Microcalorimetric studies on the interactions of lanthanide ions with bovine serum albumin

Article abstract of DOI:10.1007/s10973-006-7611-5

The interactions of lanthanide ions (Ln³⁺) with bovine serum albumin (BSA) under mimetic physiological conditions (310.15 K, pH 6.7, 0.1MNaCl) were studied by microcalorimetry. For the first time, based on Two Sets of Independent Sites Model, m

Full text of DOI:10.1007/s10973-006-7611-5

Journal of Thermal Analysis and Calorimetry, Vol. 89 (2007) 3, 899–905
MICROCALORIMETRIC STUDIES ON THE INTERACTIONS OF
LANTHANIDE IONS WITH BOVINE SERUM ALBUMIN
X. Li1*, C. Wang2, J. Li2 and Z. Wang2
1School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201, People ’s
Republic of China
2College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, People’s Republic of China
The interactions of lanthanide ions (Ln3+) with bovine serum albumin (BSA) under mimetic physiological conditions (310.15 K, pH
6.7, 0.1 M NaCl) were studied by microcalorimetry. For the first time, based on Two Sets of Independent Sites Model, molar enthalpies
(DrHm1, DrHm2) and coordination number (n1, n2) of the two sets of binding sites with different affinity were obtained directly from the
microcalorimetric results. It was shown that the interactions are endothermic and entropy-driving processes. By combining with fluo-
rescence spectroscopy, other thermodynamic parameters (DrGm1, DrSm1) were determined for high-affinity specific sites.
Keywords: bovine serum albumin (BSA), fluorescence spectroscopy, lanthanide ions (Ln3+), microcalorimetry
Introduction
ity; and the other are non-specific ones, with low af-
finity and high binding capacity. The number of
high-affinity binding sites and the binding constants
of different binding sites have been reported [15]. But
thermodynamic parameters were seldom reported.
However, these thermodynamic parameters were
indispensable because they can potentially provide
valuable insight as to the nature of the interactions
and the forces that stabilize the complexes.
Compared with classical methods, thermochemi-
cal method can provide in-situ, online, quasi-continu-
ous, non-invasive and accurate measurements of the
thermodynamic parameters of the reaction under in-
vestigation. Moreover, there is also no constraint on
both the solvent and the spectral, electrochemical, or
other properties of the reaction systems involved.
Owing to these advantages, the thermochemical
method has received increasing attention from re-
searchers in many fields. Since the absorption or pro-
duction of heat is an intrinsic property of all reactions,
calorimetry as the most powerful tool of the
thermochemical method has been extensively used
[19–22]. In particular, calorimetry has been widely
employed to the researches on the binding of metal
ions to proteins [23–26].
Serum albumins are the most abundant proteins in
blood plasma, accounting for about 60% of the total
protein [1, 2] and provide about 80% of the osmotic
pressure of blood [1]. They play an important role in
the transport and deposition of a variety of endoge-
nous and exogenous substances in blood [3] due to
the existence of a limited number of binding regions
with very different specificity [4]. In particular, its
role in the transport of essential, toxic, and therapeu-
tic metals has received considerable attention [5]. Bo-
vine serum albumin (BSA), a protein with a molecular
mass of 66 300, has been used in our study for its pri-
mary structure is very well known and its tertiary
structure was determined [1, 6].
