Interaction of bovine serum albumi with two alkylimidazolium-based ionic liquids investigated by microcalorimetry and circular dichroism

doi:10.4236/jbpc.2011.22018

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Vol.2, No.2, 146-151 (2011)

doi:10.4236/jbpc.2011.22018

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.

ABSTRACT

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

1. INTRODUCTION

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

147

([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

concentration.

2. MATERIALS AND METHODS

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-

corded.

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. RESULTS AND DIS CUSSION

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

148

Wavelength ( nm )

[]  10

–3

/degcm

dmol

–1

200 210 220 230 240 250

-12

-8

-4

BSA

50:1

150:1

200:1

-1 2

-8

-4

200 210 220230 240 250

BSA

50:1

150:1

200:1

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

BSA.

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

cKc



 (1)

L, 0LP, 0

ccc N





  (2)

where



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

QcV NH









(3)

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 (Qexp – Qcalc)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 (TSº) 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

Constants

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 ⅡA and ⅢA. A large hydrophobic

cavity is present in the ⅡA 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

149

Q/mJ

Molar Ratio

020406080100 120140

-3

-1

-4

-2

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.

[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

BF PF



. 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.

ST

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

Parameter

[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

TS1º/(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/(kJmol–1) –1.38  0.05 –1.52  0.06

G2º/(kJmol–1) –10.36  0.12 –11.38  0.18

TS2º/(kJmol–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

150

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-

spectively.).

(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 kJmol –1 ) and that

for [bmim]PF6 – BSA (–1.52 ± 0.06 kJmol –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.

4. CONCLUSIONS

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.

5. ACKNOWLEDGEMENTS

The authors are grateful to Inovation Project of Shandong Province,

LCUYZ10008.

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