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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 418–423
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectral characterization of the binding and conformational changes of bovine
serum albumin upon interaction with an anti-fungal drug, methylparaben
Keerti M. Naik, Sharanappa T. Nandibewoor ⇑
Department of Studies in Chemistry, Karnatak University, Dharwad 580 003, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" The interaction of methylparaben
The interaction between methylparaben and bovine serum albumin (BSA) has been studied by spectroscopic techniques, such as fluorescence, UV absorption and FT-IR spectral analysis under physiological
condition i.e., pH 7.4. The association constants of methylparaben with BSA were determined at different
temperatures based on fluorescence quenching results. The changes in the secondary structure of BSA
upon interaction with methylparaben were studied.
"
"
"
"
and BSA were studied by
spectroscopic methods.
The fluorescence quenching
mechanism was dynamic quenching.
The binding constants and
thermodynamic parameters were
calculated.
The interaction is driven mainly by
hydrophobic force.
The binding of methylparaben to
BSA changes the secondary structure
of BSA.
a r t i c l e
i n f o
Article history:
Received 28 September 2012
Received in revised form 20 December 2012
Accepted 22 December 2012
Available online 2 January 2013
Keywords:
Bovine serum albumin
Methylparaben
Fluorescence spectroscopy
Binding constants
a b s t r a c t
The binding of methylparaben with bovine serum albumin (BSA) was investigated by spectroscopic
methods viz., fluorescence, FT-IR and UV–vis absorption techniques under physiological conditions i.e.,
pH 7.4. Spectroscopic analysis of the emission quenching at different temperatures revealed that the
quenching mechanism of bovine serum albumin by methylparaben shows a dynamic quenching. The
binding sites number n and binding constants, K were obtained at various temperatures. The distance,
r between methylparaben and BSA was evaluated according to the theory of Förster energy transfer.
The result of FT-IR spectra and UV–vis absorption spectra showed that the conformation of bovine serum
albumin has been changed in the presence of methylparaben. The thermodynamic parameters, enthalpy
change (DH0) and entropy change (DS0) were calculated according to van’t Hoff equation, which indicated that the hydrophobic interaction was the predominant intermolecular force stabilizing the complex. The effect of common ions and site probes were also carried on the binding of methylparaben to
BSA.
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
Serum albumins are one of the most abundant proteins in blood
plasma, which are the major soluble protein constituents of the circulatory system. They play a dominant role in the transport and
⇑ Corresponding author. Tel.: +91 836 2215286; fax: +91 836 2747884.
E-mail address: [email protected] (S.T. Nandibewoor).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.12.055
deposition of endogenous and exogenous ligands in blood, since
serum albumins often increase the apparent solubility of hydrophobic drugs in plasma and modulate their delivery to cells
in vivo and in vitro. The binding ability of drug-albumin in blood
stream may have a significant impact on distribution, free concentration and metabolism of drug. Thus, it is important and necessary
to study the interaction of drug with bovine serum albumin (BSA)
(Fig. 1) at molecular level [1].
K.M. Naik, S.T. Nandibewoor / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 418–423
Fluorescence quenching is an important method to study the
interaction of substances with protein because of its accuracy, sensitivity, rapidity, and convenience of usage. Fluorescence spectroscopy is essentially a probe technique [2,3] sensing changes in the
local environment of the fluorophore, which distinguishes it from
generalized techniques, such as calorimetry, far-ultraviolet circular
dichroism (CD), and infrared (IR) spectroscopy.
Preservatives are often added to pharmaceutical and cosmetic
formulations to manage microbial contamination. Alkyl esteres of
p-hydroxybenzoic acid (parabens) are the most widely employed
preservatives in cosmetic products due to their broad antimicrobial
spectrum and effectiveness. The most common parabens used in cosmetic products are methylparaben (MePa) (Fig. 2), ethylparaben
(EtPa), propylparaben (PrPa) and butylparaben (BuPa). They are often
used in combination, since they have synergistic effects, in a wide
variety of products such as cosmetics, ointments, and suspensions
[4–6], which allows the use of lower levels while increasing preservative activity. Furthermore, parabens are often used in combination
with other types of preservatives. Parabens have multiple biological
actions, but it is generally related that their inhibitory effects on
membrane transport and mitochondrial function processes are keys
for their actions [4]. Since the presence of parabens was detected in
human breast tumors by Darbre [7,8], the use of these preservatives
in cosmetics has been discussed worldwide. It was also reported that
parabens have estrogenic activity, since the compounds have been
shown to bind to estrogen receptors from different sources [9].