With the extensive applications of rare earths in
agriculture, medicine and research in molecular biol-
ogy etc., the lanthanide elements are introduced to bi-
ological bodies by various ways. The biological effect
and mechanism of lanthanide elements have been
widely paid attention to. In particular, great interest
has been aroused in doing research on the
lanthanide-protein complexes, because lanthanide
ions possess some special functions in biochemistry
[7–12]. For example, the interactions of lanthanide
ions with serum albumins have been widely studied
by various methods [13–18], including ultrafiltration,
equilibrium dialysis, and spectroscopic measure-
ments, etc. The binding characteristics achieved were
that there are two sets of binding sites: one are spe-
cific ones, with high affinity and low binding capac-
In this work, we systemically studied the binding
reactions of lanthanide ions (Ln3+) to bovine serum al-
bumin (BSA) under mimetic physiological conditions
(310.15 K, pH 6.7, 0.1 M NaCl) on LKB-2107 batch
microcalorimeter. According to the microcalorimetric
results, number of binding sites (n1, n2) and apparent
reaction enthalpy (DrHm1, DrHm2) for different-affinity
*
Author for correspondence: lixia7467@163.com
1388–6150/$20.00
Akadémiai Kiadó, Budapest, Hungary
Springer, Dordrecht, The Netherlands
© 2007 Akadémiai Kiadó, Budapest

LI et al.
binding sites of Ln3+ on BSA were obtained directly.
calorimeter 360° clockwise and counterclockwise, re-
spectively, so as to fully mix the solutions. The heat
generated in the reaction process was detected in the
form of thermal potential by thermoelectric piles, and
the amplified output signal was recorded as the calori-
metric curve by LKB-2210 dual-pen integrating re-
corder.
In microcalorimetric experiments, all determina-
tions were made in duplicate. Moreover, a series of
lanthanide chlorides concentrations were used.
Furthermore, combining with fluorescence spectro-
scopic studies, other thermodynamic parameters
(DrGm1, DrSm1) were determined for high-affinity spe-
cific binding sites.
Experimental
Materials
BSA (fractionV, from Roche Chemical Co.) was used
without further purification. The concentration of
BSA was determined by the method as previously de-
scribed [19].
Lanthanide chlorides were prepared from their
oxides (99.9%, from China Medicine Group, Shang-
hai Chemical Reagent Corporation), and their pH val-
ues were adjusted to about 6. The concentrations of
lanthanide ions (III) were titrated with EDTA.
Other chemicals used were of analytical grade.
Doubly distilled water was used throughout. All mea-
surements were performed in 0.1 M NaCl, pH 6.7 and
310.15 K in order to simulate physiological conditions.
Fluorescence measurements
Measurements of steady-state fluorescence were made
on a Perkin-Elmer LS55 Luminescence Spectrometer
equipped with a circulating water bath at 310.15 K. The
fluorescence spectra were measured at a fixed protein
concentration of 1.0·10–5 M. Excitation and emission
slits were set at 3 and 5 nm, respectively. The spectra
were recorded in 280–550 nm range and the excitation
wavelength was set at 288 nm.
Interactions of Ln3+ with BSA were studied us-
ing a fluorescence titration method. The protein solu-
tion with fixed concentration was titrated with an in-
crement of microliter Ln3+ solution (5–50 mL each
and total volume not exceed 200 mL).
Methods
LKB-2107 batch microcalorimeter system combines
a micro-batch reactor (BR) with a conduction calo-
rimeter. All fluorescence measurements were carried
out on a Perkin-Elmer LS 55 Luminescence Spec-
trometer equipped with a Xenon discharge Lamp and
1.0 cm quartz cells and a thermostat bath. All pH mea-
surements were made with a digital pH-meter
(Orion 828) with a combined glass-calomel electrode.
Theory
Model for two sets of independent sites
The calorimetric data can be analyzed by a mass-ac-
tion approach. The underlying assumptions are as fol-
lows: i) one protein molecule has two sets of sites,
which can bind the same ligand. All sites within one
set are thermodynamically identical, and the two sets
of sites can be characterized by binding constant (K1,
K2), standard molar binding enthalpy (DH1, DH2) and
number of binding sites (n1, n2); ii) The two sets are
assumed to be completely independent; iii) One is
specific high-affinity binding sites which are prefer-
entially occupied by ligands, and the other is nonspe-
cific low-affinity binding sites which can bind ligands
only after the specific ones have been fully occupied.
Based upon the above assumptions, there are two
sets of Ln3+ binding sites on BSA that one is specific
ones (K1, DH1, n1) and the other is nonspecific ones
(K2, DH2, n2).
