368
Journal of the Chinese Chemical Society, 2009, 56, 368-373
Study on the Binding of Chloride Ion to Hemoglobin Using Electromotive
Force Method
Gong-Ke Wang (
), Yan Lu* (
) and Chang-Ling Yan (
)
College of Chemistry and Environmental Science, Henan Normal University,
Xinxiang, Henan 453007, P. R. China
The electromotive force of the concentration cell, in which both half-cells contain the same concentration of sodium chloride, and one of which also contains hemoglobin (Hb), was measured on the isoelectric
point of Hb (pH = 6.70). On the basis of Scatchard equation, a stepwise binding model and an improved
calculation method were presented. Using the new calculation method, the number of the chloride-binding sites on Hb molecule and the corresponding binding constants were calculated. The results show that
there are three classes of binding groups on a Hb molecule, the number of the binding sites and the corresponding binding constants are n1 = 1, K01 = 245; n2 = 8, K02 = 3.50; n3 = 8, K03 = 1.91, respectively. The
factors of influencing the interaction between Cl– and Hb molecule were clarified, and that the differences
between our results and the results of computer modeling, as well as the results of molecular dynamics
simulation were also discussed.
Keywords: Hemoglobin (Hb); Stepwise binding model; Binding sites; Binding constants.
INTRODUCTION
It is important to study the interactions of proteins
with ions and molecules in biochemistry and biophysics.
The relatively simple system of serum albumin and Cl– had
been mostly selected to study long before, because the natural environment of the serum proteins contains much sodium chloride. Furthermore, the special system is a representative one for investigating the relation of proteinic
physiological function and structure, and easy to establish
the model of the interactions between proteins and Cl–. In
1946, the osmotic pressure and the electromotive force of
bovine serum albumin (BSA) aqueous solutions at different concentration of sodium chloride were measured by
Scatchard1 et al. They discussed the influences of pH and
concentration of sodium chloride on the interaction between BSA and Cl–, and evaluated the molecular weight of
BSA. The further studies of Scatchard et al determined that
there are two classes of chloride-binging sites on human serum albumin (HSA), the number of the binding sites and
the corresponding binding constants are, respectively, n1 =
10, k01 = 44; n2 = 30, k02 = 1.1 by using the site-binding
model.2-4 With a more precise method, they also calculated
that there are three classes of chloride-binding sites on
BSA, which are, respectively, n1 = 1, k01 = 2400; n2 = 8, k02
= 100; n3 = 18, k03 = 3.3. Poland investigated the complete
binding polynomials for serum albumin using a new method
of analyzing the binding isotherms of biopolymers.5 Lin
Yang-Zheng6 and Lu Yan7 also established new state-equations, respectively. Using the average binding number and
the net charge, they studied the osmotic pressure of the
aqueous solutions of BSA and sodium chloride, and obtained preferable correlation and prediction results. In recent years, the extensive application of the ion-selective
electrode8-11 and the spectral technology,12-20 which is convenient for people to study the interactions of proteins with
ions and small molecules. However, there have been relatively few studies on Hb so far.
Bovine Hb has similar properties to human Hb to a
great extent, including its structure and the capability of
carrying oxygen.21 The capability of carrying oxygen of Hb
depends only on the concentration of Cl– in plasma, not on
the concentration of 2,3-diphosphoglycerate (2,3-DPG).22
Thus, it is of great importance to explain bovine Hb’s characteristic of carrying oxygen and the physical transmission
in biological systems by studying the interaction between
bovine Hb and Cl–. The computer simulation study on the
* Corresponding author. Tel: +86-373-3325249; E-mail: [email protected]
Binding of Chloride Ion to Hemoglobin
solvent accessible surface of the amino acids on bovine Hb
molecule, which was performed by Hu Yuan-Dong,21 facilitated our understanding of the reactive activity of the
amino acid residues on the surface of bovine Hb molecule.
