Interaction of 5-Fluorouracil and its derivatives with bovine serum albumin

Article abstract of DOI:10.1016/j.jphotobiol.2011.11.004

Fluorouracil (5-FU) and its derivatives are the most commonly used drugs to treat many types of cancer. Two dual functional agents, FUPAE and FUPAP, derived from 5-Fluorouracil (5-FU) have shown radiosensitizing activity but unlike their components were n

Full text of DOI:10.1016/j.jphotobiol.2011.11.004

Journal of Photochemistry and Photobiology B: Biology 107 (2012) 20–26
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Interaction of 5-Fluorouracil and its derivatives with bovine serum albumin
b
b
a
K. Abdi a, Sh. Nafisi b,c, , F. Manouchehri , M. Bonsaii , A. Khalaj
⇑
a Department of Medicinal Chemistry and Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
b Department of Chemistry, Islamic Azad University, Central Tehran Branch (IAUCTB), Tehran, Iran
c Research Institute for Islamic and Complementary Medicine, Tehran University of Medical Sciences, Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorouracil (5-FU) and its derivatives are the most commonly used drugs to treat many types of cancer.
Two dual functional agents, FUPAE and FUPAP, derived from 5-Fluorouracil (5-FU) have shown radiosen-
sitizing activity but unlike their components were not cytotoxic. This study was designed to examine the
interaction of BSA with 5-Fluorouracil (5-FU) and two of its derivatives; FUPAE and FUPAP at physiolog-
ical conditions, using a constant protein concentration and various drug contents. FTIR, UV–Vis spectro-
scopic methods as well as molecular modelling were used to determine the drugs binding mode, the
binding constants and the effects of drug complexation on BSA stability and conformation. Structural
analysis showed that 5-Fluorouracil, FUPAE and FUPAP bind BSA via polypeptide polar groups with
Received 13 September 2011
Received in revised form 14 November 2011
Accepted 17 November 2011
Available online 23 November 2011
Keywords:
Bovine serum albumin
5-Fluorouracil
FUPAE
FUPAP
FTIR
overall binding constants of K5-FU–BSA = 3.02( 0.09) ꢀ 103, KFUPAE–BSA = 1.08( 0.04) ꢀ 104, KFUPAP–BSA
=
1.21( 0.06) ꢀ 104 Mꢁ1. BSA conformation was altered by a major reduction of
a-helix from 69% (free
BSA) to 34% with 5-FU, 40% with FUPAE, 38% with FUPAP. These results suggest that serum albumins
might act as carrier proteins for FUPAE and FUPAP in delivering them to target tissues.
Ó 2011 Elsevier B.V. All rights reserved.
UV–Vis
1. Introduction
One of the important properties of a drug is the degree of
its protein binding which affects the drug effective solubility,
5-FU is a chemotherapeutic agent which has been used mainly
for colorectal and pancreatic and aggressive forms of breast
cancers. It has also been used as an anti-scarring agent in oph-
thalmic surgery and topically for treatment of actinic kurtosis
and some types of skin cancers [1–5]. 5-FU (Scheme 1A) is a
pyrimidine analogue drug which shows radiosensitizing activity.
On the basis of the radiosensitizing activities of aromatic nitro
compounds [6–9], the synthesis of 2,4-dinitrophenylamine teth-
ered to 5-fluoracil through 2 and 3 carbon atoms has been
biodistribution, half life in the body and interaction with other
endogenous or exogenous compounds. The proteins commonly
involved with drug delivery are serum albumin, lipoproteins,
and al-glycoprotein. Serum albumin is the most abundant protein
present in the circulatory system of a wide variety of organisms
and is the major macromolecule contributing to the osmotic
blood pressure [15]. The most important property of this group
of proteins is that they serve as transporters for a variety of
compounds. BSA (Scheme 2) has been one of the most extensively
studied of this group of proteins, particularly because of its struc-
tural homology with human serum albumin (HSA). The BSA
molecule is made up of three homologous domains (I, II, III) that
are divided into nine loops (L1–L9) by 17 disulphide bonds. Each
domain in turn is the product of two sub-domains (IA, IB, etc.).