Microcalorimetric measurements
The calorimetric curves were obtained at 310.15 K by
an LKB-2107 batch microcalorimeter system. Before
each calorimetric experiment, all reagent solutions
were diluted to the required concentrations with
0.1 M NaCl solution (pH 6.7).
In experiments, 2.00 mL lanthanide chlorides so-
lution was injected into reaction cell, and 4.00 mL
BSA solution (1.0·10–4 M) was injected into reaction
cell. In order to avoid the influence of the heat effect
of diluting and mixing etc., 2.00 mL 0.1 M NaCl solu-
tion was injected into reference cell, and the same ad-
dition was made to reference cell as reaction cell.
And in order to obtain the dilution heat effects of rare
earth chlorides (Qd), 2.00 mL lanthanide chlorides so-
lution was injected into reaction cell, and 0.1 M NaCl
solution was injected into other three cells. When the
microcalorimeter system had been in thermal equilib-
rium and a steady baseline was obtained on the re-
corder, the reaction run was initiated by rotating the
Methods for determination of coordination number
and thermodynamic parameters
For 1: n binding mode of protein (P) with small ligand
(L), the apparent binding constant K for a single site
can be expressed as:
900
J. Therm. Anal. Cal., 89, 2007

INTERACTIONS OF LANTHANIDE IONS WITH BOVINE SERUM ALBUMIN
[PLn ]
DG=-RT lnK
K =
(1)
(11)
(Pt -[PLn ])(n[PLn ])
DH-DG
DS =
T
where Pt, Lt refer to total concentration of protein and
small ligand, respectively; [PLn] refers to the concen-
tration of protein-ligand complex.
Given fixed protein concentration, there exists
an equation
Results and discussion
From all of the microcalorimetric curves obtained in
this work, it was shown that the interactions of BSA
with lanthanide ions are at rapid equilibrium:
Qt =D r Hm LbV
(2)
where Qi, DrHm refer to the P–L binding reaction heat
and the apparent molar binding enthalpy of a single
site, respectively; Lb concentration of the bound
ligand and V the volume of reaction system (in this
paper, V=6.00 cm3).
BSA+nLn«BSA·Lnn
(rapid equilibrium)
where Ln is lanthanide ions, and BSA·Lnn the com-
plex between BSA and Ln.
Figure 1 shows an example microcalorimetric cur-
ve for the interaction of BSA with lanthanide ions
(La3+). The calorimetric curve shows a quick endother-
mic peak, indicating that the apparent interaction
enthalpy was positive under given conditions. It can be
inferred that the interactions between BSA and Ln3+ are
largely endothermic and entropy-driven process accord-
ing to the enthalpy-entropy compensation law.
From Eq. (1), Lb can be expressed as:
Lb =Pt nKLf /(1+KLf )
(3)
where Lf refers to concentration of the unbound
ligand and K the apparent binding constant.
The relation between Lb, Lt and Lf is
Lt =Lb +Lf
(4)
Combining Eqs (2), (3) and (4), the relation be-
tween Qi and Lt can be deduced
Qi =[A-(A2 -4nPt Lt )1/ 2 ]D r HmV /2
(5)
where
A=1/ K+nPt +L t
(6)
Then, if K is more than 106, that is 1/K approxi-
mate zero, Eq. (5) can be turned into
Qi =(nPt +Lt -nPt -Lt )D r HmV /2
(7)
when protein is unsaturated complexation (nPt>Lt),
Eq. (7) can grow into
Qi =Lt D r HmV
Fig. 1 Microcalorimetric curves for the binding reaction of
That is the apparent molar binding enthalpy DrHm can
be obtained
BSA with La3+; a – C3+ =7.76·10–5 mol L–1;
La
b – C3+ =3.10·10–4 mol L–1; c – C3+ =6.21·10–4 mol L–1.
