Journal of Molecular Liquids 243 (2017) 369379

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Journal of Molecular Liquids

journal homepage: www.elsevier.com/locate/molliq

Study on interactions of cationic gemini surfactants with folded and

unfolded bovine serum albumin: Effect of spacer group of surfactants
Sonu a,1, Sayantan Halder a, Sunita Kumari a, Rishika Aggrawal a, Vinod K. Aswal b, Subit K. Saha a,
a
Department of Chemistry, Birla Institute of Technology & Science (BITS), Pilani, Pilani Campus, Rajasthan 333 031, India
b
Solid State Physics Division, Bhabha Atomic Research Centre (BARC), Mumbai 400085, India

a r t i c l e i n f o a b s t r a c t

Article history: Interactions of three cationic gemini surfactants, 12-4-12, 2Br 12-8-12, 2Br and 12-4(OH)-12, 2Br with
Received 26 April 2017 natured and denatured protein, bovine serum albumin (BSA) have been studied by means of UVVisible absorp-
Received in revised form 16 July 2017 tion, steady-state and time-resolved uorescence, and circular dichromism (CD) spectroscopy. CD spectroscopic
Accepted 28 July 2017
study shows the change in the -helix and -strand content of protein with the concentration of gemini surfac-
Available online 30 July 2017
tants. Gemini surfactant with hydroxyl group in the spacer decreases the -helix of the BSA more efciently than
Keywords:
that without hydroxyl group in the spacer. Efciency to decrease the -helix of the protein increases with de-
BSA-Gemini surfactant interaction creasing the hydrophobicity of the spacer group of the surfactants at lower concentration range following the
Gemini surfactant with various spacers order, 12-8-12, 2Br b 12-4-12, 2Br b 12-4(OH)-12, 2Br. However, at higher concentration range of surfactant,
BSA uorescence the increasing order of providing hydrophobic environment to tryptophan (Trp) and tyrosine (Tyr) residues of
Fluorescence lifetime the protein is as follows: 12-4(OH)-12 b 12-4-12 b 12-8-12. Gemini surfactant with hydrophobic spacer group
provides more hydrophobic environment around Trp and Typ residues of the protein forming micelles like struc-
tures along the protein chain. In this concentration range, 12-8-12, 2Br interacts differently as compared to
other two surfactants which are evidenced by the data on excited state lifetime of the protein. It is more efcient
to form a particular conformer and/or puckerd ring of Trp as compared to other two surfactants. The microenvi-
ronment around Trp residues of BSA is perturbed to a greater extent than that around Tyr residues in presence of
gemini surfactants. Fluorescence from Trp and Tyr are quenched by acrylamide to a greater extent in presence of
12-8-12, 2Br. Interactions of denatured BSA with gemini surfactants also have been studied. 12-8-12, 2Br even
interacts with the BSA unfolded by guanidine hydrochloride (GdHCl) to a greater extent than that by 12-4-12,
2Br and 12-4(OH)-12, 2Br.
2017 Elsevier B.V. All rights reserved.

1. Introduction
Proteins have fundamental importance in living organism and take
part in many life processes. Serum albumins are found in blood plasma
and are considered as transport proteins [1,2]. Principal function of
serum albumin is to transport general anasthetic, variety of metabolites
and fatty acids [3]. Bovine serum albumin (BSA) is one of the proteins
that is commonly used for research purposes due to its water solubility,
stability, and versatile binding capacity [1]. In the primary structure of
BSA there are nine loops which are held together by 17 disulde
bonds, resulting in three domains (I, II, III) each consists of two sub do-
mains (A and B). BSA and human serum albumin (HSA) shares 76% se-
quence homology. One of the differences between these two proteins is
that BSA has two tryptophan (Trp) residues, but HSA has only one Trp
residue [46].
Corresponding author.
E-mail address: sksaha@pilani.bits-pilani.ac.in (S.K. Saha).
1
Present address: Department of School Education, Haryana, (India).
Protein can bind with various types of ligands such as fatty acids,
metal ions, drugs, surfactants, etc. [715]. Studying protein-surfactant
interactions is an active eld of research. Interaction of proteins with
surfactants has many applications in biotechnology processes, drug de-
livery, cosmetic systems, detergents, and biosciences [1619]. Surfac-
tants unfold the native proteins structure. Proteins have both
hydrophobic and hydrophilic amino acids and due to these properties
of amino acids, surfactant molecules bind with protein [20]. At low sur-
factant concentration, surfactants bind to the protein at high energy
specic binding sites. At high concentration of surfactant, the binding
between surfactant and protein is highly specic and non-cooperative
in nature. Even at a high concentration of surfactants, three dimensional
structure of a protein is destroyed and the protein is denatured. Interac-
tion between protein and surfactant molecules depends upon surfactant
features. Protein denatured by anionic surfactant like sodium dodecyl
sulphate is used to determine the molecular weight of the protein by
electrophoresis [21]. To prevent aggregation and unwanted adsorption
of protein during ltration and storage, non ionic surfactants are used
[22]. Cationic surfactants are used as bactericide in various systems by

http://dx.doi.org/10.1016/j.molliq.2017.07.122
0167-7322/ 2017 Elsevier B.V. All rights reserved.
370 Sonu et al. / Journal of Molecular Liquids 243 (2017) 369379