X-ray crystallographic data [16] shows that the albumin structure
reported;
[3-(2-(2,4-dinitrophenylamino)ethyl)-5-fluoropyrimi-
dine-2,4(1H,3H)-dione] (FUPAE) and [3-(3-(2,4-dinitrophenylami-
no)propyl)-5-fluoropyrimidine-2,4(1H,3H)-dione] (FUPAP) [10]
(Scheme 1A). The synthesis reaction is shown in Scheme 1B. Orig-
inally it was anticipated that these compounds like 2,4-dinitro-
phenylamine mustards and dinitroaziridin have radiosensitizing
activity as well as aerobic cytotoxicity due to the parent nitro
compounds or bio-activation by NADPH: quinooxidoreductase
(DT diphrace) as an oxygen-insensitive reductase [11–14].
However, the results showed that these compounds unlike their
components were not cytotoxic but increased the sensitivity of
the normal oxygenated cells to radiation [10].
is predominantly
a-helical and the remaining polypeptide chain
occurs in turns and extended or flexible regions between sub-
domains without b-sheets. BSA has two tryptophan residues,
Trp-134 in the first domain and Trp-212 in the second domains
that possess intrinsic fluorescence [17]. Trp-212 is located within
a hydrophobic binding pocket of the protein and Trp-134 is
located on the surface of the molecule [18]. Plasma protein bind-
ing of drugs assumes great importance since it influences their
pharmacokinetic and pharmacodynamic properties, and may also
cause interference with the binding of other endogenous and/or
⇑
Corresponding author at: Department of Chemistry, Islamic Azad University,
Central Tehran Branch (IAUCTB), Tehran, Iran. Tel./fax: +1 98 21 22426554.
E-mail address: drshnafisi@gmail.com (Sh. Nafisi).
1011-1344/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2011.11.004

K. Abdi et al. / Journal of Photochemistry and Photobiology B: Biology 107 (2012) 20–26
21
Scheme 2. Bovine serum albumin (each sub-domain is marked in a different color)
with tryptophan residues shown in close up view with red color dash-line. (For
interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
concentration was determined spectrophotometrically using an
extinction coefficient of 36,500 Mꢁ1 cmꢁ1 at 280 nm [21]. Solutions
of 5-FU, FUPAE and FUPAP (1 mM) were first prepared in phos-
phate buffer/ethanol 50% and then diluted by serial dilution to
0.500, 0.125, 0.062, 0.015 mM in the same phosphate buffer. After
addition of an equal volume of 5-FU, FUPAE, FUPAP to protein solu-
tion, the final ethanol concentration was reduced to 25%. The pres-
ence of 25% of ethanol induces no major BSA structural changes
according to the literature report [22].
Scheme 1. (A) Chemical structures of 5-FU and its derivatives. (B) Synthesis
reaction of the 5-FU derivatives; FUPAE, FUPAP.
2.3. FTIR spectroscopic measurements
exogenous ligands as a result of overlap of binding sites and/or
conformational changes [19,20].
Infrared spectra were recorded on a Nicolet FTIR spectrometer
(Magna-IR 550) equipped with a liquid-nitrogen-cooled HgCdTe
(MCT) detector and a KBr beam splitter, using AgBr windows. Solu-
tions of 5-FU, FUPAE and FUPAP were added dropwise to the BSA
solution with constant stirring to ensure the formation of homoge-
neous solution and to have ligands concentrations of 0.015, 0.062,
0.125, 0.5 and 1 mM with a final protein concentration of 0.25 mM
(20 mg/mL). The concentrations of the ligands in the complex
mixtures were 7.5 ꢀ 103, 0.031, 0.0.062, 0.25 and 0.5 Mm. Spectra
were collected after 2 h of incubation of BSA with drug solution at
room temperature, using hydrated films. Interferograms were
In this report, spectroscopic analyses and molecular modelling
of the interaction of 5-FU, FUPAE and FUPAP (Scheme 1A) with
BSA has been reported in aqueous solution under physiological
conditions and using constant concentration of protein and differ-
ent concentrations of ligands. Structural information on binding
modes of 5-FU, FUPAE and FUPAP and the effects of ligand–protein
complexation on the stability and conformation of BSA are also
reported.
accumulated over the spectral range 4000–600 cmꢁ1 with
a
2. Materials and methods
nominal resolution of 2 cmꢁ1 and 100 scans. The difference spectra
[(protein solution + pyrimidine analogues solutions) ꢁ (protein
solution)] were generated using the water combination mode
around 2300 cmꢁ1, as the standard [23]. When producing differ-
ence spectra, this band was adjusted to the baseline level, in order
to normalize difference spectra.
