Báo cáo khoa học: Mutual effects of proton and sodium chloride on oxygenation of liganded human hemoglobin Oxygen affinities of the a and b subunits potx

Mutual effects of proton and sodium chloride on
oxygenation of liganded human hemoglobin
Oxygen affinities of the a and b subunits
Sergei V. Lepeshkevich and Boris M. Dzhagarov
Institute of Molecular and Atomic Physics, National Academy of Sciences of Belarus, Minsk, Belarus
Normal adult human hemoglobin (HbA) is the classic
textbook example of an allosteric protein. The HbA
molecule is a heterotetramer consisting of two a sub-
units and two b subunits, a
2
b
2
, which are arranged,
around a central water-filled cavity, as a pair of ab
dimers [1]. Each subunit carries one heme group to
which one oxygen molecule binds reversibly. Oxygen-
ation of HbA in solution or inside red blood cells is
cooperative, i.e. the oxygen affinity for each subunit
rises as the other hemes in the same tetramer became
saturated with oxygen [2,3]. This cooperative inter-
action has been explained as the result of a shift in the
equilibrium between two quaternary structures: from
the unliganded structure of the low-affinity (T-state) to
the high-affinity structure characteristic of the fully sat-
urated molecule (R-state) [1,2,4]. Recently, a new inter-
pretation of the molecular mechanism of cooperativity
and allostery of HbA has been deduced [5–7]. It was
shown that ‘stripped’ HbA is a surprisingly inert, mod-
erately cooperative O
2
carrier with limited functional

diversity if heterotropic effectors are absent. Further-
more, it was shown that HbA exhibits amazing func-
tional diversity in terms of O
2
affinity, cooperativity
and the Bohr effect only in the presence of heterotropic
allosteric effectors including hydrogen, phosphates and
chloride ions. Such functional diversity is generated
primarily by the tertiary structural constraints caused
by interaction of the effectors with HbA, especially
with oxy-HbA, rather than the T ⁄ R quaternary struc-
tural transition. Allosteric effectors allow HbA to take
up and release oxygen in response to changing physio-
logical conditions. Because tetrameric hemoglobin
consists of two types of subunits, differing in structure,
knowledge of ligand affinities for each subunit type in
Keywords
a and b subunits; affinity; human
hemoglobin; molecular oxygen; sodium
chloride
Correspondence
B.M. Dzhagarov, Institute of Molecular and
Atomic Physics, National Academy of
Sciences of Belarus, 70 Nezavisimosti Ave,
Minsk 220072, Belarus
Fax: +375 17 284 0030
Tel: +375 17 284 1620
E-mail:
(Received 1 July 2005, revised 2 October
2005, accepted 6 October 2005)

doi:10.1111/j.1742-4658.2005.05008.x
The different effects of pH and NaCl on individual O
2
-binding properties
of a and b subunits within liganded tetramer and dimer of human hemo-
globin (HbA) were examined in a number of laser time-resolved spectro-
scopic measurements. A previously proposed approach [Dzhagarov BM &
Lepeshkevich SV (2004) Chem Phys Lett 390, 59–64] was used to determine
the extent of subunit dissociation rate constant difference and subunit
affinity difference from a single flash photolysis experiment. To investigate
the effect of NaCl concentration on the association and dissociation rate
constants we carried out a series of experiments at four different concentra-
tions (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr
effect. As the data suggest, the individual properties of the a and b sub-
units within the completely liganded tetrameric hemoglobin did not depend
on pH under salt-free conditions. However, different effects NaCl on the
individual kinetic properties of the a and b subunits were revealed. Regula-
tion of the O
2
-binding properties of the a and b subunits within the ligan-
ded tetramer is proposed to be attained in two quite different ways.
Abbreviations
BR, bimolecular recombination; GR, geminate recombination; HbA, human hemoglobin.
FEBS Journal 272 (2005) 6109–6119 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6109
the different conformational forms of HbA is a key
factor in the complete description of the sigmoidal be-
havior of HbA oxygenation.
Taking advantage of the photosensitivity of the
heme Fe–ligand bond [8–14], flash photolysis has been
used extensively in kinetic studies of oxygenated hemo-