The present study focused on biophysical interactions of methylparaben with serum albumin that play an important role in drug
transport and storage in vertebrates [10]. Drug interactions at protein binding level will in most cases significantly affect the apparent distribution volume of the drugs and also affect the elimination
rate of drugs. Therefore, the studies on this aspect can provide
information of the structural features that determine the therapeutic effectiveness of drug and have been an interesting research field
in life sciences, chemistry and clinical medicine [11]. In order to
understand the binding mechanism of BSA with methylparaben
under physiological condition, we planned to carry out detailed
investigation of methylparaben–BSA association using fluorescence spectroscopy. In addition, conformational change of BSA is
discussed on the basis of UV–vis and IR spectroscopy. This study
is expected to provide important insight into the interaction of
protein BSA with methylparaben, under physiological conditions.
Experimental section
Reagents and chemicals
Bovine serum albumin (BSA) was purchased from Sigma Chemical Company, St. Louis, USA and used without purification. Meth-
419
Fig. 2. Chemical structure of methylparaben.
ylparaben was obtained from Sigma Aldrich. The solutions of
methylparaben and BSA were prepared in 0.1 M phosphate buffer
of pH 7.4. BSA solution was prepared based on their molecular
weight of 65,000. All other materials were of analytical reagent
grade and millipore water was used throughout the work.
Instrumentation and analytical procedure
Fluorescence spectra were recorded using a RF-5301 PC Hitachi
spectrofluorometer Model F-2000 (Tokyo, Japan) with a 150 W Xenon lamp, a 1 cm quartz cell and thermostatic cuvette holder. The
excitation and emission bandwidths were both 5 nm. The temperature of the sample was maintained by recycling water throughout
the experiment. The absorption spectra were recorded on a double
beam CARY 50-BIO UV–vis. Spectrophotometer (Victoria, Australia), FT-IR Nicolet-5700; USA was used to record infrared spectra.
All of the pH measurements were performed with an Elico LI120
pH meter (Elico Ltd., India).
Procedures
Methylparaben–protein interaction study
A stock solution of 250 lM of BSA and methylparaben were prepared in phosphate buffer solution (pH 7.4). An appropriate volume of BSA to obtain 5 lM and 5 lM methylparaben was mixed
and fluorescence spectra were recorded. In the next step fixing
the concentration of BSA at 5 lM and drug concentration was varied from 5 to 45 lM. Fluorescence spectra were recorded at three
temperatures (288, 298 and 308 K) in the range 300–550 nm upon
excitation at 295 nm in each case.
Fig. 1. Structure of BSA.
420
K.M. Naik, S.T. Nandibewoor / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 418–423
UV measurements
The UV measurements of BSA in the presence and absence of
methylparaben was made in the range of 230–320 nm. BSA concentration was fixed at 5 lM while the drug concentration was
varied from 5 to 20 lM in presence of phosphate buffer.
Displacement studies
The displacement experiments were performed using different
site probes viz., warfarin, ibuprofen and digitoxin for sites I, II
and III, respectively [12] by keeping the concentration of BSA and
the probe constant (5 lM each), while the concentration of methylparaben was varied from 0 to 45 lM at 298 K. The fluorescence
quenching titration was used as before to determine the binding
constant of BSA–methylparaben in presence of above site probes.
The fluorescence spectra were recorded in the range of 300–
550 nm upon excitation at 295 nm at 298 K.
FT-IR measurements
The FT-IR spectra of BSA in presence and absence of methylparaben at 298 K were recorded in the range of 500–4000 cm1. Bovine serum albumin concentration was fixed at 5 lM while that
of methylparaben was 5 lM in presence of phosphate buffer.
