Physics and Chemistry of Liquids

Vol. 41, No. 5, October 2003, pp. 509–517

CONFORMATIONAL STABILITY OF
BOVINE SERUM ALBUMIN BY CATIONIC
SURFACTANT TREATMENTS
A.A. RAFATIa,*, H. GHARIBIb and H. ILOUKHANIa
a
Department of Chemistry, Faculty of Science, Bu-Ali Sina University, Hamadan, Iran;
b
Department of Chemistry, Faculty of Science, Tarbiat Modarres University, Tehran, Iran

(Received 26 October 2001)

The chemical denaturation of bovine serum albumin (BSA) by a series of cationic surfactant was studied at pH
7.0 at two different temperatures (300 and 310 K). The conformational stability of BSA has been
explained based on
G
DðH2 OÞ . The denaturation has been considered as a two-state process and data analy-
sis was carried out based upon this consideration. The calculations show that surfactants with longer
hydrocarbon chains should denature the protein at lower concentrations. Although, UV absorbance experi-
ments showed that longer chain surfactants denatured the protein more easily than shorter ones. The enthalpy
of denaturation was also estimated by van’t Hoff method.

Keywords: Bovine serum albumin(BSA); Cationic surfactants; Spectrophotometery; Denaturation;

Conformational stability

INTRODUCTION

Many proteins spontaneously take on their secondary, tertiary, and quaternary

structures. Apparently, all of the information required for the formation of the biolog-
ically active conformation of proteins (called native form) is encoded in their amino
acid sequence. It is an important goal of biochemistry to better understand the rules
that govern protein folding and its role in biological activity [1–4].
The folding of proteins to yield their native conformations is favored under
physiological conditions. Denaturation of proteins occurs at extreme pH values;
at high temperature; or as a result of treatment with organic solvent, detergents, urea
and guanidine hydrochloride, and other denaturing agents [5–9].
The interactions, which stabilize protein conformation includes hydrogen bonds,
disulfide bridges, electrostatic interactions, complex formation with metal ions, and
hydrophobic effects. Many soluble proteins in water, fold so that the majority of the
nonpolar amino acid side chains lie within the molecule, whereas the polar side
chains face toward the solvent [10,11]. A range of experimental techniques such as

*Corresponding author. E-mail: aa-rafati@basu.ac.ir

ISSN 0031-9104 print: ISSN 1029-0451 online ß 2003 Taylor & Francis Ltd
DOI: 10.1080/00319100310001604867
510 A.A. RAFATI et al.

calorimetry, viscosity, UV spectroscopy, surface tension, potentiometry, etc. have been

used to study of protein–surfactant interactions [12–20].
Our understanding of the energetic of protein folding and conformational stability of
protein is still incomplete. One way to explain protein stability is to study the denatura-
tion process, i.e. the following reaction:

N !D

where N and D represents, native and denatured form of protein.

The conformational stability of a protein may be defined as the free energy change
for the above reaction under ambient conditions such as in water at 25
C. From
surfactant denaturation curves, conformational stability of protein is estimated and
designated
G
DðH2 OÞ [21, 22]. In this article we describe the methods used to measure

G
DðH2 OÞ .
Difference in stability of proteins might be arised from a single change in amino acid
sequence made by site-directed mutagenesis, or a change in the structure of a side chain
made by chemical modification.
This article involves with denaturation of bovine serum albumin (BSA), as a globular
protein, by a series of cationic surfactants. In this work the effect of hydrocarbon
chain length of surfactant on the denaturation process has been studied by a
spectrophotometric technique at pH ¼ 7.0 and two temperatures, 300 and 310 K.

EXPERIMENTAL

Bovine serum albumin (BSA) was a crystallized and lyophilized preparation,

essentially free from fatty acids (Sigma Chemical Co. Art No. 126F-9350). All of
the cationic surfactants, DOTAB (Dodecyltrimethyl ammonium bromide), TTAB
(Tetradecyltrimethyl ammonium bromide) and HTAB (Hexadecyltrimethyl ammonium
bromide) were obtained from Sigma Chemical Co. and used after recrystallization
in acetone/water mixture. Sodium hydrogen phosphate (Na2HPO4) and sodium
dihydrogen phosphate (NaH2PO4) were obtained from Merck and used as received.
All solutions were prepared with water, which had been double distilled. All protein
solutions were made up in a constant amount of sodium phosphate buffer (2.7 mM,
pH ¼ 7.0).
A Shimadzu spectrophotometer model 2100 was used. Absorbance measurements
were performed at
¼ 278 nm. During the absorbance measurements, the temperature
was maintained at the desired value  0.1
C by circulating thermostated water through
the cell holder used.

RESULTS AND DISCUSSION

In this work, a system of fixed BSA and phosphate buffer at concentrations below
the critical micelle concentration (CMC) of surfactants was considered. The UV
absorbance (A) data were used for the construction of denaturation curves. Figure 1
typically shows the variation A of BSA versus total concentration of surfactant.
 CONFORMATIONAL STABILITY OF BOVINE SERUM 511

FIGURE 1 TTAB denaturation curve for BSA in 27 mM phosphate buffer, pH ¼ 7 and T ¼ 300 K. Broken
lines and equations are based on a least-squares analysis of the pre and post transition baselines.