globins. Recently, the rates of O
2
association [14–16]
to both a and to b subunits within triliganded HbA
and the efficiency of O
2
escape [15,16] from these sub-
units within the completely oxygenated tetramer were
obtained using laser photolysis. In a recent study [17],
having the individual parameters of bimolecular
recombination (BR) for each subunit type within
native HbA, it has been proposed that the relative O
2
affinity for HbA subunits could be determined using a
single flash photolysis experiment. The approach is
based on determination of the bimolecular association
rate constant of O
2
rebinding and the quantum yield
of BR, c. The latter value is defined as the ratio of the
number of O
2
, which succeed in escaping into the sur-
rounding medium after photodissociation, to the num-
ber of the absorbed light quanta. It should be pointed
out that the ratio of the number of the dissociated O
2
molecules to that of the absorbed light quanta defines
the primary quantum yield of photodissociation, c
0

[16]. As soon as we can determine the a and b subunit
heterogeneity in oxygenation, it is a great importance
to find the effect of different heterotropic effectors on
the individual parameters of oxygenation for each sub-
unit type within the tetrameric protein. Recently [18],
effects on the individual properties of the a and b sub-
units within oxygenated HbA have been revealed at
the pyridoxal 5¢-phosphate modification. In this study,
we first attempt to evaluate the mutual effects of pH
(over the range of the alkaline Bohr effect) and NaCl
on the individual oxygen binding properties of the a
and b subunits within liganded tetrameric HbA. In the
current literature, there are controversial results con-
cerning the pH dependence of the fourth Adair con-
stant [19–23]. Change in pH, over the range of the
alkaline Bohr effect, does not seem to have any signifi-
cant effect on the dissociation [19] and association [20]
rate constants, suggesting pH-independent properties
for the liganded hemoglobin. This suggestion is con-
trary to kinetic results [21,22], showing that the rate
of O
2
binding to the triliganded hemoglobin is pH
dependent. The pH dependence of the fourth Adair
constant was also shown by Imai and Yonetani [23] by
determination of the hemoglobin affinity for the fourth
ligand molecule.
The purpose of this study was twofold. First, the
mutual effects of pH and NaCl on the bimolecular
association rate constant of O

2
rebinding and the
quantum yield of BR for the a and b subunits within
the liganded dimer and tetramer of hemoglobin were
determined. Second, pH and NaCl effects on the total
protein affinity to oxygen and on the subunit affinity
were studied [17]. The results indicate that the allosteric
effectors modulate the O
2
rebinding to the a and b
subunits in two quite different ways.
Results
Photo-induced HbA reoxygenation was studied when a
small amount (0.3–0.5%) of O
2
was released from fully
saturated HbA. Bearing in mind the contribution from
geminate recombination (GR), we assumed that the
primary photodissociation level did not exceed 5%
[16]. Such a photoexcitation level was used to ensure
the experimental conditions when, statistically, each
photo-deoxygenated hemoglobin molecule loses only
one molecule of oxygen after photo-irradiation and the
tetrameric protein remains in its original state [24,25].
In fact, after photodissociation in the hemoglobin solu-
tion, two reactions are initiated simultaneously. One
occurs with the participation of the a subunit within
HbA and the other with participation of the b subunit:
ðaO
2

; bO
2
ÞðaO
2
; bO
2
ÞÀ!
hv
ða; bO
2
ÞðaO
2
; bO
2
ÞþO
2
À!
k
0
a
ðaO
2
; bO
2
ÞðaO
2
; bO
2
Þ
ðaO