2.1. Materials
Bovine serum albumin fraction V (free fatty acid) was pur-
chased from Sigma Chemical Co. 5-FU was obtained from Merck
Chemical Co. and used as supplied. Other chemicals were of re-
agent grade and used without further purification. FUPAE and FU-
PAP were synthesized according to the previous literature [10].
Briefly, compound II was prepared by ultrasound promoted reac-
tion of 2,4-dinitrophenylamine (compound I) with related dibro-
moalkane. Finally FUPAE and FUPAP were synthesized by the
reaction of 5-Fluorouracil (5-FU) with related compound II
(Scheme 1B). The synthesized compounds were purified by column
chromatography and then crystallized. The purity of the synthe-
sized compounds was determined by TLC and NMR (Purity > 98%).
2.4. Analysis of protein conformation
Analysis of the secondary structure of BSA and complexes was
carried out according to the previously reported method [24]. The
protein secondary structure is determined from the shape of the
amide I band, located around 1660–1650 cmꢁ1. The FTIR spectra
were smoothed, and their baselines were corrected automatically
using Grams AI software. Thus, the root-mean square (rms) noise
of every spectrum was calculated. By means of the second derivative
in the spectral region 1700–1600 cmꢁ1, the major peaks for BSA and
the complexes were resolved. The above spectral region was decon-
voluted by the curve-fitting method with the Levenberg–Marquadt
2.2. Preparation of stock solutions
Bovine serum albumin (40 mg/mL or 0.5 mM) was dissolved in
aqueous solution containing phosphate buffer (pH 7.2). The protein
algorithm and the peaks corresponding to
a-helix (1658–

22
K. Abdi et al. / Journal of Photochemistry and Photobiology B: Biology 107 (2012) 20–26
1651 cmꢁ1), b-sheet (1640–1610 cmꢁ1), turn (1670–1665 cmꢁ1),
random coil (1648–1641 cmꢁ1), and b-antiparallel (1692–
1680 cmꢁ1) were adjusted and the area measured with the Gaussian
function. The area of all component bands assigned to a given
conformation were then summed up and divided by the total area
[25,26]. The curve-fitting analysis was performed using the
GRAMS/AI Version 7.01 software of the Galactic Industries
Corporation.
A0 ¼
e
SbSt
ð1Þ
In the presence of ligand at total concentration Lt, the absor-
bance of a solution containing the same total substrate concentra-
tion is:
AL ¼
eSb½Sꢂ þ
eLb½Lꢂ þ
e11b½SLꢂ
ð2Þ
where [S] is the concentration of the uncomplexed substrate, [L] is
the concentration of the uncomplexed ligand and [SL] is the concen-
tration of the complex) which, combined with the mass balance on
S and L, gives:
2.5. Absorption spectroscopy
AL ¼
where
e
SbSt þ
e
LbLt þ
D
e
11b½SLꢂ
ð3Þ
The absorption spectra were recorded on a Cecil BioAquarius CE
7250 double beam spectrophotometer, using a slit of 5 nm and a
scan speed of 250 nm minꢁ1. Quartz cuvettes of 1 cm were used.