Absorbance was measured at 278 nm. As previously discussed, the denaturation process
can be viewed as a process in which the native protein (N) loses its conformation and
is transferred to the denatured (D) form, as follows:

N !D KD ¼ ½D=½N ð1Þ

where KD is equilibrium constant for denaturation process, [N] and [D] are the
concentration of native and denatured form of protein, respectively.
The conformational stability of a globular protein may be defined as the free energy
change for the above reaction. This parameter is estimated from the surfactant
denaturation curve and designated
G
DðH2 OÞ .
The above mechanism has been shown to approach closely a two-state process where
the concentration of partially native molecules present at equilibrium is small enough to
be neglected. By assuming a two-state mechanism, the fraction of denatured protein,
D, may be calculated using:

Yobs  YN
D ¼ ð2Þ
YD  YN

where Yobs is the observed variable parameter (e.g. absorbance in Fig. 1) and YN and
YD are the values of Y characteristic of the native and denatured conformations,
respectively. The values of YN and YD at each point in the denaturation curve can be
512 A.A. RAFATI et al.

determined by a least-square analysis of the pre- and post-transition baseline that are
identified as broken lines. Equations of these lines are represented in Fig. 1.
Figure 2(a) and (b) shows denaturation curves for DOTAB, TTAB, and HTAB at
two different temperatures. It is well known that surfactants with longer hydrocarbon
chain have more readily denaturing proteins. By replacing [D] and [N] with D and N
in Eq. (1):

D D
KD ¼ ¼ ð3Þ
N 1  D

The differences in free energy between the native and denature conformations, i.e.,

G
D can then be calculated using:

D

G
D ¼ RT ln KD ¼ RT ln ð4Þ
1  D

where R is the gas constant and T is the absolute temperature. Combining this equation
with Eq. (2), results in:

Yobs  YN

G
D ¼ RT ln ð5Þ
YD  Yobs

Typically, Fig. 3 shows the variation of
G
D with surfactant concentration.

In general, this plot is linear and least-square analysis can be used to fit the data
from the transition region to the equation:

G
D ¼
G
DðH2 OÞ  m½D ð6Þ

where
G
DðH2 OÞ is the value of
G
D in the absence of surfactant, m is a measure of

the dependence of
G
D on denaturant concentration and [D] is the denaturant
(here surfactant) concentration. The obtained data from curves such as Fig. 3, for
different surfactants at two different temperatures are listed in Table I. Analysis
of data showed that conformational stability of BSA was about 15–20 kJ mol1.
Temperature is a factor in which affects denaturation of proteins and the slope of
the denaturation curve increased with temperature (Fig. 4).
Analysis of denaturation curves for interaction between a series of n-alkyltrimethyl
ammonium bromide and BSA indicates that the slope of the plots of
G
D versus [D]
increased on increasing of the hydrocarbon chain length of the surfactant. This
shows that hydrophobic interactions between surfactant and protein have an important
role. Although, Fig. 2 shows that HTAB initiates interaction with the protein at very
lower concentrations relative to TTAB and DOTAB.
The enthalpy of denaturation,
HD
has been calculated by the van’t Hoff method:

@ ln KD

HD
¼ RT 2 ð7Þ
@T
 CONFORMATIONAL STABILITY OF BOVINE SERUM 513

FIGURE 2 Denaturation curves for BSA by different cationic surfactants (g) DOTAB, () TTAB and
(m) HTAB at two different temperatures (a) 300 K and (b) 310 K. The concentration axis scale is specified by
arrow.
514 A.A. RAFATI et al.

FIGURE 3 Typical plot of
G
D vs. denaturant concentration for DOTAB/BSA interaction at 300 K.
The line was obtained by a least square fit of data to equation.

TABLE I Denaturation parameters for BSA-cationic surfactant interaction at different temperatures

Surfactant DOTAB TTAB HTAB

Temperature (K) 300 310 300 310 300 310

G
DðH2 OÞ ; kJ mol1 8.54 15.52 15.3 21.5 16.96 23.76
m 2123 4841 25950 38110 51950 78350

If the equilibrium constant is measured as a function of temperature, the enthalpy

change can be calculated using the above equation. The obtained data were plotted
in Fig. 5. For BSA denaturation,
HD
is found to increase markedly with surfactant
concentration. As the protein denaturation is an endothermic process, so
HD
increase
with increasing concentration of denaturant, i.e. [D]. The intramolecular bonds in the
protein break on interaction with the surfactant. Increasing the surfactant concentra-
tion caused more intermolecular interactions and higher values of
HD
. As the
intermolecular interaction status is hydrophobic, so the slope of curves
HD
versus
[D] increases with increasing length of the hydrocarbon chain of the surfactant.
Although the variation of
HD
with [D] has sharpened with increasing surfactant
chain length or hydrophobicity of surfactant. This is very important role in protein
structuring.

CONCLUSION

The results of protein denaturation can be treated according to a two-state process and
a linear dependence of
G
D on the denaturant concentration. We have noted that
 CONFORMATIONAL STABILITY OF BOVINE SERUM 515

FIGURE 4 Effect of temperature on denaturation process [(g) 300 K and () 310 K] for different
surfactant (a) DOTAB (b) TTAB and (c) HTAB.
516 A.A. RAFATI et al.

FIGURE 4 Continued.

FIGURE 5 The enthalpy of denaturation derived from van’t Hoff method for different surfactants (g)
DOTAB, () TTAB and (m) HTAB. The concentration axis scale is specified by arrow.
 CONFORMATIONAL STABILITY OF BOVINE SERUM 517

cationic surfactants induced transitions are generally closed to two-state process,

and this is especially true for BSA. Obtaining a reliable value for thermodynamic
stability from denaturation transition curves will be possible for globular proteins.
The denaturation curve analysis show that BSA is more unstable than other globular
proteins (the value of
G
DðH2 OÞ for BSA is 15 kJ mol1 approximately whereas for
other globular protein, such as RNase, it is 39 kJ mol1).

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