Vol.2, No.2, 146-151 (2011)
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
Journal of Biophysical Chemistry
Interaction of bovine serum albumin with two
alkylimidazolium-based ionic liquids investigated by
microcalorimetry and circular dichroism
Lan-Ying Zhu1*, Guang-Qian Li2, Fu-Yin Zheng1
1College of Life Science and Bioengineering, Liaocheng University, Liaocheng, China;
*Corresponding Author: zhulanyingzly@163.com or zhulanying@lcu.edu.cn
2College of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng, China.
Received 17 January 2011; revised 2 March 2011; accepted 28 March 2011.
The interactions of bovine serum albumin (BSA)
with two alkylimidazolium-based ionic liquids,
1-butyl-3-methylimidazolium tetrafluoroborate
([bmim]BF4) and 1-butyl-3-methylimidazolium
hexafluorophosphate ([bmim]PF6), in buffer so-
lutions at pH 7.0 were investigated by isothermal
titration calorimetry (ITC) and circular dichroism
(CD). CD spectra showed that the two ionic liq-
uids changed the secondary structure of BSA.
Data process w as based on the supposition that
there were several independent types of binding
sites on each BSA molecule for the two ligand
molecules. The results obtained by using this
supposition combined w ith Langmuir adsorption
model showed that there w ere two types of such
binding sites. One was the high affinity binding
site, and the other was the low affinity binding
site. The binding constants, changes in enthalpy,
entropy and Gibbs free energy for the two types
of binding were obtained, w hich showed that the
two types of binding were driven by a favorable
entropy increase. Furthermore, for either the
ionic liquids, the number of the high affinity
bindin g sites i s much sma ller th an that of th e lo w
affinity ones. These res ults were interpreted w ith
the molecular structure of BSA and the different
substituent groups on imidazole ring of the two
ionic liquid molecules.
Keywords: Isothermal Titration Calorimetry;
Circular Dichroism Spectra;
Alkylimidazolium-Based Ionic Liquid s; Bovine
Serum Albumin
Protein pharmaceuticals are subjected to a number of
stresses during production, storage, and shipping, result-
ing in loss of the protein concentration and activities or
formation of soluble and insoluble aggregates. The gen-
eral method for stabilizing liquid protein pharmaceuti-
cals is the use of formulation excipients. Surfactants are
indispensable as solubilizing agents in the isolation and
purification of proteins. To use them correctly, it is nec-
essary to have an idea of how and in which amounts they
interact with proteins. Although surfactant-protein inter-
actions have been widely studied for half a century [1-5],
the mechanism of interaction is not well understood.
Knowledge of the interactions is not only fundamental in
theoretics, but also practical in industrial applications. In
the cosmetic and food fields, protein function is largely
influenced by an added surfactant [6].
In the studies of surfactant-protein interactions, the
serum albumin, e.g., human serum albumin (HSA) or
bovine serum albumin (BSA), is commonly used as
model protein due to its well-established primary struc-
ture, stability, water solubility and versatile binding ca-
pacity [7-9]. Ionic liquids (ILs) are a class of organic
molten electrolytes at or near ambient temperature [10].
Their physical and chemical properties can be tailored
by judicious selection of cation, anion, and substituent.
They have no significant vapor pressures, outstanding
catalytic properties, high ion-conductivity, non-flamma-
bility, and are relatively inexpensive to manufacture [11].
Thus ILs have attracted much attention as electrolytes
and solvent media for reactions and extractions [12-14].