La
La
T=310.15 K, pH=6.7, [NaCl]=0.1 mol L–1,
D r Hm =Qi /(LtV )
(8)
CBSA=6.06·10–5 mol L–1
when protein is saturated complexation (nPt<Lt),
Eq. (7) can grow into
Determination of apparent molar reaction enthalpy
DrHm and coordination number n
Qi =nPt D r HmV
(9)
Then combining with acquired DrHm, coordination
number n can be determined by
Under given conditions, the reaction heat effects of
Ln3+ with BSA were obtained by fixing the protein
concentration and varying the concentration of Ln3+.
According to microcalorimetric results, Fig. 2
was obtained by plotting the pure reaction heat effect
(Qi) vs. the Ln3+/BSA molar ratio (R). Obviously, all
of curves occur in a biphasic manner with a character-
istic inflection between the two sets of binding sites
on BSA for Ln3+. Qualitative similar binding curves
obtained for all other Ln3+ are also biphasic.
n=Qi /(Pt D r HmV )
(10)
Moreover, from the binding constants (K) and
the standard molar binding enthalpy (DH), the stan-
dard molar binding free energy (DG) and the standard
molar binding entropy (DS) can be calculated by the
following relations, respectively:
J. Therm. Anal. Cal., 89, 2007
901

LI et al.
Fig. 2 The graphs of BSA-Ln3+ pure binding heat effects (Qi) changing with Ln3+/BSA molar ratio (R) increase; a – BSA-La3+;
b – BSA-Er3+; c– BSA-Gd3+; d – BSA-Yb3+
Based upon the Two Sets of Independent Sites
Model, it can be concluded that at low Ln3+ concen-
tration, Ln3+ binds to the specific high-affinity sites
(sites I) on BSA with Qi increasing; and at certain
concentrations (about one to two Ln3+ ions per protein
molecule) these specific binding sites are fully satu-
rated and Qi approximately arrive at a fixed value
(platform II); then at even higher concentrations, Ln3+
binds to the nonspecific low-affinity sites (sites II)
with Qi increasing; finally, these low affinity sites ap-
pear saturated when the total Ln3+ concentration ex-
ceeds a certain value and Qi arrive at a new fixed
value (platform II).
Similarly, apparent molar reaction enthalpy
DrHm2 and coordination number n2 of sites II can be
obtained by calorimetric data before platform I and
the heat effect of platform II (QII).
Table 1 presents the apparent molar reaction
enthalpy (DrHm1, DrHm2) and coordination number
(n1, n2) for La3+ binding to BSA.
The apparent molar reaction enthalpy (DrHm1,
DrHm2) and coordination number (n1, n2) for different
Ln3+ binding to BSA were obtained directly under mi-
metic physiological conditions (310.15 K, 0.1 mol L–1
NaCl, pH 6.7). Results are listed in Table 2.
Then, apparent molar reaction enthalpy DrHm1 of
sites I can be determined by calculating several calo-
rimetric data before platform I by Eq. (8)
Other thermodynamic parameters for the binding of
lanthanide ions (Ln3+) to BSA
Steady-state fluorescence spectroscopic studies was
carried out by exciting the protein at 288 nm, moni-
toring the BSA intrinsic fluorescence intensity at
345 nm as a function of added Ln3+ with a view to elu-
cidating the binding stoichiometry. Figure 3 showed
that by plotting F0/F (F0 and F refer to the fluores-
cence intensity of BSA at 345 nm without Ln3+ and
DrHm1= Qi /(CLnV)
Subsequently, coordination number n1 of
sites¢ñcan be obtained by Eq. (10)
n1=QI/ (CBSADrHm1V)
where QI refers to the heat effect of platform I.