protein denaturation [2325]. Various techniques have been used for
the study of protein surfactant interactions [2633]. BSA has two Trp
residues at position 134 and 213 of amino acid sequence. Trp 134 locat-
ed on the surface of BSA [34]. Intrinsic uorescence of Trp is helpful to
study the structure and dynamics of BSA and also to investigate the iso-
therm of protein and surfactant interactions [35].
Many reports are available for study on interaction of various types
of single chain surfactants with protein [3639]. Another class of surfac-
tant called gemini surfactant has gained now-a-days more importance
over conventional monomeric surfactants. Gemini surfactants are
attracting special attention in material sciences, biological sciences,
nanotechnology, supramolecular chemistry etc. [40]. Gemini surfactants
are made up of two hydrophobic tails and two hydrophilic headgroups,
covalently joined by a spacer group at their headgroups. Some proper-
ties of gemini surfactants are superior to their conventional counterpart
[41]. The spacer part plays a signicant role for the aggregation proper-
ties of a gemini surfactant. The critical micelle concentration (cmc),
counter ions binding, thermodynamic properties, microviscosity and
micropolarity, rheological behavior, aggregation number etc. of gemini
surfactants vary with any change in the spacer part [40,4247]. The
spacer group of a gemini surfactant can be long or short, hydrophilic
or hydrophobic, exible or rigid [40].
Few reports are available for the study on interaction of gemini sur-
factant with proteins. Xu et al. have studied the interaction between BSA
and gelatine with gemini surfactant [48]. Jiange et al. [49] also have
studied the interaction of gemini surfactants with BSA. They have re-
ported the effect of spacer chain length of gemini surfactant. The gemini
surfactant with longer spacer chain length interact strongly with BSA
due to stronger hydrophobic interaction. Sinha et al. [50] have reported
the effect of hydroxyl group substituted spacer group of gemini surfac-
tant on protein-surfactant interactions. Moya et al. studied the binding
of cationic single chain and dimeric surfactant with BSA [51]. Kabir-
ud-din et al. have reported the comparative interaction of cationic single
chain and gemini surfactant with HSA [20]. These studies have showed
that -helical content of BSA decreases with increasing the concentra-
tion of gemini surfactant. Folding of denatured protein into its native
structure is important at fundamental and biotechnology level. Geneti-
cally engineered cells produced protein in non native form. The native
conformation is required for basic and biotechnological applications
[52].
In the present work we have demonstrated the effect of spacer
group of gemini surfactants, 12-4-12, 12-8-12 and 12-4(OH)-12
(Scheme 1) on interactions of surfactants with folded and unfolded
BSA. Although there are reports on the effect of spacer group on interac-
tions of gemini surfactants with folded or natured proteins, but to the
best of author's knowledge there are no such systematic reports on in-
teractions of gemini surfactants with unfolded or denatured proteins.
It has been reported that the effect of conventional surfactant on bind-
ing with BSA is less as compared to gemini surfactant [49,51]. Rather
et al. [52] have reported the refolding of BSA by gemini surfactants
assisted by articial chaperones. Mukherjee et al. [53] have studied
the effect of -cyclodextrin on BSA unfolded by sodium dodecyl sul-
phate. In the present work we have focused on the effect of spacer
group of gemini surfactants on their interactions with natured protein
and protein denatured by GdHCl using steady-state and time resolved
uorescence methods in absence of any articial chaperones.
2. Materials and methods
Gemini surfactants, 12-4-12, 12-8-12 and 12-4(OH)-12 were syn-
thesized according to the reported method [44,47]. The synthesized
compounds were recrystallized several times with the mixture of

2Br-
CH3
H3C
+
+ N
N CH3
H3C C12H25
C12H25

1,4-bis(dodecyl-N,N-dimethylammonium bromide)-butane (12-4-12)

2Br
H3C CH3
N
N
H3C CH3
C12H13
C12H13

1,8-bis(dodecyl-N,N-dimethylammonium bromide)-octane (12-8-12)

2Br-
CH3
H3C
+
+ N
N CH3
H3C C12H25
C12H25 OH

Scheme 1. Chemical structure of gemini surfactants.