So study on binding of ionic liquid-type surfactants to
bovine serum albumin would be very necessary to fur-
ther understand the structural and functional information
of surfactant-protein interactions. Among various ILs,
the alkylimidazolium salts which belong to ionic liq-
uid-type surfactants have been extensively studied in the
field of colloid and interface science [15-17]. In this pa-
per, 1-butyl-3-methyl imidazolium tetrafluoroborate
L.-Y. Zhu et al. / Journal of Biophysical Chemistry 2 (2011) 146-151
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
([bmim]BF4) and 1-butyl-3-methylimidazolium hexa-
fluorophosphate ([bmim]PF6) (see Scheme 1) were se-
lected as ligands to the protein. Isothermal titration calo-
rimeter (ITC) was used to determine the thermodynamic
parameters (enthalpy, binding site number and binding
constant, etc) of the interactions of the both alkylimida-
zolium-based ionic liquids, [bmim]BF4 and [bmim]PF6,
with BSA at the temperature of 298.15 K. CD spectros-
copy was also employed to determine the dependence of
-helical content in the protein molecules on ionic liquid
2.1. Materials
Bovine serum albumin was purchased from Acros,
which was used without further purification. The con-
centration of BSA was determined by using the extinc-
tion coefficient at 280 nm of 44720 M–1cm–1 at pH 7.0
[18]. 1-butyl-3-methylimidazolium tetrafluoroborate
([bmim]BF4) and 1-butyl-3-methylimidazolium hexa-
fluorophosphate ([bmim]PF6) were both obtained from
Aldrich (mass fraction > 99%). All solutions were pre-
pared with thrice distilled water in calorimetric experi-
ment. Tris (hydroxymethyl) aminomethane (Tris-HCl) used
in the preparation of the buffer was of analytical grade.
a) (b)
Scheme 1. Molecular structure of two ionic liquids (a)
1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4);
(b) 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim]PF6).
2.2. Circular Dichroism (CD) Measurements
CD measurements of BSA in tris-HCl buffer solutions
of pH 7.0 were performed using a Jasco J-810 spectro-
polarimeter (Japan) at the temperature 298.2 K. The light
source system was protected by nitrogen (flow rate: 5 L
min–1). The spectra of protein solutions (2 M) were
determined in 1 mm cells. The wavelength region of
scanning was 190 - 250 nm. The solutions were scanned
at 100 nm min–1 using a 1 s time constant with step reso-
lution of 0.1 nm. The average of three scans was re-
2.3. Microcalorimetric Measurements
Titration microcalorimetry was performed on a nano-
watt-scale isothermal titration micorcalorimeter sup-
ported by Thermal Activity Monitor TAM 2277 (Ther-
mometric, Sweden), which was controlled by Digitam
4.1 software. This instrument has an electrical calibra-
tion with a precision better than 1% that can be deter-
mined by measuring the dilution enthalpy of a concen-
trated sucrose solution [19]. Each channel is a twin
heat-conduction calorimeter where the heat-flow sensor
is a semiconducting thermopile (multi junction thermo-
couple plates) positioned between the vessel holders and
the surrounding heat sink. For the measurement of the
protein (BSA)—ligand ([bmim]BF4 or [bmim]PF6) solu-
tions, the 1 mL sample cell of the calorimeter made from
stainless steel were initially loaded with 800 L BSA
solution whose concentration was 100 M. 30.00 mM
[bmim]BF4 or [bmim]PF6 solution was injected into the
stirred sample cell in 30 portions of 12 L using a 500
L Hamilton syringe controlled by a Thermometric 612
Lund Pump. The interval between two injections was 35
min., which was sufficiently long for the signal to return
to the baseline. The system was stirred at 30 rpm with a
gold propeller. All experiments were performed at a
fixed temperature of (298.15 0.01) K and repeated
thrice. To deduct the dilution heats of ionic liquid and
BSA solutions, we performed titration experiments of
ionic liquid solution into buffer solution and buffer solu-
tion into BSA solution, respectively.
3.1. Circular Dichroism Studies
Figure 1 shows the CD spectra of BSA under the co-
existence of one of the two ionic liquids at different
concentrations. [bmim]BF4 and [bmim]PF6 do not pre-
sent any CD signal in the spectral range 190 - 250 nm.
This indicates that the observed CD signal is only pro-
duced by BSA. The CD spectrum of BSA exhibits two
negative bands in the ultraviolet region at 208 (π→π*
transition) and 222 nm (n→π* transition), which is
characteristic of the α-helical structure of a protein [20].
