N groups in the protein
polypeptides. This is in accordance with H- bonding results obtained from fluorescence studies.
The HSA–Pd (II) complexes caused the rearrangement of the polypeptide carbonyl hydrogen
bonding network, and finally, the reduction of the protein α-helical structure. Similar
observations were made by S. Shahrakiet. al. (Shahraki, Shiri, Mansouri-Torshizi, & Shahraki,
2
016).
Figure. 15.
4
. Conclusion
In summary, we have designed and prepared two new, water soluble, neutral, and isomeric
Pd(II) complexes, [Pd(Gly)(Leu)](I) and [Pd(Gly)(Ile)](II) ( Gly, Leu and Ile are glycine,
leucine and isuleucine amino acids, respectively). Their molecular structures were characterized
1
by elemental analysis, conductivity measurement, FT-IR, UV-Vis and H-NMR techniques.
Structural studies showed that amino acids coordinated as bidentate chelate with the Pd(II)
center (Fig.1). The Invitro cytotoxic activities were studied by MTT assay against the cancer
model's cell line K562, indicating that isomer I is more active than that of II, but both have good
activity potential and possible future improvements. The interactions of these water soluble
complexes with CT-DNA and HSA were carried out by using absorption spectroscopy,
fluorescence titration spectra, Ethidium bromide titration, and gel electrophoresis studies. The
results suggest that: i) both isomeric complexes exothermically interact with CT-DNA and HSA
at very low concentrations, ii) interaction affinity of isomer I to DNA is more than that of
isomer II and the reverse was observed for HSA interaction, iii) thermodynamic parameters
indicated that I and II spontaneously interact with CT-DNA by groove binding and probably get
loaded in main binding site of HSA via H-binding and Van der Waals forces, iv) quenching
mechanism of both CT-DNA-EB and HSA solutions by the isomeric Pd(II) complexes seems to
be statically quenching, and v) number of binding sites for both I and II on CT-DNA and HSA
is approximately equal to unity. On the basis of the above results, it was concluded that these
types of isomeric complexes may be useful in development of drug formulation.
2
6

Acknowledgment
Funding of our research from the university of Sistan and Baluchestan (USB) is gratefully
acknowledged.
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