 Sonu et al. / Journal of Molecular Liquids 243 (2017) 369379
methanol and ethylacetate and their structures were conrmed by FT-
IR and 1H NMR data (Table S1, Supporting information). BSA was pur-
chased from Sigma Chemical Company and was used as received.
GdHCl and acrylamide were purchased from Sd-ne Chemical Compa-
ny, India and were used as received. All solutions were prepared in
25 mM Na-phosphate buffer of pH = 7.25. Concentration of BSA in all
measurements was kept at 5.0 M.
The absorption spectra were recorded using Shimadzu (UV-1800)
UVVisible spectrophotometer with the sample in a 1 cm path length
quartz cuvette. The steady-state uorescence spectra were measured
using a Horiba Jobin Yvon Fluoromax-4 scanning spectroourimeter
with a 1 cm path length quartz cuvette. The slit width of 3 nm was
kept for all the uorescence measurements. In steady-state uorescence
experiments, all the samples were excited at 295 nm. Synchronous uo-
rescence spectra were recorded on the same spectrouorimeter. The
differences between excitation and emission wavelength () were
kept at 15 nm and 60 nm to get the contribution of Tyrosin (Try) and
Trp residues, respectively [48].
For time-resolved uorescence measurements, Horiba Jobin Yvon
Fluorocube-01-NL picosecond time-correlated single-photon counting
(TCSPC) experimental setup was used. A diode of wavelength 300 nm
(NanoLED 300, IBH, UK) was used as a light source. The uorescence de-
cays were collected at a magic angle (54.7) with a vertically excitation
beam using a TBX photon detection module (TBX-07C). The typical Full
Width at Half Maximum (FWHM) after deconvolution using a liquid
scatterer of the system response was about 900 ps for 300 nm diode.
IBH DAS-6 decay analysis software has been used for the analysis of de-
cays. All the decays were tted with a biexponential function. The good-
ness of ts was analyzed by both 2 criterion and visual inspection of the
residuals of the tted function to the data.
The far UV circular dichromism (CD) spectra of all the studied sys-
tems were performed using Chirascan CD spectropolarimeter (Applied
Photophysics Ltd.) in the wavelength range of 200260 nm. To ll the
sample a cuvette of path length of 0.1 cm was used. Each CD spectrum
was average of three scans with a scan speed of 200 nm min1 with a
spectral bandwidth of 10 nm. Surfactant buffer spectra were subtracted
from the recorded spectra of BSA and gemini surfactant for background
correction.
The UVVisible absorption, steady-state and time-resolved uores-
cence and circular dichroism (CD) spectra of BSA in presence of gemini
surfactants at concentrations below and well above their respective cmc
values have been recorded. The cmc values of 12-4-12, 12-8-12 and 12-
4(OH)-12 are 1.17 mM, 0.72 mM, and 0.78 mM, respectively [46,47].The
changes in the spectroscopic properties of BSA due to the presence of
gemini surfactants have been discussed in respective sections. All the
measurements were performed at 298 0.1 K.
3. Results and discussions
3.1. UVVisible absorption study
Surfactants bind strongly to proteins and bring a substantial change
in the protein conformation. Fig. 1 shows the UVVisible absorption
spectra of BSA in absence and presence of gemini surfactant, 12-4-12
as a representative one. An absorption spectrum of pure BSA shows
two absorption peak maxima one at 210 nm and other at 279 nm. The
absorption peak maximum at 210 nm corresponds to transition
of characteristic of C_O group in polypeptide backbone structure [54]
and the absorption peak maximum at 279 nm corresponds to n
transition of aromatic amino acids, i.e., Phenylalanine (Phe), Trytophan
(Trp), and Tyrosine (Tyr). In presence of a gemini surfactant, the absorp-
tion spectra of BSA show changes in absorption peak maxima and absor-
bance values i.e. UVVisible absorption properties are very much
sensitive to the concentration of gemini surfactants. The absorption
peak maximum for pure BSA located at 210 nm shifts towards longer
wavelength with increasing the concentration of gemini surfactant.
371
4.0
Pure BSA
3.5
[12-4-12]
3.0 0.08 mM
0.60 mM
Absorbance
2.5 8.00 mM
20.0 mM
2.0
1.5
1.0
0.5
0.0
180 210 240 270 300 330 360
Wavelength (nm)
Fig. 1. Absorption spectra of BSA at different concentrations of 12-4-12. [BSA] = 5.0 M.
Absorbance at 210 nm decreases continuously with increasing the con-
centration of gemini surfactant.
The observed changes in the spectroscopic properties of BSA are due
to the interaction of gemini surfactant with BSA. In presence of gemini
surfactant the microenvironment around amide group of BSA is differ-
ent as compared to the native protein. When amide groups of BSA are
exposed to water environment the energy of transition is de-
creased. The chromophore, C_O has higher polarity in the excited
state than that in the ground state. Polar water molecules stabilize the
energy of excited state more than that of ground state and that is why
absorption spectrum of BSA shows bathochromic shift with increasing
the concentration of gemini surfactant. Absorption maximum at
279 nm shows very little change in absorbance with increasing the con-
centration of gemini surfactant. Similar behaviors in the absorption
spectra are observed in presence of 12-8-12 and 12-4(OH)-12 gemini
surfactants as well.
3.2. Steady-state uorescence study
BSA has three types of aromatic amino acids residues, Phe, Trp, and
Tyr. These amino acids give intrinsic uorescence of BSA. The intrinsic
uorescence of BSA is mainly contributed by Trp and Tyr residues. Phe
has very low quantum yield and therefore uorescence from Phe can
be ignored [55]. BSA has two Trp amino acid residues at 134 and 213
(Trp 134 and Trp 213) positions of amino acid sequence in domains I
and II, respectively. Trp 134 is located at the protein surface in domain
I (subdomain IC), and Trp 213 is located in the hydrophobic binding
pocket of protein in domain II (subdomain IIA). The intrinsic uores-
cence of BSA is sensitive towards the change in microenvironment.
Thus, uorescence analysis is used as an effective method for the
study of interaction of BSA with surfactant molecules [48,49,51,54,56].
Fig. 2 shows the emission spectra of BSA in absence and presence of
gemini surfactant, 12-4-12 as a representative one. Spectrum of pure
BSA shows uorescence peak maximum at 347 nm. Fig. 3 shows the
variation in uorescence intensity of BSA with changing the concentra-
tion of all three surfactants. With increasing the concentration of gemini
surfactant (12-4-12) upto 0.2 mM, the uorescence intensity of BSA is
decreased with a shift in the uorescence peak maximum towards
shorter wavelength at 335 nm (Fig. 4). Further increase in the concen-
tration of 12-4-12 upto 4.0 mM, a rise in uorescence intensity has
been observed with a red shift in uorescence peak maximum. Beyond
4.0 mM, uorescence intensity remains almost constant. Similar type of
variation in the uorescence intensity of BSA has been observed for
other gemini surfactants, 12-8-12 and 12-4(OH)-12 as well. At very
low concentration (0.0 mM0.005 mM) of all three gemini surfactants,
372 Sonu et al. / Journal of Molecular Liquids 243 (2017) 369379
Fluorescence Intensity (a.u.)
320 340 360 380 400
Wavelength (nm)
Fig. 2. Steady-state uorescence spectra of BSA in presence of various concentrations
(0.001 mM to 6.0 mM) of 12-4-12. [BSA] = 5.0 M. ex = 295 nm.
the uorescence intensity of BSA is increased without much change in
the uorescence peak maximum. The rise in uorescence intensity is
due to the interaction of gemini surfactant at very high energy binding
sites of BSA which gives rise to compactness to the BSA native structure
[53,57]. It indicates that at a low concentration of surfactant (1:1 molar
ratio of BSA and gemini) the secondary structure of BSA remains unal-
tered and the tertiary structure gets affected [53]. The high energy bind-
ing interaction is found to be more signicant in case of 12-4(OH)-12 as
compared to other two surfactants, 12-4-12 and 12-8-12.
These observations indicate that gemini surfactants, 12-4-12, 12-8-
12 and 12-4(OH)-12 interact with BSA and alter the native structure
of BSA. Variation in the uorescence intensity of uorophores indicates
that microenvironment around uorophores change with the concen-
tration of gemini surfactant. Surfactant molecules unfold the native
structure of protein [58]. Ionic surfactants interact strongly with protein
by hydrophobic interaction between surfactant tail and nonpolar amino
acids, and by electrostatic attraction of headgroups of surfactant and op-
positely charged amino acids. The secondary structure of BSA is altered
at high concentration of surfactant [48]. It has been observed that the
percentage of -helix decreases with increasing the amount of surfac-
tant [48]. As mentioned above for 12-4-12 surfactant, the uorescence
1.2
1.1
1.0
0.9
F/F0
0.8
0.7
0.6
0.5
0.4
0 2 4 6
[Gemini Surfactant]/mM
Fig. 3. Plot of uorescence intensity of BSA with different concentration of gemini
surfactants. [BSA] = 5.0 M. ex = 295 nm.
Pure BSA 350 12-4-12
[12-4-12] 348 12-4(OH)-12
0.001 mM 12-8-12
346
0.01 mM
344
0.08 mM
0.2 mM 342
max
fl
0.8 mM 340
6.0 mM 338
336
334
0 2 4 6 8 10
[Gemini Surfactant]/mM
420 440
Fig. 4. max of BSA in presence of various concentration of 12-4-12, 12-4(OH)-12 and 12-
8-12 at ex = 295 nm. [BSA] = 5.0 M.
intensity is decreased upto 0.2 mM which is due to the unfolding of
BSA. In the unfolded state of BSA, Trp and Tyr residues are exposed to
the polar environment and that is why uorescence intensities of Trp
and Tyr residues are reduced [7]. Enhancement in the uorescence in-
tensities at comparatively higher concentration of gemini surfactant in-
dicates that Trp and Tyr residues get located in the nonpolar
environment [51]. Fig. 4 shows variations of uorescence peak maxima
of BSA at various concentrations of gemini surfactants. These variations
are in accordance with the change in uorescence intensities.
It is very clear from the Fig. 3 that up to 0.2 mM concentration of sur-
factant, the change in uorescence intensity is lowest with 12-8-12 and
the same is highest with 12-4(OH)-12 i.e. the increasing order of inter-
actions of gemini surfactants with protein is 12-8-12 b 12-4-12 b 12-
4(OH)-12. It indicates that the chemical nature of spacer group of gem-
ini surfactant has effect on the denaturation of protein i.e. to decrease
the -helix. Sinha et al. [50] have also indicated the similar effects. The
present study demonstrates that the extent of denaturation of protein
increases with decreasing hydrophobicity of the spacer group of gemini
surfactants at lower concentration range keeping the hydrocarbon tail
lengths of surfactants xed. We have also observed that 12-4(OH)-12
even shows comparatively stronger high energy binding site interac-
tion. This is due to the presence of one hydroxyl group in the spacer
part of 12-4(OH)-12. Gemini surfactant, 12-4(OH)-12 may interact
with BSA through hydrogen bonding and through lone pair of oxygen
atom [59]. It is noteworthy that the concentrations of 12-8-12, 12-4-
12 and 12-4(OH)-12 at which the minimum of F/Fo occurs in Fig. 3 are
0.04 mM, 0.20 mM and 0.30 mM, respectively. It infers that the spacer
chain length has control over the efciency of a surfactant molecule to
decrease the -helix. 12-8-12 with maximum hydrophobicity in the
spacer group is least efcient and 12-4(OH)-12 with minimum hydro-
phobicity in the spacer group is most efcient to decrease the -helix.
However, 12-8-12 surfactant at a concentration above 0.5 mM (Fig. 3)
provides an environment around uorophore which is more hydropho-
bic than that in folded protein. Thus the micelles of 12-8-12 formed
along the protein chain provides comparatively more hydrophobic en-
vironment to the uorophore because the spacer group of 12-8-12 is
12-4-12 more hydrophobic than that of 12-4-12 and 12-4(OH)-12.
12-4(OH)-12
12-8-12 3.3. Synchronous uorescence spectroscopy study
8 10 Synchronous uorescence spectroscopy is an effective technique to
explore the microenvironment of amino acid residues [48,60]. Synchro-
nous uorescence technique involves simultaneous scanning of excita-
tion and emission monochromators while maintaining a constant
wavelength interval between them. When the wavelength interval
 Sonu et al. / Journal of Molecular Liquids 243 (2017) 369379 373