The UV absorption of BSA in the presence and absence of 5-FU,
FUPAE, FUPAP solutions were measured at pH 7.2 by keeping the
concentration of BSA constant (0.025 mM), while varying the
D
e11
=
e11
ꢁ
eS
ꢁ
eL (eL molar absorptivity of the ligand). By
measuring the solution absorbance against a reference containing
ligand at the same total concentration Lt, the measured absorbance
becomes:
A ¼
e
SbSt þ
D
e
11b½SLꢂ
ð4Þ
concentration of the drug (1
250–550 nm.
lM–1 mM), in the range of
Combining Eq. (4) with the stability constant definition
The binding constants of the drugs–BSA complexes were
calculated as reported [27]. It is assumed that the interaction
between the ligand L and the substrate S is 1:1; for this reason, a
single complex SL (1:1) is formed. It was also assumed that the
sites (and all the binding sites) are independent and finally the
Beer’s law is followed by all species. A wavelength is selected at
which the molar absorptivities, eS (molar absorptivity of the
substrate) and e11 (molar absorptivity of the complex) are differ-
ent. Then at total concentration St of the substrate, in the absence
of ligand and the light path length is b = 1 cm, the solution
absorbance is:
K11 = [SL]/[S][L], gives:
D
A ¼ K11D
e11b½Sꢂ½Lꢂ
ð5Þ
where
D
A = A ꢁ A0. From the mass balance expression St = [S] + [SL],
we get [S] = St/(1 + K11[L]), which is Eq. (5), giving Eq. (6) at the rela-
tionship between the observed absorbance change per centimeter
and the system variables and parameters.
D
b
A
StK11De11½Lꢂ
1 þ K11½Lꢂ
¼
ð6Þ
Fig. 1. FTIR spectra in the region of 1800–600 cmꢁ1 of hydrated films (pH 7.2) for free BSA (0.5 mM), (A) free 5-FU (1 mM), (B), free FUPAE (1 mM), (C) free FUPAP (1 mM) and
their BSA complexes with difference spectra (diff.) (bottom two curves) obtained at different drug concentrations (indicated on the figure).

K. Abdi et al. / Journal of Photochemistry and Photobiology B: Biology 107 (2012) 20–26
23
Table 1
Eq. (6) is the binding isotherm, which shows the hyperbolic
dependence on free ligand concentration. The double-reciprocal
form of plotting the rectangular hyperbola, is based on the
Secondary structure analysis (infrared spectra) from the free BSA and its complexes in
hydrated film at pH 7.2.
f
1
1
e
Complex (ligand
concentration (mM))
a
( 3%)
-Helix
b-Anti
( 1%)
b-Sheet
( 2%)
Turn
( 2%)
Random
( 1%)
¼
d ꢃ x þ d, linearization of Eq. (6) according to the following
y
equation:
Free BSA
5-FU–BSA (1 mM)
69
34
40
40
39
38
39
3
3
5
6
4
5
3
12
9
14
40
31
31
34
37
43
2
3
2
8
7
6
3
b
1
1
tDe11
¼
þ
ð7Þ
5-FU–BSA (15
FUPAE–BSA (1 mM)
FUPAE–BSA (15 M)
FUPAP–BSA (1 mM)
FUPAP–BSA (15 M)
l
M)
16
15
16
13
12
D
A
StK11De11½Lꢂ
Thus the double reciprocal plot of 1/
S
D
A versus 1/[L] is linear and
l
the binding constant can be estimated from the following
equation:
l
intercept
slope
K11
¼
ð8Þ
and templateproteinswas 0.20 Å for positionsof backboneatoms, as
calculated with DeepView/Swiss-PdbViewer 4.0.1. By using the
structure and model assessment tools of SWISS-MODEL workspace,
it was found that the quality of the predicted BSA structure is similar
to the structure of the free HSA which was used as a template.
2.6. Molecular modelling and docking
2.6.1. Model of bovine serum albumin
Structure of BSA was predicted by automated homology model-
ling using SWISS-MODEL Workspace [28,29]. The structure of free
HSA (PDB id: 1AO6, chain A) obtained by X-ray crystallography
[30] was used as a template. BSA and HSA proteins share 78.1% of se-
quenceidentity, whichis sufficientto obtainreliable sequencealign-
ment [17,31]. Images of the structures were generated using UCSF
Chimera 1.5.3 (Web address: http://www.cgl.ucsf.edu/chimera)
(Scheme 2). Root Mean Standard Deviation (RMSD) between model
2.6.2. Molecular docking
To determine the preferred binding sites on BSA, the docking
studies were performed by AutoDock 4.2.3 software (http://
autodock.scripps.edu).