Wang He-Yao et al.23 studied the secondary structure of
polymerized bovine Hb with Fourier transform infrared
spectrometer, and primarily analyzed the effects of the
polymerization on Hb’s conformation and physiological
function. Rosa and coworkers24 studied the chloride-binding sites on the surface of bovine Hb with molecular dynamics simulation and discussed the structural basis for an
adaptive mechanism. The purpose of the present study is to
investigate the interaction of Hb with Cl – and the chloride-binding sites on Hb molecule using electromotive
force method, and to present an improved method for
calculating the number of the chloride-binding sites and the
corresponding binding constants.
EXPERIMENTAL
Materials and Apparatus
Hemoglobin (molecular weight of 65,000 Da; from
bovine blood) was purchased from Sigma Chemical Co.
Sodium chloride and potassium chloride were kept over
P2O5 in a desiccator prior to use. All solutions were prepared in sodium phosphate buffers of pH 6.70 and an ionic
strength of 0.10 mol kg-1. The deionized water used throughout the experiments was doubly distilled over KMnO4.
SDC digital potential-meter (Nanjing, China) with an
accuracy of 10-5 V and a pair of Ag-AgCl electrodes (Shanghai, China) were used to measure the electromotive force of
the cell. Before each experiment, the Ag-AgCl electrodes
were stored in the solutions with the approximately same
concentration as those to be used in the experiments. All of
the chemical reagents were weighed on an electronic balance (Sartorius, Germany) with a sensitivity of 10-5 g. Precise pH meter (PHS-2C, Shanghai, China) was used to adjust pH of Hb solutions.
Measurements of the Electromotive Force
Since Hb can bind Cl–, the concentration of Cl– in
aqueous solution of NaCl without Hb must be different
from that with Hb. A pair of Ag-AgCl electrode was inserted in each of the two solutions and connected to a salt
bridge of saturated potassium chloride to form a concentration cell for Cl–. Let S1 denote the aqueous solution of NaCl
without Hb and S2 with Hb. The concentration of NaCl in
solutions S1 and S2 is expressed as c¢ and c, the activity coefficients of chloride ions as g¢ and g. The concentration of
J. Chin. Chem. Soc., Vol. 56, No. 2, 2009
369
Hb in solution S2 is expressed as c1, which is always kept in
a low concentration of 7 ´ 10-4 mol kg-1. In equilibrium, the
concentration of unbound Cl– in solution S2 is expressed as
[Cl–]F. Then the electromotive force E of the cell is
E = (RT/F)ln(c¢g¢/[Cl–]Fg)
(1)
where R is the gas constant, T the absolute temperature and
F the faraday constant. As mentioned above, the concentration of Hb in solution S2 is always kept in a low concentration. When the concentration of NaCl is small, the activity
coefficients of NaCl in both solutions S1 and S2 are close to
unit. When the concentration of NaCl is large, the amount
of Cl– bound to Hb is relative small, which will not make
evident effect on the activity coefficient of NaCl. Therefore, it is reasonable to assume that the activity coefficients
g¢ and g of NaCl in both solutions S1 and S2 are the same
when c¢ and c are close within 1%. At the same time, it was
assumed that the effect of Hb on g is negligible. From equation (1), [Cl–]F may be calculated as follows:
[Cl–]F = c¢ ´ exp(–EF/RT)
(2)
The average binding number B for Cl– is
B = (c – [Cl–]F)/c1
(3)
In the course of experiments, we firstly test the stability of the electrodes. When the system reaches equilibrium,
the electromotive force can be measured to an accuracy of
0.01 mV.
RESULTS AND DISCUSSION
Model of the Interaction of Chloride Ion with Protein
In general, the interactions between proteins and
small anions are essentially electrostatic interaction. If
each of n groups on a protein molecule (P) binds to an small
anion (I) with the same intrinsic binding constant (K0), the
average binding number (B) can be given by Scatchard
equation2,3
B = nK0exp (-2wZpZI)aA/
[1 + K0 exp (-2wZpZI)aA]
(4)
where w is the electrostatic coefficient, Zp is the average
370
J. Chin. Chem. Soc., Vol. 56, No. 2, 2009
Wang et al.
charge of the protein, and ZI is the charge of I ion. On the
isoelectric point of protein, Zp is equal to BZI. If the concentration of unbound I ion can be expressed as [I]F, the activity aA of unbound I ion should be g[I]F, in which g is the
activity coefficient.