(, difference between excitation and emission wavelength) is kept at
15 nm and 60 nm, synchronous uorescence spectra give information
about Tyr and Trp residues, respectively [48,60]. Fig. 5 shows the syn-
chronous uorescence spectra of BSA for = 60 nm in presence of
12-4-12, 12-8-12 and 12-4(OH)-12. With increasing the concentration
of each of 12-4-12, 12-8-12 and 12-4(OH)-12, there is a tendency for
decrease in uorescence intensity with blue shift in uorescence peak
maxima. It indicates that the conformation of BSA is changed in the
presence of gemini surfactant. Fig. 6 shows the synchronous uores-
cence spectra of BSA for = 15 nm in presence of each of 12-4-12,
12-8-12 and 12-4(OH)-12. At = 15 nm, uorescence intensity in-
creases with increasing the concentration of gemini surfactants [48].
In addition, the uorescence intensity for Trp is higher than that for
Tyr with greater extent of variation in the uorescence intensity of the
former as compared to the latter. These results can be depicted based
on discussion made by Ruiz et al. [61] in their work on protein-surfac-
tant interactions as follows: (i) Major intrinsic uorescence is contribut-
ed by the Trp residue of BSA, (ii) There is resonance energy transfer
from Phe to Tyr to Trp, (iii) With increasing concentration of surfactant
the conformation of BSA is changed. As a result of that the distance be-
tween donor and acceptor increases. Thus the energy transfer from Tyr
to Trp is reduced leading to enhancement in Tyr uorescence and
quenching of Trp uorescence. It is noteworthy that when =
60 nm, the synchronous uorescence peak maximum (337 nm) is
close to that as observed in Fig. 2 (347 nm). In fact in case of 128-12
the peak maximum is exactly 347 nm. From these observations it is
clear that the microenvironment around Trp residues of BSA is
perturbed to a greater extent than that around Tyr residues in presence
of gemini surfactants and 12-8-12 has strongest effect on it.
3.4. Binding of gemini surfactant with BSA: Study of binding isotherm
The nature of protein-surfactant interaction can be understood by
using the binding isotherm of protein and surfactant. If a protein (BSA
in the present study) has n0 available sites for binding of a given surfac-
tant, and at a certain stage surfactant molecules bind to n sites at BSA,
then the fraction () of BSA occupied by surfactant is given by = n/
n0 [56,61]. At saturation binding condition, when all the possible sites
are occupied by surfactant then = 1, and in the absence of the surfac-
tant, = 0. The fractional change in the uorescence of BSA () due to
the binding of gemini surfactant has been calculated by Eq. (1) [56,61,
62]


IIo
1
Imin Io
where I is the uorescence intensity at any surfactant concentration, Io is
the uorescence intensity in the absence of surfactants and Imin is the
uorescence intensity at saturation level. Fig. 7 displays the variation
in with the concentration of studied gemini surfactants, 12-4-12,
12-8-12 and 12-4(OH)-12. With increasing the concentration of surfac-
tant, generally the protein-surfactant isotherm shows three regions
prior to the saturation region [50,56] viz. (1) specic binding, (2) non-
cooperative binding, and (3) co-operative binding. In Fig. 7, region 1
(not shown) includes the total surfactant concentration from 0 mM to
0.03 mM. In this concentration region the binding isotherm rises very
slowly due to ionic binding of surfactant molecules with protein at spe-
cically high energy binding sites [56] (Scheme S1). Region 2 shows the
non-cooperative binding region. In this region, the binding isotherm
rises sharply with surfactant concentration from 0.03 mM to 0.4 mM.
As mentioned above in this concentration range the uorescence inten-
sity is dropped drastically and BSA conformation is altered. Above
0.4 mM the binding isotherm rises gradually. This represents the mas-
sive cooperative binding between BSA and surfactant molecules [51].
In this region, the surfactant molecules form micelles like structure
along the protein chain (Scheme S1). Beyond region 3, a plateau has
been observed which indicates the saturation of binding of gemini sur-
factant. Hence, binding of surfactants with BSA passes through four dif-
ferent stages. Das et al. [56] also observed four regions of binding of
surfactant with protein.

Pure BSA
Fluorescence Intensity (a.u.)

60000000
a 2000000
Fluorescence Intensity (a.u.)

[12-4-12] Pure BSA

50000000 0.01 mM [12-8-12]
0.05 mM b 0.01 mM
1500000
40000000 0.08 mM 0.04 mM
0.10 mM 0.3 mM
30000000 1000000 0.5 mM

20000000
500000
10000000

0 0
310 320 330 340 350 360 370 320 330 340 350 360 370 380
Wavelength(nm)
Wavelength (nm)
Fluorescence Intensity (a.u.)

60000000 pure BSA

C [12-4(OH)-12]
50000000 0.01 mM
0.04 mM
40000000 0.20 mM
0.50 mM
30000000

20000000

10000000

0
310 320 330 340 350 360 370 380
Wavelength (nm)

Fig. 5. Synchronous uorescence spectra of BSA at = 60 nm in presence of (a) 12-4-12, (b) 12-8-12 and (c) 12-4(OH)-12. [BSA] = 5.0 M.
374 Sonu et al. / Journal of Molecular Liquids 243 (2017) 369379

16000000 Pure BSA

Pure BSA 600000
b

Fluorescence Intensity (a.u.)