By using genetics algorithm (GA) for the local search, the so-
called pseudo-Solis and Wets algorithm was applied using a max-
imum of 300 iterations per local search [32].
Fig. 2. Second derivative resolution enhancement and curve-fitted amide-I region (1700–1600 cmꢁ1) for free BSA and its drug adducts in aqueous solution with 1 mM drug
for 5-FU, FUPAE and FUPAP concentrations at pH 7.2.

24
K. Abdi et al. / Journal of Photochemistry and Photobiology B: Biology 107 (2012) 20–26
torsional term, and desolvation energy of oligonucleotide–ligand
In AutoDock, the overall docking energy of a given ligand mol-
ecule in its active site is expressed as follows:
complex, respectively. rij, Aij, Bij, Cij, and Dij represent the interatomic
distance, the depths of energy well, and the equilibrium separations
between the two atoms, respectively. The first three terms are in va-
cuo force field energies for intermolecular interactions. The fourth
term accounts for the internal steric energy of the ligand molecule.
The energies used and reported by AutoDock should be distin-
guished since there are docked energies, which include the inter-
molecular and intramolecular interaction energies, and are used
during dockings to predict free energies; including the intermolec-
ular energy and torsional free energy, and are only reported at the
end of a docking [33].
!
!
X
X
Aij Bij
rij
Cij Dij
D
G ¼
D
G
12 ꢁ
þ
D
Ghbond
12 ꢁ 10 þ Ehbond
v
dW
r6ij
rij
rij
i;j
i;j
X
X
qi ꢁ qj
2ij=2r2
Þ
þ
D
Gelec
þ
D
GtorNtor
þ
D
Gsol
SiVjeðꢁr
ð9Þ
eðrijÞrij
i;j
i
C ;j
where
DGvdW, DGhbond, DGelec, DGtor, and DGsol are free energy coef-
ficients of van der Waals, hydrogen bond, electrostatic interactions,
The relationship between binding constant, Kbinding and the
binding free energy change of binding,
D
Gbinding is as follows:
D
Gbinding ¼ ꢁRT ln Kbinding
ð10Þ
where R is the gas constant, 1.987 cal Kꢁ1 molꢁ1, and T is the abso-
lute temperature, 298.15 K.
AutoDock Tools1.5.4 (ADT) (Web address: http://mgl-
tools.scripps.edu) and UCSF Chimera 1.5.3 were used for the anal-
ysis of display docking results.
3. Results and discussion
3.1. FTIR spectra of 5-FU, FUPAE and FUPAP–BSA complexes
The 5-FU, FUPAE, FUPAP–BSA interaction was characterized by
infrared spectroscopy and its derivative methods. Since there was
no major spectral shifting for the protein amide I band at
1653 cmꢁ1 (mainly C@O stretch) and amide II band at 1541 cmꢁ1
(C–N stretching coupled with N–H bending modes) [24,34] upon
pyrimidine analogues interaction, the difference spectra [(protein
Fig. 3. UV–Vis results of free BSA and its drug complexes in aqueous solution
spectra of (a) free drug (0.21 mM); (b) free BSA (0.025 mM); (1–7) drug–BSA
complexes; 1 (0.21 mM), 2 (0.084 mM), 3 (0.071 mM), 4 (0.054 mM), 5 (0.035 mM),
6 (0.021 mM) and 7 (0.01 mM). Plot of 1/(A ꢁ A0) versus (1/drug concentration),
where A0 is the initial absorbance of BSA (279 nm) and A is the recorded absorbance
at different 5-FU, FUPAE and FUPAP concentrations (0.21–0.01 mM) with constant
BSA concentration of 0.025 mM at pH 7.2.
Fig. 4. Docking structure between ligands and BSA. (A) Ribbon representation of
BSA complexes with 5-FU. 5-FU was shown in red surface (display side). (A0) Close
up view of BSA complexes with 5-FU. Outcome is represented by stick style.