In the general case, there is more than one class of
binding groups on a protein molecule. If each of n1 sites in
group 1 with the same intrinsic constant K01, and each of n2
sites in group 2 with K02, etc., equation (4) should be replaced by
B = B1 + B2 + …
= n1K01 exp(-2wB1Z2I) g[I]F/[1
+ K01 exp (-2wB1Z2I) g[I]F]
+ n2K02 exp (-2wB2 Z2I) g[I]F/[1
+ K02 exp (-2wB2Z2I) g[I]F] + …
(5)
If we define q as
q = g[I]F exp(-2wBZ2I)
(6)
spectively, n and nK0, and the slope is -K0. If there is more
than one class of binding groups, the plot will give a concave curve.3
Determination of the Parameters of Scatchard Equation
On the isoelectric point of Hb, pH = 6.70, the concentration of NaCl solution falls in the range from 0.5 ´ 10-3
mol kg-1 to 120 ´ 10-3 mol kg-1. The electromotive force E
measured in the experiment, [Cl–]F and B calculated from
equation (2) and (3), and the temperature T of each experiment are given in Table 1.
As mentioned above, if we want to obtain the number
of chloride-binding sites, n, on Hb molecule, the w and g
must be determined. In view of the simplified model of a
spherical protein molecule, the total charge of the protein
molecule is distributed symmetrically over the surface of
the sphere.2,25 If the radius of the protein molecule is b, an
unbound small ion can approach its center to a distance, a,
the Debye theory gives
w = e2/2DkT[1/b – k/(1 + ka)]
then equation (1) can be rewrited as
B/q = K0(n – B)
(7)
If there is only one class of binding groups on a protein
molecule, a plot for B/q versus B should be a straight line,
its intercepts on the horizontal and vertical axes are, re-
(8)
where e is the protonic charge, D is the dielectric constant
of the solvent, k is Boltzmann’ constant, T is the absolute
temperature, and k has its usual meaning in the Debye theory. We can assume that the values of b and a are, respectively, 30 Å and 32.5 Å.2,4 Thus, for the system, equation
Table 1. The parameters of Scatahard equation for the first class of binding between chloride ion and Hb in
sodium phosphate buffers of pH 6.70
c¢ ´ 103
c ´ 103 [Cl–]F ´ 103 E (mv)
0.50
1.00
2.00
3.01
5.08
8.00
10.00
11.99
16.01
20.00
30.00
50.00
70.01
90.00
119.99
00.50
01.00
02.00
02.99
05.08
08.01
10.00
11.98
16.02
20.01
30.01
50.00
70.02
90.00
120.050
000.428
000.868
01.75
02.68
04.62
07.36
09.20
11.10
14.90
18.68
28.21
47.29
66.57
86.08
115.280
3.92
3.56
3.34
2.88
2.39
2.10
2.08
1.93
1.80
1.70
1.54
1.39
1.26
1.11
1.00
B
w
g
q
B/q
T (K)
0.10
0.19
0.35
0.44
0.61
0.93
1.14
1.26
1.60
1.90
2.57
3.87
4.93
5.60
6.81
0.0979
0.0914
0.0836
0.0786
0.0716
0.0654
0.0624
0.0599
0.0560
0.0530
0.0478
0.0417
0.0380
0.0354
0.0326
0.9757
0.9663
0.9538
0.9448
0.9307
0.9163
0.9085
0.9018
0.8902
0.8808
0.8623
0.8370
0.8194
0.8050
0.7900
0.00041
0.00081
0.00158
0.00237
0.00361
0.00596
0.00726
0.00863
0.01111
0.01348
0.01900
0.02867
0.03763
0.04667
0.05821
244
234
222
186
169
156
157
146
144
141
136
135
131
120
117
293
293
293
292
291
290
290
290
290
291
290
290
290
291
290
Binding of Chloride Ion to Hemoglobin
J. Chin. Chem. Soc., Vol. 56, No. 2, 2009
(8) can be given as follows3
w = 2.303[0.0517 – 0.5085 I 0 /(1 + 10.6 I 0 )]
(9)
in which I0 is the ionic strength. For the activity coefficient
of Cl–, considering its diameter, we have used the correctional form of Debye-Huckel expression
somewhat greater than – n1. It is same to say that the intercept is equal to n1K01 and the slope is equal to – K01, just
like what the equation (7) expressed. If n1 is considered to
be an integer, it may be determined with some precision,
and K01 may be determined immediately. According to the
form of the curve in Fig. 1, we can express it as
B/q = A + C exp(–B/D)
logg = –A|z+z-| I 0 /(1 + aB I 0 )
(11)
The values of q, w and g calculated with equation (6),
(9) and (11), respectively, are also listed in Table 1.