[12-8-12]
a

Fluorescence Intensity (a.u.)

14000000 [12-4-12] 0.01mM
0.01 mM 500000 0.04 mM
12000000 0.3 mM
0.05 mM
2 mM
0.08 mM 400000 6 mM
10000000
0.60 mM 10 mM
8000000 3.00 mM 300000
8.00 mM
6000000
15.0 mM 200000
4000000
100000
2000000

0 0
280 290 300 310 320 330 280 290 300 310 320 330
Wavelength (nm) wavelength(nm)

18000000
Fluorescence Intensity (a.u.)

16000000 Pure BSA

[12-4(OH)-12]
14000000 c 0.01 mM
0.04 mM
12000000
0.20 mM
10000000 2.00 mM
6.00 mM
8000000 12.0 mM
6000000

4000000

2000000

0
280 290 300 310 320 330
Wavelength (nm)

Fig. 6. Synchronous uorescence spectra of BSA at = 15 nm in presence of (a) 12-4-12, (b) 12-8-12 and (c) 12-4(OH)-12. [BSA] = 5.0 M.

3.5. Circular dichroism study
Circular dichroism (CD) study reveals the informations about the
change in the conformation of the protein. The far-UV CD spectra in
the range of 200 nm to 260 nm have been recorded for pure BSA and
BSA in presence of gemini surfactants. The representative CD spectra
are given in Fig. S1. CD spectra show two characteristic negative bands
for -helix of BSA at 210 nm and 223 nm [63]. There is a decreasing ten-
dency of negative molar ellipticity with increasing the concentration of
gemini surfactant which shows that the secondary structure of BSA is al-
tered in the presence of a gemini surfactant. The changes in the -helical
and -sheet content upon addition of gemini surfactant have been cal-
culated using K2D3 secondary structure analysis software and the data
1.0
4 3.6. Time-resolved uorescence spectroscopic study
3
obtained are given in Table 1. In case of pure BSA the -helix content
is 67.74% which is comparable to the reported value [63,64]. The de-
crease in the values of % of -helix of BSA with gemini surfactant con-
centration indicates that gemini surfactant interacts with BSA and
unfolds it. In case of 12-8-12 we could not calculate the values of % of
-helix and % of content beyond 1.00 mM of its concentration because
of appearance of excessive noise in the CD spectra at those concentra-
tions. However, while comparing these values at 1.00 mM concentra-
tions of all three surfactants and at 5.00 mM concentrations of 12-4-
12 and 12-4(OH)-12, it is revealed that the increasing order of alteration
of secondary structure of BSA is as follows: 12-8-12 b 12-4-12 b 12-
4(OH)-12. The presence of hydroxyl group in the spacer group of 12-
4(OH)-12 enhances the unfolding of BSA to a greater extent than that
by the spacer groups present in other two surfactants without any hy-
droxyl group in them.

0.8 Time resolved uorescence measurement is an effective method to

study protein conformational dynamics. Trp exists in various rotational
conformers and due to this Trp exhibits multiple exponential decays
0.6
BSA + 12-4-12 Table 1
-Helix and strand content of BSA in the presence and absence of gemini surfactant.
BSA + 12-4(OH)-12
0.4 BSA + 12-8-12 BSA/gemini surfactant system [Gemini surfactant] % of -helix % of strand
2 (mM)

BSA/12-4(OH)-12 0.00 67.74 8.74

0.2 0.10 67.79 8.47
1.00 66.58 9.07
5.00 3.28 32.3
BSA/12-4-12 0.10 67.60 8.69
0.0 1.00 66.62 9.08
0.000 0.005 0.010 0.015 0.020 0.025 5.00 18.95 28.74
BSA/12-8-12 0.01 67.73 8.72
[Gemini surfactant] /M 0.05 67.45 8.61
0.20 67.54 8.37
1.00 66.74 8.86
Fig. 7. Binding isotherms of 12-4-12, 12-8-12 and 12-4(OH)-12 with BSA.
 Sonu et al. / Journal of Molecular Liquids 243 (2017) 369379 375

[55,65]. The three rotational conformers (1, 2 and 3) of Trp are shown by Table 2
Scheme S2. It is believed that the conformer 3 is quite stable and conver- Excited state lifetimesa,b of BSA in presence of 12-4-12.