Hydrogen bond between BSA and 5-FU are represented by Pale Turquoise line. (B)
and (C) Ribbon representations of BSA complexes with FUPAE and FUPAP. (For
interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)

K. Abdi et al. / Journal of Photochemistry and Photobiology B: Biology 107 (2012) 20–26
25
Table 2
Amino acid residues involved in drug–BSA interaction with the free binding energy for the best-selected docking positions from docking study and experimental data from UV–
Vis spectroscopy.
Complex
Residues involved in the interaction
DGbinding (kcal/mol)
Calculated
Experimental
BSA–5-FU
BSA–FUPAE
BSA–FUPAP
Try30, Leu31, Gln32, Gln33, Met87, Lis106, Asp107
Arg186, Lys 190, Pro421, Arg428, Lys432
Asn109, Arg186, Lys190, Pro421, Arg428, Lys432
ꢁ4.39
ꢁ5.3
ꢁ4.75
ꢁ5.5
ꢁ5.6
ꢁ5.53
solution + pyrimidine analogues solutions) ꢁ (protein solution)]
were obtained, in order to monitor the intensity variations of these
vibrations and the results are shown in Fig. 1. Similarly, the infrared
self deconvolution with second derivative resolution enhancement
and curve-fitting procedures [24] were used to determine the pro-
tein secondary structures in the presence of pyrimidine analogues–
BSA complexes (Fig. 2 and Table 1).
binding site was observed for each ligand–protein complex with
the overall binding constants of K5-FU–BSA = 3.02( 0.09) ꢀ 103,
KFUPAE–BSA = 1.08( 0.04) ꢀ 104, KFUPAP–BSA = 1.21( 0.06) ꢀ 104 Mꢁ1
(Fig. 3). The order of binding is FUPAP–BSA > FUPAE–BSA > 5-FU–
BSA. Similar binding constants were observed for tamoxifen and
its metabolites protein complexes [39]. The association constants
calculated for the tested compounds–BSA complexes suggest a
low affinity for complex formation, compared to strong ligand–
protein complexes, with binding constants ranging from 106 Mꢁ1
to 108 Mꢁ1 [39–41].
At different pyrimidine analogues concentrations (15 lM,
1 mM), decrease in intensity of the amide I band at 1653 cmꢁ1
(BSA) was observed with features at 1654 cmꢁ1 (5-FU–BSA,
FUPAE–BSA) and at about 1652 cmꢁ1 (FUPAP–BSA) for 15
lM and
at 1655 cmꢁ1 (5-FU–BSA), 1654 cmꢁ1 (FUPAE–BSA) and 1652
(FUPAP–BSA) for 1 mM.
3.3. Docking
In the difference spectra of pyrimidine analogues–BSA
complexes (Fig. 1, diff, 15 lM, 1 mM), negative features are due
to the reduction of intensity due to loss of protein structure
(Fig. 1) of the amide I band and suggests a major reduction of
5-FU, FUPAE, and FUPAP molecules were docked to BSA to
determine the preferred binding sites on the protein and the re-
sults are shown in Fig. 4 and Table 2. Data drived from docking
of the tested compounds to BSA, showed different moods of inter-
actions. The free energy of the binding of the most compatible
structure with FTIR and UV results are shown in Table 2. The mod-
els show that 5-FU is surrounded by Try30, Leu31, Gln32, Gln33,
Met87, Lis106, and Asp107 (subdomains of IA and IB) with a bind-
ing energy of ꢁ4.39 kcal/mol. The 5-FU–BSA docking results indi-
cated the presence of hydrogen bond between O4 of 5-FU and
nitrogen of the amine in Gln33 of BSA and H3 of 5-FU and oxygen
of amide, in Tyr30, which stabilizes 5-FU–BSA complexes (Fig. 4A0).
The results showed different mechanisms of the interaction of 5-
FU and its derivatives with BSA. FUPAE and FUPAP mainly interact
with the positively charged amino acids and the binding sites are
located at the surface between subdomains IB and IIIA (Fig. 4B0
and C0). The orders of the stability of drug–BSA complexes obtained
from docking study is consistent with those of the spectroscopic
results showing FUPAP–BSA > FUPAE–BSA > 5-FU–BSA (Fig. 3 and
Table 2).
protein
a-helical structure at different pyrimidine analogues
concentrations. Similar infrared spectral changes were observed
for the protein amide I band in several ligand–protein complexes,
where major protein conformational changes occurred [35,36].