2
; bO
2
ÞðaO
2
; bO
2
ÞÀ!
hv
ðaO
2
; bÞðaO
2
; bO
2
ÞþO
2
À!
k
0
b
ðaO
2
; bO
2
ÞðaO
2
; bO
2
Þ

ð1Þ
where (aO
2
, bO
2
)(aO
2
, bO
2
) denotes the oxyhemoglob-
in molecule. In Scheme 1, the oxygenated subunits
are shown together with O
2
. The central terms in
Scheme 1 represent the case of free O
2
motion in the
solution. Here k¢
a
and k¢
b
are, respectively, the rate
constants of BR for the a and b subunits within tri-
liganded HbA.
Time courses for O
2
rebinding are shown in Figs 1
and 2. The transient absorption decays were analyzed
using a standard least-squares technique using home-
made software for PC. After kinetic normalization,

analysis showed that the time courses for the HbA re-
oxygenation over the microsecond (0–4000 ls) time
range are fitted with a biexponential function:
DA
norm
¼ a
a
Á expðÀk
0
a
Á½O
2
ÁtÞþa
b
ÁexpðÀk
0
b
Á½O
2
ÁtÞ
ð2Þ
where DA
norm
is a normalized change in optical density
of the sample and a
a
, a
b
, k¢
a

and k¢
b
are the ampli-
tudes and rate constants of BR. The quantity [O
2
]is
the concentration of molecular oxygen dissolved in the
pH and NaCl effects on HbA subunits oxygenation S. V. Lepeshkevich and B. M. Dzhagarov
6110 FEBS Journal 272 (2005) 6109–6119 ª 2005 The Authors Journal Compilation ª 2005 FEBS
buffer. Based on considerations described previously
[13–16], these two exponential processes are assigned
to BR of the a and b subunits within HbA (Model 1).
The quantum yield of these processes are defined as
c
a(b)
¼ 2Æa
a(b)
Æc. Here c, the quantum yield of BR for
tetrameric HbA, is determined using a relative method
discussed previously [15]. HbA in 10 mm Tris ⁄ HCl,
pH 7.4, buffer is used as a reference standard, for
which c ¼ 0.023 ± 0.003 was obtained [16]. Also, the
efficiency of O
2
escape from the protein matrix after
photodissociation, d, is calculated as the ratio of the
quantum yield of BR, c, to that of the primary quan-
tum yield of photodissociation, c
0
.

Having the individual parameters of bimolecular
oxygenation for each subunit type within HbA, the
extent of subunit dissociation rate constant difference
(k
2
/k
1
) and the magnitude of subunit affinity difference
(K
2
/K
1
) [17] was calculated using the formulas:
k
2
k
1
¼
k
th2
c
02

Á
k
th1
c
01

À1

Á
c
2
c
1
ð3Þ
and
K
2
K
1
¼
k
th2
c
02

Á
k
th1
c
01

À1
Á
k
0
2
k
0

1
Á
c
1
c
2
ð4Þ
respectively. Here the subscripts 1 and 2 correspond to
two compared subunits within the tetramers as well as
within the dimers in similar or different conformations.
The kinetic rate, k
th
, represents the thermal bond-
breaking rate. As concluded previously [17], for each
oxygenated subunit type in the different conformational
forms of the protein, the thermal bond-breaking rate,
Fig. 1. Effect of chloride on hemoglobin oxygenation at (A) pH 8.5,
(B) pH 7.4 and (C) pH 6.8. Time courses for the recombination of
hemoglobin with oxygen in the absence of NaCl (a) and at a NaCl
concentration of 0.5
M (b), and 2.0 M (c). Insets shows residuals (a)
(b), and (c) from the double exponential fits of the curve (a) (b), and
(c), respectively. Excitation wavelength, k
exc
¼ 532 nm; detection
wavelength, k
det
¼ 430 nm. Conditions: 10 mM Tris ⁄ HCl buffer, at
21 °C. Protein concentration, 100 lm in heme.
Fig. 2. Normalized time courses for the oxygenation of hemoglobin