sion from 3 to either 1 or 2 and vice versa is difcult in nanosecond time [12-4-12] (mM) a1 1 a2 2 2
scale. The shorter excited state lifetime of Trp is due to the existence of (ns) (ns) (ns)
conformer 3 and the longer excited state lifetime arises due to the rapid 0.0 0.29 3.05 0.71 6.80 6.22 1.01
inter-converting conformers, 1 and 2. It is also believed that in the 0.0005 0.32 2.34 0.68 6.29 5.70 1.00
ground state the indole ring is slightly puckered, but it becomes planer 0.001 0.29 2.66 0.71 6.38 5.84 1.05
0.005 0.32 2.42 0.68 6.24 5.65 1.05
in the excited state, which leads to the delocalization of lone pair of elec-
0.01 0.41 2.57 0.59 6.35 5.52 1.02
tron from nitrogen atom to the aromatic ring. Upon unfolding process of 0.05 0.51 2.35 0.49 5.84 4.81 1.01
protein, the interacting ligand approaches to the Trp residues of protein 0.08 0.60 2.34 0.40 5.98 4.63 1.04
and distorts the planarity of indole ring. Trp residue is also exposed to 0.1 0.58 2.23 0.42 5.73 4.51 1.01
the bulk solvent and that is why the excited state lifetime of Trp is de- 0.2 0.62 2.62 0.38 6.09 4.66 1.00
0.4 0.63 2.73 0.37 6.51 4.93 1.05
creased [54,57]. We have measured the excited state lifetime of Trp 0.5 0.51 2.61 0.49 6.17 5.08 1.04
moiety of BSA by exciting the sample using a 300 nm light emitting 0.6 0.52 2.63 0.48 6.23 5.10 1.04
diode (LED). Bi-exponential decays of BSA were observed in presence 0.8 0.45 2.22 0.55 5.90 5.03 0.96
and absence of studied gemini surfactants. Fig. 8 shows uorescence de- 0.9 0.52 2.61 0.48 6.20 5.08 1.02
1.0 0.56 2.78 0.44 6.31 5.04 0.98
cays of native BSA and BSA in presence of various concentration of 12-4-
2.0 0.49 2.45 0.51 5.92 4.93 1.05
12 as representative. Tables 2 and 3 represent the excited state lifetime 4.0 0.54 2.62 0.46 5.99 4.85 1.08
values of BSA in presence of various concentrations of 12-4-12 and 12- 5.0 0.50 2.28 0.50 5.72 4.74 0.99
8-12, respectively and Table S2 represents the same for 12-4(OH)-12. 6.0 0.53 2.68 0.47 5.86 4.78 1.01
Average excited state lifetime, has been calculated by using the 8.0 0.47 2.37 0.53 5.68 4.79 1.00
10.0 0.52 2.39 0.48 5.52 4.52 1.05
following Eq. (2) [55,61]:
a
ex = 300 nm.
X b
em is the uorescence peak maximum in respective system.
ai 2i
X
i
2
ai i decreased. It means that in this concentration range due to unfolding
i of BSA the relative population of conformer 3 is increased and that of
interconverting conformers, 1 and 2 is decreased. Similar behavior
where ai is the pre-exponential factor of i-th component and i is the also has been observed in presence of 12-4(OH)-12 as well (Table S2).
lifetime of the i-th component. Fig. 9 represents the variation in average However, it is different with 12-8-12 surfactants. Enhancement in the
excited state lifetime of BSA in presence of the gemini surfactants. Excit- excited state lifetime has been observed with further increasing the
ed state lifetime of pure BSA is close to the reported lifetime value [53, concentration of gemini surfactants from 0.2 mM to 1.0 mM for 12-4-
57]. Excited state lifetime decreases upto 0.1 mM concentration of all 12. Above 0.4 mM of 12-4(OH)-12 the excited state lifetime values re-
three surfactants. It can be noted here that the steady-state uorescence main almost steady. It can be seen in Fig. 9 that at higher concentration
intensity of Trp is decreased in this concentration range due to the con- of 12-4(OH)-12, the average excited state lifetime values of BSA are
formational change in the native structure of BSA. Lowering of excited lower than that in presence of 12-4-12 gemini surfactant. The effect of
state lifetime of BSA also supports the conformational change of BSA hydroxyl group of gemini surfactant, 12-4(OH)-12 on interaction with
in presence of gemini surfactants. BSA is also evidenced by the excited state lifetime data. It is noteworthy
As mentioned above the shorter lifetime is for the conformer 3 and that the effect of concentration of gemini surfactants on steady-state
the longer lifetime is due to the rapid interconverting conformers, 1 uorescence intensities of BSA (Fig. 3) is consistent with that on the ex-
and 2. The contributions of decay components represent the relative cited state lifetime of BSA (Fig. 9) in cases of 12-4-12 and 12-4(OH)-12
populations of various conformers. The contribution of fast component i.e. the former one provides more hydrophobic environment around Trp
is increased from concentration 0.01 mM to 0.1 mM for 12-4-12 gemini
surfactant and simultaneously the contribution of slow component is
Table 3
Excited state lifetimesa,b of BSA in presence of 12-8-12.
10000 [12-8-12] (mM) a1 1 a2 2 2
Prompt
(ns) (ns) (ns)
Native BSA
0.0 0.29 3.05 0.71 6.80 6.22 1.01
8000 [12-4-12]
Counts (a.u.)

0.001 0.66 1.44 0.34 6.05 4.59 1.12

0.2 mM 0.005 0.61 1.57 0.39 6.09 4.79 1.17
2 mM 0.01 0.56 1.34 0.44 5.82 4.80 1.19
6000 0.04 0.56 1.34 0.44 5.82 4.80 1.19
8 mM 0.1 0.62 0.54 0.38 3.15 2.58 1.13
10 mM 0.3 0.56 1.44 0.44 5.51 4.49 1.18
4000 0.4 0.37 1.38 0.63 4.25 3.79 1.10
0.5 0.58 1.62 0.42 5.77 4.61 1.08
0.6 0.58 1.90 0.42 6.00 4.75 1.14
2000 0.8 0.56 1.48 0.44 5.61 4.57 1.17
0.9 0.54 1.75 0.46 5.81 4.75 1.19
1.0 0.60 1.33 0.40 5.54 4.43 1.19
0 2.0 0.56 1.29 0.44 5.22 4.28 1.18
4.0 0.61 0.80 0.39 4.54 3.73 1.10
12 14 16 18 20 5.0 0.62 0.78 0.38 4.52 3.70 1.15
Time (ns) 6.0 0.64 0.65 0.36 4.08 3.32 1.18
8.0 0.66 0.64 0.34 4.17 3.36 1.19
10.0 0.60 0.64 0.40 3.90 3.26 1.19
Fig. 8. Fluorescence decays of native BSA and BSA in presence of various concentration of
a
12-4-12. ex = 300 nm, em is uorescence peak maximum in respective system, [BSA] = ex = 300 nm.
b
5 M. em is the uorescence peak maximum in respective system.
376 Sonu et al. / Journal of Molecular Liquids 243 (2017) 369379
12-4-12
6 BSA + 12-4(OH)-12
12-4(OH)-12
12-8-12
5 BSA
< > / ns
4 3
3
2
0 2 4 6 8 10
[Gemini Surfactant]/mM
Fig. 9. Variation of excited state average lifetime of BSA with the concentration of gemini
surfactants. ex = 300 nm, em is the uorescence peak maximum in respective system,
[BSA] = 5.0 M.
as compared to the latter. However, lifetime values are lower in case of
12-8-12 as compared to that of other two surfactants. This is in contrary
to that is observed in the variation of uorescence intensities with the
concentration of gemini surfactants. In addition to that the data in
Tables 2, 3 and S2 show that the contributions of the faster components
are mostly lower than that of the slower components in case of 12-4-12
and 12-4(OH)-12. However, the contribution of the faster component is
greater than that of the slower components in case of 12-8-12. Also life-
time values of fast components for 12-8-12 are shorter than that for 12-
4-12 and 12-4(OH)-12 with similar lifetime values for longer compo-
nents for all three surfactants. The synchronous uorescence spectra
at = 60 nm in case of 12-8-12 is also quite different from that of
12-4-12 and 12-4(OH)-12. It infers that the hydrophobic spacer group
interacts strongly with the Trp residues. Although 12-8-12 is not suf-
ciently effective to decrease the -helix of protein, but has profound ef-
fect to make the indole ring puckered. Thus these results indicate that
12-8-12 surfactant is not efcient to expose the Trp ring to the bulk sol-
vent, but has potential effect to make the indole ring non-planer. More-
over, the comparatively greater contribution of the faster component
and shorter lifetime of fast component in case of 12-8-12 might indicate
that possibility of having conformer 3 and/or puckered Trp ring is more
with 12-8-12 than that with 12-4-12 and 12-4(OH)-12. Zana et al. [44]
have reported that the gemini surfactants with spacer group containing
N56\\CH2 unit form a loop extended towards the hydrophobic core of
the micelle. Probably 12-8-12 with a loop in its spacer might be effective
to form a particular conformer and/or puckerd ring of an amino acid res-
idue of protein i.e. Trp in the present case.
3.7. Fluorescence quenching of BSA by acrylamide
Fluorescence spectra of BSA have been recorded at various concen-
trations of acrylamide as a quencher in presence and absence of gemini
surfactants (5.0 mM). The Stern-Volmer plot in case of pure BSA is devi-
ated from the linearity at high concentration of acrylamide (Fig. 10). The
upward curvature might indicate that the quenching of pure BSA is dy-
namic as well as static in nature [55]. However, both uorescence inten-
sity and uorescence lifetime of BSA in presence of gemini surfactants
(5.0 mM) continuously decreases with increasing the concentration of
acrylamide following the Stern-Volmer [55] Eq. (3):
F o = F o = 1 K SV Q  1 kq o Q  3 creases and uorescence maximum shifts towards longer wavelength
6
5 BSA + 12-4-12
BSA + 12-8-12
4
F0/F
2
1
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
[Acrylamide] /M
Fig. 10. Stern-Volmer plot of BSA and BSA -12-4-12/12-8-12/12-4(OH)-12 systems. [BSA]
= 5.0 M [12-4-12] = [12-4(OH)-12] = [12-8-12] = 5.0 mM.
where Fo and F are the uorescence intensities of BSA in absence and
presence of acrylamide. KSV and kq are the Stern-Volmer and bimolecu-
lar quenching rate constants, respectively. [Q] is the concentration of
quencher. o is the excited state lifetime in absence of quencher. These
results infer that the quenching mechanism is only dynamic in nature
in presence of surfactants (Fig. 10). The Stern-Volmer quenching con-
stant (KSV) values in all the studied systems have been calculated
using Eq. (3). For the calculation of KSV, the linear part of the quenching
data has been used. While the KSV value for pure BSA was found to be
4.70 M1, the KSV values for BSA in presence of each of 12-4-12, 12-8-
12 and 12-4(OH)-12 were found to be 4.78 0.40 M 1, 4.28
0.33 M1 and 4.02 0.35 M1, respectively. The values of bimolecular
quenching rate constant (kq) for pure BSA and in presence of each of 12-
4-12, 12-8-12 and 12-4(OH)-12 have been calculated using equation, kq
= KSV / o and found to be 7.56 108 M1 s1, 1.01 109 M1 s1, 1.16
109 M1 s1 and 9.33 0.14 108 M1 s1, respectively. kq for BSA
in presence of each of gemini surfactants is higher than that in pure BSA.
In absence of gemini surfactant, Trp and Tyr residues of pure BSA those
are accessible to the acrylamide get quenched. In presence of gemini
surfactants, there is a change in the conformation of BSA. The buried
Trp and Tyr residues are more exposed to the quencher in presence of
a gemini surfactant and that is why quenching rate constant increases.
The increasing order of quenching rate constant is 12-4(OH)-12 b 12-
4-12 b 12-8-12. This is also the order of hydrophobicity provided to
Trp and Tyr residues at higher concentration range of surfactant. The
surfactant molecules at 5.0 mM form micelles along the BSA chain.
Even if the hydrophobicity of environment around Trp and Tyr residues
is high with 12-8-12, the quenching occurred by acrylamide is also high.
This might be indicating that the acrylamide molecules rst get solubi-
lized in micelles and then quench the uorescence. It might be that the
number of acrylamide molecules solubilized in micelles is maximum for
12-8-12 and minimum for 12-4(OH)-12.
3.8. Interaction of gemini surfactant with unfolded BSA
Interaction of gemini surfactant with unfolded BSA has been studied
by steady-state and time resolved uorescence methods. BSA has been
denatured by guanidine hydrochloride (GdHCl). Fluorescence spectra of
BSA have been recorded in presence of various concentration of GdHCl.
Recorded uorescence spectra of BSA in presence of GdHCl are similar
to that reported in the literature [57]. Intensity of uorescence de-
 Sonu et al. / Journal of Molecular Liquids 243 (2017) 369379 377