It can be seen from Figure 1 that the interaction of
[bmim]BF4 or [bmim]PF6 with BSA caused a slight de-
crease in band intensity at all wavelengths of the far UV
CD without any significant shift in the peak position.
This clearly indicates the minor changes in the protein
secondary structure, namely the decrease in the
α-helical content in protein. This maybe caused by the
interaction between the ionic liquid and BSA which
leads to a swelling of the biomacromolecule and expos-
ing of the hydrophobic residues [21]. Thus some of the
original α-helices are broken to give a more open dis-
ordered structure. However, it can be seen from Figure
1 that the change of secondary structure is quite small in
L.-Y. Zhu et al. / Journal of Biophysical Chemistry 2 (2011) 146-151
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
Wavelength ( nm )
[] 10
200 210 220 230 240 250
-1 2
200 210 220230 240 250
Figure 1. Circular dichroism spectra of BSA in Tris-HCl buffer (10 mM) at pH 7.0 as a function of ionic liquid
concentration: (a) BSA + [bmim]BF4; (b) BSA + [bmim]PF6 . (The ratio in this figure is the analytic concentra-
tion ratio of ionic liquid to BSA).
the molar ratio range of titration experiment (ionic-BSA:
135 : 1). So we can conclude that the heat effect caused
by conformational change of BSA can be neglected.
That is, the measured enthalpy changes are mainly
caused by the binding of the ionic liquid molecules to
3.2. Thermodynamic Data Analysis
3.2.1. Titration Calorimetric Model for the
Binding of Ionic Liquid to BSA
A multiple-site model has been proposed for the
binding of ligand molecules to protein, which can be
regarded as adsorption of the ligand particles on the sites
belonging to different classes and abiding Langmuir
isotherm [4,22-23]. For the i-th class of binding sites,
there are:
(1 )
ii iL
 (1)
L, 0LP, 0
ccc N
 (2)
i is the degree of occupancy of the i-th types of
sites. cL,0 and cP,0 indicate total concentration of ligand
and protein, respectively. cL is concentration of free
ligand molecule at equilibrium state and m is the number
of binding classes.
The net heat of interaction between the ligand mole-
cule and the biomacromolecule evolved from the j-th
injection in an ITC experiment can be expressed
P, 0P, 0
ii i
where VP,0 is the volume of the protein solution as titrand
in the calorimeter cell and
i is the change in occu-
pancy from the (j – 1)th injection to the jth one.
Eq.1 and Eq.2 indicate that cL is the function of Ni
and Ki when cL,0 and cP,0 are known. So there are 3 m
unknown parameters in (3), which are Ki, Hiº and N
These parameters for single-class (m = 1), two-class (m
= 2) and three-class (m = 3) binding model were com-
puted from the actual calorimetric data with an iterative
non-linear least-square regression program for minimiz-
ing the value of (QexpQcalc)2 by using of software
MATLAB 7.01. The coincidence degree between calcu-
lated curve and experimental integrate heat indicates that
existence of two types of binding sites is most reason-
able when either [bmim]BF4 or [bmim]PF6 binds to BSA.
The nonlinear fitting curves of integrated heat versus the
mixed ligand/BSA molar ratio are shown in Figure 2.
According to the thermodynamic formula:
lnGRTK oo
GHTS (4)
Standard changes of Gibbs free energy (Gº) and en-
tropy effect (TSº) for the binding process of ionic liquid
to BSA can be derived. The thermodynamic results for
the two ligand molecules + BSA complexes are listed in
Table 1.