Results and discussion
Fluorescence studies
The conformational changes of BSA were evaluated by the measurement of intrinsic fluorescence intensity of protein before and
after addition of drug. Fluorescence measurements give information about the molecular environment in a vicinity of the fluorophore molecules. The effect of drug (methylparaben) on BSA
fluorescence intensity was shown in Fig. 3. When different concentration of methylparaben solution was titrated into a fixed concentration of BSA, a remarkable decrease of fluorescence intensity of
BSA was observed, which indicated that methylparaben could
interact with BSA. Furthermore, from Fig. 3, the maximum wavelength of BSA shifted from 343 to 338 nm after the addition of
methylparaben, so a slight blue shift of the maximum emission
wavelength was observed and it could be deduced that the fluorophore of protein was placed in a more hydrophobic environment
after the addition of methylparaben. It was probably owing to
the loss of the compact structure of hydrophobic subdomain IIA
where tryptophan is placed [13]. The quantitative analysis of the
binding of methylparaben to BSA was carried out using the fluorescence quenching at 343 nm at various temperatures. With the increase of the concentration of methylparaben, the fluorescence
intensity of system gradually decreased, and with the further addition of methylparaben, the fluorescence intensity of system decreased tardily in each titration curve which indicates the
beginning of saturation of the BSA binding site.
Dynamic and static quenching are the different mechanisms of
quenching and they can be distinguished by their differing dependence on temperature [14]. The quenching rate constants decreased with increasing temperature for static quenching, but the
reverse effect is observed in the case of dynamic quenching [15].
Fig. 3. Fluorescence spectra of BSA (5 lM) in presence of methylparaben: (a) 0 lM,
(b) 5 lM, (c) 10 lM, (d) 15 lM, (e) 20 lM, (f) 25 lM, (g) 30 lM, (h) 35 lM, (i) 40 lM
and (j) 45 lM. Inset plot of fluorescence intensity vs. [methylparaben].
A possible quenching mechanism is evident from the Stern–Volmer plots (Fig. 4) of BSA with methylparaben at different temperatures (288, 298 and 308 K). The quenching data were subjected to
Stern–Volmer equation [14]
F 0 =F ¼ 1 þ K SV ½Q ¼ 1 þ K q Co ½Q ð1Þ
where F and F0 are the fluorescence intensity of BSA with and without quencher (drug), respectively. Kq, KSV, C0 and [Q] are the
quenching rate constant of the biomolecule, the dynamic quenching
constant, the average lifetime of the biomolecule without quencher
and the concentration of quencher, respectively. Obviously,
K q ¼ K SV =C0
ð2Þ
The fluorescence lifetime of the biopolymer is 108 s [16]. In the linear range of Stern–Volmer regression curve (Fig. 4), the average
quenching constants for methylparaben at 288, 298 and 308 K temperatures were given in Table 1. That indicates the dynamic
quenching interaction between methylparaben and BSA.
Binding constant and number of binding sites
Fluorescence intensity data can also be used to obtain the binding constant, K and the number of binding sites, n. When small
molecules bind independently to a set of equivalent sites on a mac-
30
(c)
25
20
(b)
Fo/F
Effects of some common ions
The effects of some common ions viz., Co2+, Cu2+, Ni2+, Ca2+ and
2+
Zn were investigated on methylparaben–BSA interactions. The
fluorescence spectra of methylparaben–BSA system were recorded
in presence of above ions at 343 nm upon excitation at 295 nm at
298 K. The overall concentration of BSA and that of the common
ions was fixed at 5 lM, while the concentration of methylparaben
was varied from 0 to 45 lM at 298 K.
15
(a)
10
5
0
0
10
20
30
40
50
[Methylparaben] × µM
Fig. 4. The Stern–Volmer curves for quenching of methylparaben with BSA 288 K
(a), 298 K (b) and 308 K (c). kex = 295 nm; kem = 343 nm and [BSA] = 5 lM.
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K.M. Naik, S.T. Nandibewoor / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 418–423
romolecule, the equilibrium between free and bound molecules is
given [17] by the following equation
logðF 0 FÞ=F ¼ log K þ n log½Q The values of K and n were obtained from the intercept and slope of
the plot of log[(F0 F)/F] vs. log[Q] (Fig. 5). The values of K and
those of n are given in Table 1. The increasing temperature may result in the increasing diffusion coefficient, and also it leads to the
lower stability of the methylparaben–BSA system. The competition
of the diffusion coefficient and stability of the methylparaben–BSA
system with increased temperature may induce the above results.
The value of n is helpful to know the number of binding sites. In
the present study, n was obtained nearly 2. There may be two binding sites of methylparaben on BSA, one of which is a strong binding
site and the other is a weak one. This may be due to the methylparaben involved other sites with higher binding affinity and selectivity. Although in most of the studies on binding of drug–protein
interactions, the value of n obtained was unity, however, there are
also a few reports [17,18] where the value of n was more than unity.