at pH 6.8 (a, b) and pH 8.5 (c) in the presence of 2.0
M NaCl. Heme
concentration: 100 l
M (a, c), and 20 lM (b). Excitation wavelength,
k
exc
¼ 532 nm; detection wavelength, k
det
¼ 430 nm. Conditions:
10 m
M Tris ⁄ HCl buffer, at 21 °C.
S. V. Lepeshkevich and B. M. Dzhagarov pH and NaCl effects on HbA subunits oxygenation
FEBS Journal 272 (2005) 6109–6119 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6111
k
th
, can be considered constant with an accuracy of
9%. Dzhagarov et al. [26] determined the value, c
0
, for
the a and b subunits within oxygenated HbA to be
equal to that for the isolated chains, c
0
¼ 0.23 ± 0.03.
Therefore, the ratio of k
th
/c
0
in Eqns (3) and (4) can
be considered constant for each oxygenated subunit
type in different conformational forms of tetrameric

and dimeric HbA. Knowledge of the association rate
constants, k¢
2(1)
, and the quantum yields of BR, c
2(1)
,
is required only to find simultaneously the extent of
subunit dissociation rate constant difference and the
magnitude of subunit affinity difference from a single
flash photolysis experiment.
As soon as we are able to determine the magnitude
of the a and b subunit affinity difference (Eqn 4), it
seems very important to introduce the total tetramer
(dimer) affinity, K
t
, for the last ligand binding step:
K
t
¼
K
1
Á K
2
K
1
þ K
2
ð5Þ
Here, K
1

and K
2
correspond to the affinity of O
2
bind-
ing to the a and b subunits within the triliganded
(monoliganded) tetramer (dimer), respectively. Hence
it is straightforward to show that the extent of protein
total affinity difference can be determined as:
K
t
ðp
1
Þ
K
t
ðp
2
Þ
¼
1 þ
K
1
ðp
2
Þ
K
2
ðp
2

Þ
K
1
ðp
2
Þ
K
1
ðp
1
Þ
þ
K
1
ðp
2
Þ
K
2
ðp
1
Þ
ð6Þ
Here, p
1
and p
2
correspond to two compared proteins.
Subscripts 1 and 2 correspond to two different types
of subunits within considered proteins.

Rates of O
2
binding to the a and b subunits
within liganded hemoglobin measured over the
pH range of the alkaline Bohr effect
The bimolecular oxygenation parameters measured at
different proton concentrations are given in Table 1.
In this set of experiments, the HbA concentration is
100 lm in heme. At such a concentration no more
than 10%, by weight, of the hemoglobin is in the
dimer form [27–30]. However, dimer formation does
not appear to affect the measured values of hemo-
globin oxygenation because under these pH conditions
the kinetic parameters for the last step in ligand bind-
ing to tetrameric HbA and those for binding to dimer-
ic HbA are almost identical [13–15]. As seen from
Table 1, the individual properties of the a and b sub-
units do not depend on pH. Small nonprincipal scat-
tering of the kinetic parameters, observed at a number
of pH values, can be considered ‘error bars’ for the
results. Therefore, for later use, the averaged bimole-
cular oxygenation parameters in the salt-free buffers
(Table 1, Average) can be considered as follows. The
BR rate constant for the a subunits within triliganded
HbA and the BR quantum yield for the a subunits
within completely oxygenated HbA fall in the range
30±3lm
)1
Æs
)1