3.2.2. Binding Site Number and Binding
Nonlinear fitting curves of integrated heat versus the
mixed ionic liquid/BSA molar ratio are shown in Figure
2. It can be seen that the binding of the two ionic liquids
to BSA is exothermic in the range of selected ionic liq-
uid concentration. Crystal structure analyses have re-
vealed that the binding sites on BSA molecules are lo-
cated in subdomains A and A. A large hydrophobic
cavity is present in the A subdomain [24]. Moreover,
due to the presence of amino acid residues on the sur-
face of HSA, most authors agree on the existence of a
limited number of binding sites on the surface of protein
molecules [25]. The optimum simulated results show
that there are two types of binding sites on BSA mole-
cules for the binding of the two ionic liquids to BSA: 1)
binding to ionic sites on BSA surface driven by hydro-
gen bonding interaction of anions of [bmim]BF4 or
(a) (b)
L.-Y. Zhu et al. / Journal of Biophysical Chemistry 2 (2011) 146-151
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
Molar Ratio
020406080100 120140
Figure 2. Non-linear fitting curve of the binding heat
versus the molar ratio of the two ionic liquid to BSA,
where points are gotten from experiments and the solid
line is the result of simulation, : [bmim]BF4; :
[bmim]PF6 with amino acid residues on BSA surface,
which corresponds to the high affinity sites; 2) hydro-
phobic interaction of imidazole ring and the hydrophobic
chain on either of the two ionic liquids with the hydro-
phobic cavities of BSA molecules, which corresponds to
the low affinity sites. The work of Swati De et al. indi-
cates that high affinity sites corresponding to the ionic
sites on protein surface, and low affinity sites corre-
sponding to the hydrophobic cavities of protein mole-
cules [26]. From the data in Table 1, we can reach two
conclusions, which are: when the same ionic liquid binds
to BSA, the high affinity site number (N1) is smaller than
the low affinity site number (N2), while the binding con-
stant for the high affinity sites is evidently larger than
that for the low affinity sites. This difference may be
explained as follows. The isoelectric point of serum al-
bum is at pH 4.70 [27] and BSA molecule is negatively
charged at pH 7.0. The actually ligand particles are
[bmim]+ cations, for which the mainly binding force to
the first type of sites is electrostatic interaction. This can
also explain the fact that the two values of the equilib-
rium constant K1 respectively for [bmim]BF4 and
[bmim]PF6 are almost the same. As to the difference
between the two values of N1 respectively corresponding
to [bmim]BF4 and [bmim]PF6 might be interpreted from
the structures of the two anions, 4
and 6
. The
size of the former is smaller than the later, and so there
is more opportunity for the former to approach the bio-
macromolecule, which obstructs the ligand cation i.e.
decrease the binding site. The relative differences be-
tween values of K2, N2,
, respectively cor-
responding to [bmim]BF4 are all not evident. This phe-
nomenon indicate that the actually ligand is also the
cation in the second type of binding process driven by
hydrophobic interaction.
3.2.3. Enthalpy and Entropy Effects
The standard enthalpy effects, standard entropy effects
Table 1. Thermodynamic parameters for the binding of
[bmim]BF4 and [bmim]PF6 to BSA at 298.15 Ka.
ionic liquid
[bmim]BF4 [bmim]PF6
High affinity sites
N1 1.0 0.2 3.1 0.1
K1/(M–1) (1.12 0.04) 103 (1.48 0.07) 103
H1º/(kJ mol–1) –2.14 0.08 –2.46 0.09
G1º/kJ mol–1) –17.40 0.19 –18.09 0.28
TS1º/(kJ mol–1) 15.26 0.27 15.63 0.37
Low affinity sites
N2 25.0 0.9 27.0 1.0
K2/(M–1) 65.20 2.60 98.50 3.50
H2º/(kJmol–1) –1.38 0.05 –1.52 0.06
G2º/(kJmol–1) –10.36 0.12 –11.38 0.18
TS2º/(kJmol–1) 8.98 0.17 9.86 0.24
aData are expressed as mean S. D. (N = 3).
and changes in standard Gibbs free energies for the
binding of [bmim]BF4 and [bmim]PF6 to BSA are shown
in Figure 3. It is necessary to know the main existing
form of the two ionic liquids in sample cell in order to
interpret the above experimental results. The critical
micelle concentrations (CMC) of BF4 and [bmim]PF6
are about 0.8 M [28] and 0.031 M [29] respectively,
which exceed the initial concentration (30.00 mM) of the
two ionic liquids in the syringe. Therefore, the two ionic
liquids in the sample cell are both existent as monomers
instead of micelles. In other words, there is no demicel-
lization of the ionic-liquid-type surfactant in the experi-
mental concentration ranges. Furthermore, as described
above, the heat effect caused by conformational change
of BSA can be neglected. It can be seen from Figure 3
that the standard enthalpies of formation for the binding
of the two ionic liquids to the both types of binding sites
of BSA are negative, which indicates that the binding
process are both exothermic. The reasons for the nega-
tive values of o
are manifold. Firstly, the electro-
static attraction of the cation, [bmim]+, with the nega-
tively charged protein molecule is an exothermic process.