902
J. Therm. Anal. Cal., 89, 2007

INTERACTIONS OF LANTHANIDE IONS WITH BOVINE SERUM ALBUMIN
Table 1 Results of the apparent molar reaction enthalpy and coordination number for La3+ binding to BSA*
CLa/010–5 mol L–1
R
Qi/J
DrHm,i/kJ mol–1
DrHm/kJ mol–1
ni
n
5.64
6.21
0.93
1.02
1.28
1.71
2.05
2.56
3.41
5.12
6.83
10.24
0.01024
0.01399
0.01408
0.01485
0.01731
0.02485
0.03146
0.03707
0.04382
0.04400
30.48
30.25
DrHm1=30.37
7.76
1.28
1.34
n1=1.3
Platform I
10.34
12.41
15.51
20.69
31.03
41.37
62.06
23.27
26.72
25.33
19.91
DrHm2=23.81
5.06
5.08
n2=5.1
Platform II
*Annotation: R refers to Ln3+/BSA molar ratio; Qi the pure heat effect of reaction (Qi=Q'–Qd, where Q' refers to the total heat effect
i
i
of reaction and Qd the corresponding dilution heat effect of rare earth chlorides); DrHm,i is the calculated apparent molar reaction
enthalpy corresponding to a given R; The apparent molar reaction enthalpy of sitesland sites II (DrHm1 and DrHm2) equal to the
average value of DrHm,i before Platform l and II, respectively; ni the calculated number corresponding to a given R of Platform; The
coordination number of sitesland sitesII (n1 and n2) equal to the average value of ni corresponding to Platformland II, respectively
Table 2 Apparent molar reaction enthalpy (DrHm1, DrHm2) and coordination number (n1, n2) for 11 kinds of Ln3+ binding to BSA
Specific high-affinity sites (sites I)
Nonspecific low-affinity site (sites II)
Ln3+
ZRE
n1
DrHm1/kJ mol–1
n2
DrHm2/kJ mol–1
La3+
Pr3+
57
59
60
62
63
64
65
66
68
69
70
1.3
1.8
3.0
3.2
1.7
1.5
2.2
1.8
2.4
2.1
1.6
30.37
42.56
31.98
35.65
33.38
42.94
36.10
33.61
43.47
43.20
45.90
5.1
4.9
6.8
7.6
6.6
5.6
5.7
6.6
6.8
6.5
6.1
23.81
33.95
23.74
24.65
23.16
33.82
31.65
28.50
31.51
29.98
34.52
Nd3+
Sm3+
Eu3+
Gd3+
Tb3+
Dy3+
Er3+
Tm3+
Yb3+
with Ln3+, respectively) against the Ln3+/BSA molar
ratio, the fluorescence intensity at 345 nm increased
linearly with Ln3+ addition until the Ln3+/BSA molar
ratio reached a certain value where the titration curves
break off. Qualitative similar titration curves were ob-
tained for all other Ln3+. Collectively, it demonstrates
that there are at least two sets of Ln3+-binding sites on
BSA as previously described [27]. The coordination
number of specific binding sites (n1) corresponds to
the Ln3+/BSA molar ratio of break off points (shown
in Table 3). It was shown that the values of n1 corre-
spond to those obtained by microcalorimetry.
It can be obtained that
F/F0 = ([BSA]total – [BSA·Lnn1])/[BSA]total
[Ln3+] = [Ln3+]total – n1[BSA·Lnn1]
[BSA] = [BSA]total – [BSA·Lnn1]
Then, the apparent binding constants K1 for spe-
cific binding sites can be expressed as
K1 =[BSA Ln n1 ]/([Ln 3+ ]n1[BSA])=(1-F /F0 )/
([Ln 3+ total -n1[BSA]total +n1FF0-1[BSA]total )n1 FF0-1
]
{
}
(12)
Based upon the two sets of independent sites
model proposed in this paper, the specific binding
sites were saturated and the BSA·Lnn1 complex can be
formed at the break off point:
By Eq. (12), the apparent binding constants K1 of
specific sites (sites I) under mimetic physiological
conditions (310.15 K, pH 6.7, 0.1 M NaCl) were ob-
tained (Table 3). Then, other thermodynamic parame-
BSA+ n1Ln3+®BSA·Lnn1
J. Therm. Anal. Cal., 89, 2007
903

LI et al.