The correlation coefficients are nearer to 0.9990, indicating that the
interaction between methylparaben and BSA agrees well with the
site binding model underlying the Eq. (3).
Thermodynamic parameters and the nature of binding forces
Considering the dependence of binding constant on temperature, a thermodynamic process was considered to be responsible
for the formation of the complex. Therefore, the thermodynamic
parameters (enthalpy change DH0, entropy change DS0 and free
energy change DG0 (Table 1) dependent on temperatures were
analyzed in order to further characterize the acting forces between
methylparaben and BSA, as these are the main evidences to propose the binding mode. Primarily, four types of forces take part
in drug–protein interaction, viz., electrostatic forces, hydrophobic
forces, Vander Waals force of interactions and hydrogen bonding
[17]. Signs and magnitudes of thermodynamic parameters determine the nature of forces actually taking part in protein–drug
interaction. The binding studies were carried out at three different
temperatures viz., 288, 298 and 308 K and thermodynamic parameters were evaluated using the following van’t Hoff equation and
Gibbs–Helmholtz equation:
log K ¼ DH0 =2:303RT þ DS0 =2:303R
ð4Þ
and
DG0 ¼ DH0 T DS0
0
ð5Þ
log [(Fo-F)/F]
ð3Þ
(a)
(b)
(c)
-4.0
-4.2
-4.4
-4.6
-4.8
-5.0
-5.2
-5.4
-5.6
log [Methylparaben]
Fig. 5. The plot of log (F0 F)/F vs. log [methylparaben] for quenching of BSA by
methylparaben at 288 K (a) 298 K, (b) and 308 K (c) (kex = 295 nm; kem = 343 nm.
[methylparaben] = 5–45 lM; [BSA] = 5 lM).
DH0 and DS0 values arise from typical hydrophobic interaction [19].
Obviously, it is not possible to account for the thermodynamic
parameters of methylparaben–BSA complex based on a single intermolecular force model. Therefore, the binding interaction of methylparaben to BSA might involve the hydrophobic interaction
strongly as evidenced by the positive DS0 value and the electrostatic
interaction could also not be excluded.
Energy transfer between methylparaben and BSA
There is a considerable overlap between absorption spectrum of
methylparaben (acceptor) and fluorescence spectrum of BSA (donor) which forms the basis of fluorescence resonance energy transfer (FRET) (Fig. 6). Energy transfer phenomena have wide
applications in energy conversion process [20]. Photodynamic action, which is often used in the treatment of cancer, is also a consequence of energy transfer [21]. According to Forster’s non
radiative energy transfer theory [22], the energy transfer will happen under the following conditions: (i) the donor can produce fluorescence light, (ii) fluorescence emission spectrum of the donor
and UV absorption spectrum of the acceptor have more overlap
and (iii) the distance between the donor (BSA) and the acceptor
(methylparaben) is lower than 8 nm. The fluorescence quenching
of BSA upon binding with methylparaben indicated the energy
transfer between methylparaben and BSA. The efficiency of energy
transfer, E, was calculated using the equation
R60
0
The values of DH and DS were obtained from the slope and intercept of the plot of log K vs. 1/T (Fig. S1). The values of DH0, DG0 and
DS0 are listed in Table 1. The negative sign of DG0 values supported
the assertion that all binding processes are spontaneous and it was
exothermic reaction. Thus, the formation of the methylparaben–
BSA complex was an exothermic reaction accompanied by a positive value of DS0 value. From the point of view of water structure,
a positive value of DS0 is frequently taken as a typical evidence
for the hydrophobic interaction. Furthermore, specific electrostatic
interaction between ionic species in aqueous solution are characterized by the negative DH0 and positive DS0 values, while the positive
-1.3
-1.0
-0.7
-0.4
-0.1
0.2
0.5
0.8
1.1
1.4
1.7
2.0
E¼
R60
þ r6
¼
F0 F
F0
ð6Þ
where F and F0 are the fluorescence intensities of BSA in presence
and absence of methylparaben, r the distance between acceptor
and donor and R0 the critical distance when the transfer efficiency
is 50%. The value of R0 is calculated using the equation
2
R60 ¼ 8:8 1025 k N 4 UJ
ð7Þ
2
where k is the spatial orientation factor of the dipole, N is the
refractive index of the medium, U the fluorescence quantum yield
Table 1
Interaction parameters of methylparaben–BSA system at different temperatures.