with increasing the amount of GdHCl. At 4.0 M GdHCl, the uorescence

1.5
peak maximum of BSA appears at 358 nm. Fig. 11 shows the variation in
average excited state lifetime of BSA with increasing concentration of
1.4
GdHCl. Excited state lifetime initially increases and then decreases
with increasing the concentration of GdHCl. These results clearly show
1.3
the unfolding of BSA in presence of GdHCl.
Mukherjee et al. [53] have studied the interaction of BSA by -cyclo- 1.2
dextrin. They have reported that -cyclodextrin remains inactive for

refolding of BSA denatured by GdHCl. Present study have demonstrated
the interaction of gemini surfactants, 12-4-12, 12-8-12 and 12-4(OH)-
12 with unfolded BSA. BSA is rst denatured by using 4.0 M GdHCl
and then a gemini surfactant of varying concentrations is added. Circu-
lar dichroism (CD) spectra do not show any change in peak position
with changing concentration of gemini surfactant (spectra not
shown). So, refolding does not takes place in presence of gemini surfac-
tants. Fig. S2 shows the steady-state uorescence spectra to demon-
strate the interaction of unfolded BSA with various concentrations of
12-4-12, 12-8-12 and 12-4(OH)-12. Although for 12-4-12 we could go
maximum concentration up to 20.0 mM, but for 12-8-12 and 12-
4(OH)-12 the sample with maximum concentration prepared were
8.0 mM and 10.0 mM, respectively beyond which the precipitation in
the solutions was observed. Fluorescence intensity of BSA progressively
increases with increasing the concentration of gemini surfactants.
Therefore, gemini surfactant molecules create hydrophobic environ-
ment around Trp and Try residues along the protein chain, may be by
forming micelle-like structures. The increasing order of enhancement
in uorescence intensity is as follows: 12-4(OH)-12 b 12-4-12 b 12-8-
12 (Fig. 12) which is similar to that in absence of GdHCl (Fig. 3). Fluores-
cence peak maximum is shifted from 358 nm to shorter wavelength
with increasing the concentration of 12-4-12, 12-8-12 and 12-4(OH)-
12. In presence of 8.0 mM of each of 12-4-12, 12-8-12, and 12-4(OH)-
12, the uorescence peak maximum appears at 348 nm, 343 nm and
353 nm, respectively. At 10.0 mM of each of 12-4-12 and 12-4(OH)-
12, the uorescence peak maximum appears at 347 nm and 353 nm, re-
spectively. The uorescence peak maximum of pure BSA is located at
347 nm. This result shows that 12-8-12 provides an environment
around Trp and Tyr residues in the unfolded BSA chain which is more
hydrophobic than that with other two surfactants. For 12-8-12, it is
even more hydrophobic than that in pure BSA. Thus hydrophobic envi-
ronment created by gemini surfactants in presence of GdHCl is similar to
that in absence of GdHCl.
F/F0
1.1
1.0 12-4-12
12-4(OH)-12
0.9
12-8-12
0.8
0 2 4 6 8
[Gemini Surfactant]/mM
Fig. 12. Plot of variation of uorescence intensity of BSA unfolded by 4.0 M GdHCl with
increasing concentration of gemini surfactants.
In presence of 20.0 mM and 10.0 mM concentration of 12-4-12, the
intensity gains are 68% and 63%, respectively. For 12-4(OH)-12 at
10.0 mM concentration, the gain in intensity is about 51%. With
8.0 mM of 12-8-12, the intensity was greater than that for pure BSA.
In fact in absence of GdHCl also one can see that at higher concentration
of 12-8-12 the uorescence intensity is higher than that for pure BSA
(Fig. 3) which is in contrast to that in presence of other two surfactants.
These observations indicate that in presence of gemini surfactants, the
denatured BSA regain substantial amount of hydrophobic environment
and gain in that environment is more in presence of 12-8-12 than that of
12-4-12 and 12-4(OH)-12. In fact it is substantially different in case of
12-8-12. Excited state lifetimes also have been measured to support
the above mentioned process. Fig. S3 shows the uorescence decays of
unfolded BSA in absence and presence of 12-4-12 as representative.
Similar decays have been recorded for other three surfactants as well.
Lifetime values are tabulated in Tables S3S5. Fig. 13 shows the varia-
tion of excited state lifetime for interaction with various concentrations
of gemini surfactants. This variation is similar to that in uorescence in-
tensities (Fig. 12). As far as 12-8-12 is concerned, it is different from that
in absence of GdHCl at higher concentration of surfactant. Different
trends in lifetime data for 12-8-12 in presence of GdHCl might be indi-
cating that to what extent the conformation of Trp can be changed by