Investigation of the Binding Parameters by the Stepwise Binding Model
Using the data in Table 1, B/q is plotted against B in
Fig. 1 and the curve obtained is obviously not a straight
line, which indicates that there is more than one class of
chloride-binding sites on Hb molecule. Scatchard 3 had
pointed out that the product of the intercept and the limit
slope of the curve in Fig. 1 at B = 0 is approximately equal
to – n1(K01)2, and the ratio of the intercept to the slope was
Fig. 1. The Scatchard plot of B/q versus B for the first
class of binding between chloride ion and Hb of
isoelectric point.
(12)
(10)
where z+ is 1, z- is –1, the average effective diameter of Cl–,
a, is 6.1 Å,2,4 the constants A and B are, respectively, 0.505
and 0.328.26 Then equation (10) can be expressed as the
following approximate form
logg = –0.505 I 0 /(1 + 2 I 0 )
371
where A, C and D are constants. Making a tangent on the
curve at B = 0, the intercept of the tangent on the vertical
axe is A + C, and its slope is – C/D. In order to obtain n1 and
K01, the data of B/q and B in Table 1 is fitted according to
equation (12). The results show A1 = 128.64, C1 = 138.75,
D1 = 0.62. Therefore, the binding parameters of the first
class of group, n1 = 1 and K01 = 245 may be determined.
The chloride-binding sites on Hb molecule may be
classified as first class groups, second class groups, third
class groups, and so on, according to the values of biding
constant. After the first class of binding parameters has
been determined, we wish to further study the binding parameters for other class of groups. Therefore, a stepwise
binding model is presented here. The model is based on an
assumption that the binding of the latter class of Cl– will
take place when the binding equilibrium of the former class
of Cl– has reached. According to this model, we can take
the protein and the bound Cl– as a whole when considering
the further binding of Cl–. This means that we can use the
same method to investigate the second class of chloridebinding sites on Hb molecule as the first class determined.
The calculation method is as follows. With n1 and K01 obtained above, the average binding number of the first class
of binding, B1, can be determined by using equation (4) for
every experiment point listed in Table 1. With B1, we can
calculate the concentration of unbound chloride ions,
[Cl–]F1, of the solution which is in the equilibrium of the
first class of groups with Cl–. With [Cl–]F1, we can determine the corresponding w1 and g1 by using equation (9) and
(11). Substituting [Cl–]F1, w1 and g1 into equation (6) and
using (B – B1) instead B, we can calculate a set of q1 for every experiment point. According to the method for determine n 1 and K 01 mentioned above, a plot of (B – B1)/q1
against (B – B1) are made with the data calculated, which
are given in Fig. 2. It can be seen that Fig. 2 neither gives a
straight line, which indicates that there is other class of
chloride-binding groups on the whole. The data can also be
fitted to equation (12) and obtain A2 = 26.93, C2 = 1.48 and
372
J. Chin. Chem. Soc., Vol. 56, No. 2, 2009
Wang et al.
Ability of the Binding of Hb to Chloride Ion
As calculated above, there are three classes of chloride-binding sites on Hb molecule, and the total number of
binding sites is 17. The values of the binding constants of
the three classes are in the order K01 > K02 > K03, which can
be explained by the electrostatic repulsion originated from
the negative charge of Cl–. The binding constant of the first
class is much lager than others, which indicate that it is the
strongest binding among the three classes. The binding
constants of the second and third classes are, respectively,
3.50 and 1.91, which shows that the two classes of binding
are comparatively weak. It has been assumed above that the
binding of Cl– to the binding groups of Hb molecule is in
the order of the first class, the second class and the third
class. The values order of the binding constants for the
three classes is consistent with the binding order.