Temp.
(K)
KSV 105 M1
Kq 1013 M1 S1
R2
Binding constant
K 109 M1
R2
No. of binding
sites n
D H0
(k J mol1)
DS0
(J K1 mol1)
DG 0
(k J mol1)
288
298
308
2.05
3.59
5.74
2.05
3.59
5.74
0.9990
0.9990
0.9940
4.58
3.32
1.75
0.9960
0.9990
0.9910
2.01
1.92
1.82
35.4 ± 3.0
62.5 ± 1.0
54.1 ± 3.0
422
K.M. Naik, S.T. Nandibewoor / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 418–423
4000
0.1
(a)
3500
Intensity (F)
2500
0.06
2000
0.04
1500
1000
Absorbance
0.08
3000
0.02
(b)
500
0
0
300
350
400
450
Wavelength (nm)
Fig. 6. The overlap of the fluorescence spectrum of BSA (5 lM) (a) and the
absorption spectrum of methylparaben (5 lM), (b) (kex = 295 nm; kem = 343 nm.
[BSA]/[methylparaben] = 1:1).
of the donor and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the
acceptor. J is given by the following equation
P
J¼
FðkÞðkÞk4 Dk
P
FðkÞDk
ð8Þ
where F(k) is the fluorescence intensity of the fluorescent donor of
wavelength, k, e(k) is the molar absorption coefficient of the acceptor at wavelength, k. For ligand–BSA interaction, k2 = 2/3, N = 1.336
and U = 0.15 [21]. The values of J, R0, E and r were calculated to be
5.2702 1014 cm3 L mol1, 3.3659 nm, 0.1690 nm, 4.2677 nm
respectively at 298 K. The donor to acceptor distance, r < 7 nm indicated that the energy transfer from BSA to methylparaben occurs
with high possibility [23].
Absorption spectroscopic studies
UV–vis absorption measurement is a very simple method and
applicable to know the change in hydrophobicity [17] and to know
the complex formation [24]. In the present study, we have observed the change in UV absorption spectra of methylparaben,
BSA and methylparaben–BSA system (Fig. 7). It is suggested possibly that the complex was formed between methylparaben and BSA
[25]. It is evident that the UV absorption intensity of BSA increased
regularly with the variation of methylparaben concentration. The
maximum peak position of methylparaben–BSA was shifted
slightly towards lower wavelength region. The change in kmax indicates the change in polarity around the tryptophan residue and the
change in peptide strand of BSA molecules and hence the change in
hydrophobicity [23]. These above observations signify that with
the addition of methylparaben, the peptide strands of BSA molecules were extended more and hydrophobicity was decreased [17].
Fig. 7. Absorption spectra of methylparaben, BSA and methylparaben–BSA system.
[BSA] = 5 lM (a) methylparaben concentration for methylparaben–BSA system was
at, (b) 5, (c) 10, (d) 15 and (e) 20 lM. A concentration of 5 lM methylparaben (x)
was used for methylparaben only.
reveals that the peak position of amide I was shifted from 1652
to1647 cm1 in the IR spectrum of BSA upon interaction with
methylparaben. This indicated that the methylparaben interacted
with BSA and the secondary structure of BSA was changed.
Site probe studies
Sudlow et al. [28] have suggested two main distinct binding
sites (sites I and II) in BSA. Site I of BSA has affinity for warfarin,
phenylbutazone, etc. and site II for ibuprofen, flufenamic acid,
etc. It is reported that digitoxin binding is independent of sites I
and II [29] and binds to site III. In order to establish the binding site
in BSA for methylparaben, competitive binding studies were performed using site probes, warfarin, ibuprofen, and digitoxin. For
this, emission spectra of ternary mixtures of methylparaben, BSA
and site probes were recorded separately. The corresponding binding constant values were evaluated and these are recorded in Table
2. The binding constant of methylparaben–BSA decreased remarkably in presence of warfarin while this value remained almost
same in presence of ibuprofen and digitoxin. These results revealed
FT-IR spectroscopic studies
Additional evidence for methylparaben–BSA interaction was
obtained from FT-IR spectra. Infrared spectrum of protein exhibited a number of amide bands due to different vibrations of the
peptide moiety. Of all the amide modes of the peptide group, the
single most widely used one in studies of protein secondary structure is the amide I. The amides I and II peaks occurred in the region
of 1600–1700 cm1 and 1500–1600 cm1, respectively (Fig. 8).