Fig. 3 Fluorescence titration curves (lex=288 nm): the fluorescence intensity of BSA at 345 nm; a – with the La3+/BSA molar ra-
tio and b – with the Gd3+/BSA molar ratio
Table 3 Coordination number n1, apparent binding constants lgK1 and other thermodynamic parameters of specific sites for 11
kinds of Ln3+*
Ln3+
n1
lgK1
DrGm1/kJ mol–1
DrSm1/J K–1 mol–1
La3+
Pr3+
1.9
2.2
3.5
3.0
2.1
2.1
2.0
2.2
2.1
1.9
2.0
7.90
8.98
–46.91
–53.32
–70.06
–71.90
–50.23
–40.37
–52.31
–85.26
–52.66
–57.83
–63.47
249.17
309.14
329.00
346.77
269.58
268.61
285.06
383.27
309.95
325.75
352.69
Nd3+
Sm3+
Eu3+
Gd3+
Tb3+
Dy3+
Er3+
11.80
12.11
8.46
6.80
8.81
14.36
8.87
Tm3+
Yb3+
9.74
10.69
*Annotation: DrGm1 and DrSm1 refer to the standard molar binding free energy and the standard molar binding entropy for the
specific high-affinity sites (sites I)
ters (DrGm1 and DrSm1) for specific sites were calcu-
lated by Eq. (11) (shown in Table 3).
A reasonable analysis for DrHm all positive can
be reached with the assumption that the total heat ef-
fect of coordination reaction between Ln3+ and BSA
divides into two parts, namely the hydration-
bond-breaking heat effect and the coordination-
bond-forming heat effect. The former dehydration
process is endothermic, while the latter coordination
process is exothermic. When the positive effect of the
former process on the enthalpy change exceeds the
negative effect of the latter one, the overall enthalpy
change DH is positive. It is confirmed that the coordi-
nation reaction is an entropy-driven process accord-
ing to the enthalpy-entropy compensation law.
Results showed that the apparent molar reaction
enthalpy DrHm are all positive ones. Then it was fur-
ther demonstrated that the binding process of Ln3+ to
BSA is endothermic, and is accompanied by entropy
increasing (as shown in Table 3).
As Evans [28] pointed out that in aqueous solu-
tion Ln3+ generally exist as complex ions. For H2O
tend to coordinate with Ln3+, the ubiquitous complex
is [Ln(H2O)n]3+. With other ligands coexisting, they
will probably compete with H2O for Ln3+. While only
under appropriate concentration, the oxo-ligands or
chelates can complex with Ln3+, and the complex
formed often contain water molecules. And it was fur-
ther indicated that the properties of complex ions are
important for lanthanide ions in aqueous solution.
And as Zhang [15] described that Ln3+ exist in
the form of hydrated ions in aqueous solutions, and
that the outer-sphere of BSA molecules combine with
one hydrated layer. Besides, the ligand-forming radi-
904
J. Therm. Anal. Cal., 89, 2007

INTERACTIONS OF LANTHANIDE IONS WITH BOVINE SERUM ALBUMIN
8 K. Chiba, T. Chyashiki and T. Mohri, J. Biochem.,
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In this work, the interactions of lanthanide ions (Ln3+)
with bovine serum albumin (BSA) under mimetic physi-
ological conditions (310.15 K, pH 6.7, 0.1 M NaCl)
were systemically studied on LKB-2107 batch
microcalorimeter. According to the microcalorimetric
results, number of binding sites (n1, n2) and apparent re-
action enthalpy (DrHm1, DrHm2) for different-affinity
binding sites of Ln3+ on BSA were obtained directly for
the first time. It was shown that the interactions are en-
dothermic and entropy-driving processes. Furthermore,
combining with fluorescence spectroscopic studies,
other thermodynamic parameters (DrGm1, DrSm1) were
determined for specific binding sites.
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Received: March 23, 2006
Accepted: August 7, 2006
DOI: 10.1007/s10973-006-7611-5
J. Therm. Anal. Cal., 89, 2007
905