5.0
6
4.5

4.0
5 3.5
/ ns

< > / ns

3.0

2.5
4
<

2.0
12-4-12
1.5 12-4(OH)-12
12-8-12
3 1.0
0 2 4 6 8
0 1 2 3 [Gemini Surfactant]/mM
GdHCl / M
Fig. 13. Average excited state lifetime of unfolded BSA for surfactant interaction process in
Fig. 11. Variation of excited state lifetime of BSA in presence of GdHCl. [BSA] = 5.0 M, ex presence of 12-4-12, 12-8-12 and 12-4(OH)-12. ex = 300 nm, em is the uorescence
= 300 nm. peak maximum in respective system, [BSA] = 5.0 M, [GdHCl] = 4.0 M.
378 Sonu et al. / Journal of Molecular Liquids 243 (2017) 369379
the spacer group of a gemini surfactant depends on the extent of dena-
turation of the protein. In presence of 4.0 M GdHCl, the extent of dena-
turation of protein is quite high.
Like isotherm plot (Fig. 7) the interaction of denatured BSA with sur-
factant at higher concentration range also occurs in step wise manner.
Upto 0.5 mM concentration of each of gemini surfactants, there is a sud-
den increase in lifetime. In between 0.5 mM to 1.0 mM, some irregular-
ity in the change in lifetime has been noted. Above 1.0 mM, the order of
increment in the lifetime values is as follows: 12-4(OH)-12 b 12-4-12
b 12-8-12. Average excited state lifetime for pure BSA is 6.22 ns. In pres-
ence of 4.0 M GdHCl the average excited state lifetime is 1.52 ns. During
interaction process with 8.0 mM of each of 12-4-12, 12-8-12 and 12-
4(OH)-12 the average excited state lifetime are found to be 3.38 ns,
3.49 ns and 2.26 ns, respectively. It shows that the gain in excited
state lifetimes for 12-4-12, 12-8-12 and 12-4(OH)-12 are 54.34%,
56.11%, and 36.33%, respectively. It has been mentioned above that
12-4(OH)-12 is more effective for denaturation of native protein be-
cause of hydrogen bonding interaction with protein due to presence of
\\OH group in the spacer group. However, these lifetime values depict
that high concentration of 12-8-12 is more effective in creating hydro-
phobic environment around Trp and Tyr residues could be because of
presence of more hydrophobic spacer group in this case as compared
to that in other two surfactants.
3.9. Conclusions
This work has demonstrated the interactions of different gemini sur-
factants with natured and denatured BSA. Effect of spacer group of gem-
ini surfactants has been studied. All gemini surfactants interact with BSA
in a sequential manner. Gemini surfactant with hydroxyl group in the
spacer part (12-4(OH)-12) interacts with native BSA comparatively
stronger than that without hydroxyl group (12-4-12 and 12-8-12) in
the spacer to decrease the -helix of the protein. However, at higher
concentration range, the gemini surfactant with hydrophobic spacer
(12-8-12) provides more hydrophobic environment around Trp and
Tyr residues forming micelle like structures along the protein chain as
compared to other two surfactants (12-4-12 and 12-4(OH)-12). The mi-
croenvironment around Trp residues of BSA is perturbed to a greater ex-
tent than that around Tyr residues in presence of gemini surfactants.
Fluorescence lifetime data of Trp depict that a gemini surfactant with
long hydrophobic spacer like that in 12-8-12 is more efcient to form
conformer 3 and/or puckerd ring of Trp. To what extent the conforma-
tion and/or ring puckeredness of Trp residue changes in presence of
12-8-12 surfactant that depends on the extent of denaturation of pro-
tein. 12-8-12 with a loop in its spacer group might be effective to form
a particular conformer and/or puckerd ring of an amino acid residue of
protein i.e. Trp in the present case. Fluorescence from Trp and Tyr are
quenched by acrylamide to a greater extent in presence of 12-8-12.
Steady-state uorescence and excited state uorescence lifetime data
of Trp residues of BSA also show that 12-8-12 even interacts with the
BSA unfolded by GdHCl to a greater extent than that by 12-4-12 and
12-4(OH)-12. 12-8-12 creates more hydrophobic environment around
Trp and Try residues of BSA than that by 12-4-12 and 12-4(OH)-12
which is similar to that in absence of GdHCl.
Acknowledgement
S.K.S. acknowledges the UGC-DAE Consortium for Scientic Re-
search, Mumbai Centre (UGC-DAE CSR) [UDCSR/MUM/AO/CRS-M-
219/2016/720, UDCSR/MUM/AO/CRS-M-219/2017/975] and the Coun-
cil of Scientic and Industrial Research (CSIR) [(01(2839)/16/EMR-II)]
for nancial support, the University Grants Commission (UGC) for spe-
cial assistance program [F.540/14/DRS/2007(SAP-I)], and Department
of Science and Technology (DST) FIST program, Government of India.
S.K. acknowledges the UGC-BSR and Birla Institute of Technology &
Science (BITS), Pilani, S.H. acknowledges UGC-DAE CSR and R.A.
acknowledges CSIR for fellowship and S. acknowledges UGC for nan-
cial support under the senior research fellowship. We also thank AIRF,
JNU, New Delhi for helping us with the CD experiments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.molliq.2017.07.122.
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