Amide I band is more sensitive to changes in protein secondary
structure compared to amide II. Hence, the amide I is more useful
for studies of secondary structure [26,27]. The FT-IR spectrum
Fig. 8. FT-IR spectra of (a) BSA and (b) BSA–methylparaben.
K.M. Naik, S.T. Nandibewoor / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 418–423
Table 2
Effect of warfarin, ibuprofen, digitoxin on binding constant of methylparaben–BSA
system.
Systems
Binding
constant
(M1)
No. of binding
sites (n)
R2
BSA + methylparaben
BSA + methylparaben + warfarin
BSA + methylparaben + ibuprofin
BSA + methylparaben + digitoxin
3.32 109
2.45 108
3.23 109
2.89 109
1.92
1.64
1.94
1.87
0.9990
0.9940
0.9810
0.9910
Table 3
Effect of common ions on binding constant of methylparaben–BSA system.
Systems (cations)
Binding constant
(M1)
No. of binding
sites (n)
R2
BSA + methylparaben
BSA + methylparaben + Co2+
BSA + methylparaben + Ni2+
BSA + methylparaben + Ca2+
BSA + methylparaben + Zn2+
BSA + methylparaben + Cu2+
3.32 109
2.00 109
2.10 1010
1.73 109
3.05 109
1.48 109
1.92
1.84
2.06
1.81
1.86
1.81
0.9990
0.9980
0.9990
0.9860
0.9980
0.9970
that the warfarin displaced methylparaben from the binding site
while ibuprofen and digitoxin had a little effect on the binding of
methylparaben to BSA. Hence, it is concluded that the methylparaben was bound to site I of BSA.
Effect of metal ions on the interactions of methylparaben with BSA
In plasma, there are some metal ions, which can affect the interactions of the drugs and serum albumins. Trace metal ions, especially the bivalent type are essential in the human body and play
an important structural role in many proteins. It is reported [30]
that Cu2+, Zn2+, Ni2+, Co2+ and Ca2+ and other metal ions can form
complexes with serum albumins. Hence, the effects of some metal
salt solutions viz., CuCl2, ZnCl2, NiCl2, CoCl2 and CaCl2 on the binding of methylparaben with BSA were investigated in the present
study. Under the experimental conditions, none of the cations gave
the precipitate in phosphate buffer. The binding constants of methylparaben–BSA in presence of above ions were evaluated and the
results are shown in Table 3. The binding constant of methylparaben–BSA system decreased in presence of Cu2+, Ca+2, Zn+2 and Co2+.
This was likely to be caused by a conformational change in the
vicinity of the binding site. The decrease in the binding constant
in presence of above metal ions would shorten the storage time
of the drug in blood plasma and hence more amount of free drug
would be available in plasma [23]. This led to the need for more
doses of drug to achieve the desired therapeutic effect in presence
of above ions. The binding constant increased in presence of Ni2+,
thereby indicating the formation of metal ion–methylparaben
complex via metal ion bridge. This led to the need for less dose
of drug for desired therapeutic effect. The larger stability of methylparaben–BSA complex is due to the larger number of binding
sites per BSA (n) (Table 3). This may prolong storage period of
methylparaben in blood plasma and enhance its maximum effects.
Conclusion
The present work provides an approach for studying the interactions of BSA with methylparaben using absorption, fluorescence
and FT-IR techniques under physiological conditions. The results
423
showed that BSA fluorescence was quenched by methylparaben
through dynamic quenching mechanism. Methylparaben interacted with BSA through hydrophobic forces. This work also reports
the distance between BSA and bound methylparaben based on Forster’s energy transfer theory. The remarkable change of amide I
peak position in the BSA infrared spectrum after interaction with
methylparaben indicated that secondary structure of BSA has been
changed. Since, the pharmaceutical firms need standardized
screens for protein binding in the first step of new drug design, this
kind of study of interaction between BSA with methylparaben
would be useful in pharmaceutical industry, life sciences and clinical medicine.
Acknowledgment
Keerti M. Naik thanks UGC, New Delhi for the award of Research
Fellowship in Science for Meritorious Students (RFSMS).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2012.12.055.
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