A quantitative analysis of the protein secondary structure for
the free BSA and its pyrimidine analogues adducts in hydrated
films has been carried out, and the results are shown in Fig. 2
and Table 1. The ꢁfr1ee protein was 69%
a
-helix (1653 cmꢁ1), 12%
b-sheet (1620 cm ), 14% turn structure (1676 cmꢁ1), 3% b-anti-
parallel (1690 cmꢁ1), and 2% random coil (1637 cmꢁ1) (Fig. 2 and
Table 1). The results are consistent with the spectroscopic studies
of bovine serum albumin previously reported [37,38]. Upon pyrim-
idine analogues interaction, a major decrease of a-helix from 69%
(free BSA) to 34% (5-FU–BSA,1 mM), 40% (FUPAE–BSA, 1 mM),
and 38% (FUPAP–BSA, 1 mM) with changes in b-sheet from 12%
(free BSA) to 9% (5-FU–BSA,1 mM), and 15% (FUPAE–BSA, 1 mM)
and 13% FUPAP–BSA were observed (Fig. 2 and Table 1). A similar
increase was also observed for the turn structure from 14% (free
BSA) to 40% (5-FU–BSA, 1 mM), 31% (FUPAE–BSA, 1 mM), and
37% (FUPAP–BSA, 1 mM) (Fig. 2 and Table 1). These results are
consistent with the decrease in intensity of the protein amide I
4. Conclusion
Results of this study present important quantitative data on the
binding affinity of 5-FU and its derivatives to bovine serum albu-
min, a drug carrier protein. It was also showed distinct differences
in the mode of protein binding between the parent drug and its
derivatives. The interaction of 5-FU and its derivatives with BSA
can be used to gain insight into the mechanism of action of 5-FU
in cancer therapy and synthesizing new drugs. Based on our spec-
troscopic data, pyrimidine analogues binding to BSA occurs and
causes a partial protein destabilization. The affinity of pyrimidine
analogues–protein complexation is FUPAP > FUPAE > 5-FU with
the binding constants of K5-FU–BSA = 3.02( 0.09) ꢀ 103, KFUPAE–BSA
band discussed above. The decrease in
a-helix structure and
increase in b-sheet and turn structures is indicative of protein
destabilization upon 5-FU and its derivatives interaction.
3.2. UV spectra and stability of pyrimidine analogues–BSA complexes
An increase in the 5-FU, FUPAE, FUPAP concentrations resulted
in an increase in UV light absorption and shifting of BSA band at
279–274 nm that can be related to complex formation (Fig. 3).
The binding constants of the complexes were also determined
using UV–Visible spectroscopic method (described in Materials
and methods). The double reciprocal plot of 1/(A ꢁ A0) versus 1/(li-
gand concentration) is linear and the binding constant (K) can be
estimated from the ratio of the intercept to the slope (Fig. 3). A0
is the initial absorbance of the free BSA at 280 nm and A is the re-
corded absorbance at different drug concentrations (Fig. 3). One
= 1.08( 0.04) ꢀ 104,
KFUPAP–BSA = 1.21 (0.06) ꢀ 104 Mꢁ1
.
The
pyrimidine analogues–BSA binding site is mainly in the vicinity
of the non-acidic amino acids and positively charged amino acid
located in the protein domains I and III in two 5-FU derivatives.
It is important to note here that the low affinity binding is consis-
tent with the role of serum proteins as carrier molecules for the
delivery of the parent drug and its derivatives to target tissues.

26
K. Abdi et al. / Journal of Photochemistry and Photobiology B: Biology 107 (2012) 20–26
[18] S. Hamdani, D. Joly, R. Carpentier, H.A. Tajmir-Riahi, The effect of methylamine
Acknowledgements
on the solution structures of human and bovine serum albumins, J. Mol. Struct.
936 (2009) 80–86.
This work was supported by a research grants from center of
excellence of medicinal chemistry, Tehran University of Medical
Sciences. We thank, Ms. Shoukofeh Hassani (Lab Supervisor) for
allowing us to use their lab facilities.
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