Secondly, the repulsion force between the anions of the
ionic liquid and the negatively charged BSA surface
makes positive contribution to o
. Thirdly, destroy of
iceberg structure surrounding hydrophobic chains of the
ionic liquid is an endothermic process. The negative
values of 1
indicate that the hydrogen bonding in-
teraction is predominant over the electrostatic repulsion
interaction and the destroy effects of iceberg structure.
As for
, there are also several types of weak inter-
actions simultaneously contribute to its negative value,
including the expulsion of high energy water molecule
from hydrophobic cavity of the protein molecule to bulk
solution (exothermic process), destroy of iceberg struc-
ture surrounding hydrophobic groups of the ionic liquid
L.-Y. Zhu et al. / Journal of Biophysical Chemistry 2 (2011) 146-151
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
Figure 3. Proportion of the thermodynamic parameters for the
binding of bmim]BF4 and [bmim]PF6 to BSA (Superscript 1
and 2 represent high affinity sites and low affinity sites, re-
(endothermic process), and the hydrophobic interaction
of imidazole ring and the hydrophobic chain on it with
the hydrophobic cavities of BSA molecules (exothermic
process) [30]. Among them, the hydrophobic interaction
should be the main driving force, evidenced by the ex-
perimental results that the difference between the o
for [bmim]BF4 – BSA (–1.38 ± 0.05 kJmol –1 ) and that
for [bmim]PF6 – BSA (–1.52 ± 0.06 kJmol –1) is subtle
because the two ionic liquids contain the same hydro-
phobic chain.
It can be seen from Figure 3 that the entropy effects
for the binding of [bmim]BF4 and [bmim]PF6 to BSA are
all positive, which is beneficial for this interacting proc-
ess. This may be due to the total result of the binding of
the two ligand ions to BSA molecule (negative contribu-
tion to entropy) and the releasing of water molecules
from cavity (positive contribution to entropy) as well as
the disruption of hydration layer structure on the surface
of BSA molecule (positive contribution to entropy). In
addition, we can infer from Figure 3 that the binding
process is predominantly entropy driven.
ITC experiments demonstrate that there are two types
of binding sites on BSA molecules for the Alkylimida-
zolium cation in the both ionic liquids, [bmim]BF4 and
[bmim]PF6. One type of binding with high affinity bind-
ing is caused electrostatic interaction of the cation with
negatively charged sites on BSA molecules, and the
other one is low affinity binding due to the hydrophobic
interaction of imidazole ring and the hydrophobic chain
on it of the both ionic liquids with the hydrophobic cavi-
ties of the protein molecules. The thermodynamic results
obtained from the calorimetric data with an iterative
non-linear least-square regression program show that
when the same ionic liquid cations bind to BSA mole-
cules, the number of high affinity sites (N1) is smaller
than that of the low affinity sites (N2). On the contrary,
the binding constant for the high affinity sites (K1) is
evidently larger than that for the low affinity sites (K2).
The entropy effects for the two binding sites are both
negative while the entropy effects for the two binding
sites are both positive. Circular dichroism (CD) spectra
show that the two ionic liquids change the secondary
structure of BSA. These results can be understood by
considering several weak interactions of the biomacro-
molecule with the cation, the anion as well as the solvent
effect of water on the protein-ligand interaction system.
The data and message obtained in this study may be im-
portant for understanding the influence of pro-
tein-surfactant interactions on the functionality of
globular proteins in the biochemical systems.
The authors are grateful to Inovation Project of Shandong Province,
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