Hb molecule is a tetramer, which is composed of two
same dimers, a1b1-subunit and a2b2-subunit, and each peptide chain contains 70c/o a-helix content. The molecular
dynamics simulation 24 shows that the chloride-binding
sites on Hb molecule are Val-a11, Asn-a1131, Arg-a2141,
Lys-b8, Lys-b76 and His-b77. With this study, the number
of the chloride-binding sites on Hb molecule is 6, which is
less than that of this work. The different number should be
caused by the difference of pH used in the two works. The
pH in our experiments is 0.7 less than that of used by
Rosa. 24 This small change in pH will induce a large decrease in the affinity of Hb molecule for oxygen, and cause
the conformation of Hb molecule to change from R state to
T state.24,27 This allosteric effect will enhance the affinity of
Hb molecule for Cl–,23 thus more Cl– can be bound to Hb
molecule.
Hb molecule has many kinds of groups, but the binding sites for Cl– should be the positively charged groups.
Hu Yuan-Dong et al. studied the solvent accessible surface
for the ab-subunit of Hb molecule.21 Their results indicated that there are 24 lysine residues in Hb molecule, but
only 12 of them have bigger exposure extent and better reactive affinity. The present work is to study the Hb molecule with two ab-subunits. According to the results of Hu
Yuan-Dong, the Hb molecule should have at least 24 active
sites which have the ability of binding to Cl–. Although this
number is some bigger than that of this work, the two results are not contradictory and the difference is easy to ex-
Fig. 2. The Scatchard plot of (B – B1 )/q1 versus (B –
B 1 ) for the second class of binding between
chloride ion and Hb of isoelectric point.
Fig. 3. The Scatchard plot of (B – B1 – B2)/q versus (B
– B1 – B2) for the third class of binding between
chloride ion and Hb of isoelectric point.
D2 = 0.43. According to the same method used above, the
binding parameters of the second class of group with Cl–
are calculated as n2 = 8 and K02 = 3.50. Again, we take the
protein and the Cl– bound by the two classes of groups as a
new whole, and calculate the corresponding parameters
with the same method just used for the binding of second
class group. Following the same procedure, a plot of (B –
B1 – B2)/q2 versus (B – B1 – B2) is given in Fig. 3. Since the
curve in Fig. 3 is a straight line with the experimental errors, it means that there is not other class of chloride-binding sites on the protein molecule. According to equation
(7), the data is linearly regressed and the results indicate n3
= 8, K03 = 1.91.
Binding of Chloride Ion to Hemoglobin
plain. The exposure extent of a amino-acid residue is a necessary condition for binding to ion, but it is not sufficient.
There are many other factors which can influence the binding, such as temperature, pH, charge density of the group
and the electrostatic repellency of the bounded Cl– to the
Cl– which will be bounded next. Ayranci has studied the
binding of iodide ion to protamine.10 He pointed out that although all the reactive sites in protamine are accessible to
iodide ion, protamine does not contain an easily available
positively charged centre. Therefore, 24 should be the upper limit of the number of sites which can bind to Cl–. It is
reasonable that the total binding number of Hb molecule
for Cl– is 17.
CONCLUSIONS
In this paper, the binding of Cl– to Hb on the isoelectric point was studied with the method of electromotive
force measurement. Based on Scatchard equation, a stepwise binding model and an improved calculation method
were presented. Using the new calculation method, the
number of the chloride-binding sites on Hb molecule and
the corresponding binding constants were calculated. The
calculating results showed that there were three classes of
binding sites, their numbers are n1 = 1, n2 = 8 and n3 = 8, respectively. The values of the binding constants of the three
classes are in the order K01 > K02 > K03. This order is consistence with their binding sequence. Comparing with the
studies of computer modeling and molecular dynamics
simulation, the total binding number, 17, of Hb molecule
for Cl– is reasonable.
ACKNOWLEDGEMENTS
The authors acknowledge financial support from the
National Natural Science Foundation of China (No.
20673034) and the Research Fund for the Doctoral Program of Higher Education of China (No. 20060476001).
Received October 16, 2008.
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