and 0.012 ± 0.003, respectively. The
association rate constant and the BR quantum yield
for the b subunits are found to lie in the range
66±3lm
)1
Æs
)1
and 0.036 ± 0.006, respectively. The
data show an essential ligand-rebinding difference
between the a and b subunits. On average, one in
every 10 photodissociated O
2
molecules succeeds in
escaping from the protein matrix of the triliganded
HbA (Table 1, <d>), but only one in every 20 ligands
leaves the a subunits (Table 1, <d
a
>), and in every
six ligands leaves the b subunits (Table 1, <d
b
>).
Using Eqns (3) and (4), the dissociation rate con-
stant, k, and the O
2
affinity, K , can be derived for both
the a and b subunits from the averaged parameters of
HbA oxygenation (Table 1, Average). The association
and dissociation rate constants for the b subunits
are found to exceed 2.2 ± 0.3- and 3.1 ± 0.9-fold,
respectively, the corresponding values obtained for the

a subunits within HbA. We also found that the O
2
affinity for the a subunits is 1.4 ± 0.3 times higher
than that for the b subunits. The data are in a good
agreement with previous data [13,14].
Mutual effects of pH and NaCl on the total
protein affinity
To investigate the effect of NaCl on O
2
binding to the
a and b subunits within liganded HbA we carried out
a series of experiments at four NaCl concentrations of
0.1, 0.5, 1.0 and 2.0 m. The rate constant of BR and
the quantum yield of BR, in the presence of NaCl,
gave a direct evidence of significant functional hetero-
geneity for the a and b subunits in the last ligand-
binding step (Table 2).
The change in the total protein affinity to oxygen is
derived from the rate constant and quantum yield of
BR using Eqns (4) and (6). The NaCl effect is seen at
a concentration of 0.1 m (results not shown). At both
pH 6.8 and 7.4, the total protein affinity to oxygen
decreases as the NaCl concentration is increased up to
0.5 m with respect to the absence of NaCl (Fig. 3A1
and A2), the largest change being at pH 6.8. However,
as the salt concentration continues to be increased up
to 2.0 m at these pH values (Fig. 3, B1 and C1; B2
and C2), the affinity does not decrease further but
increases. At pH 8.5 (Fig. 3, A3, B3 and C3), there is
a constant increase in the affinity as a function of

increasing NaCl concentration. The tendency for an
pH and NaCl effects on HbA subunits oxygenation S. V. Lepeshkevich and B. M. Dzhagarov
6112 FEBS Journal 272 (2005) 6109–6119 ª 2005 The Authors Journal Compilation ª 2005 FEBS
increase by a factor of 1.35 ± 0.37 in the oxygen affi-
nity is found at 2.0 m NaCl.
Effect of NaCl concentration on the rates of O
2
binding to the a and b subunits within liganded
dimer
Sodium chloride is known to promote the dissociation
of liganded hemoglobin [2]. In addition to tetramer–
dimer dissociation, the dimer oxygenation is assumed
to be altered with increasing ionic strength of the sol-
vent [31]. Therefore, to investigate the effect of NaCl
on O
2
binding to tetrameric HbA, the contribution of
O
2
rebinding with dimer to the total protein oxygen-
ation must be taken into account and the effect of
NaCl concentration on the rates of O
2
binding to the
a and b subunits within the dimer must be found. It is
well known [31] that an increase in the dimer fraction
with protein dilution at a fixed high salt level can
imply a change in the time course for total protein
oxygenation. We took this as a starting point for our
investigation. Thus, to estimate the contribution of O

2
rebinding with dimer to the total protein oxygenation
we performed the following experiment. At pH 6.8 and
8.5 with 2.0 m NaCl (Fig. 2), the HbA concentration is
reduced from 100 to 20 lm in heme. Under these con-
ditions, the fraction of monomers in solution can be
neglected [32]. However, there is an appreciable
amount of dimer. Such dilution, at pH 6.8, must lead
to an increase in the dimer fraction from 40 ± 75 to
75 ± 90% [27,28,33].
As it can be seen from Table 2, this expected
increase in dimer fraction at pH 6.8 leads to a notice-
able increase in the association rate constant for a sub-
units, k¢
a
. It should be emphasized that the protein
solution after dilution at pH 6.8 (Figs 3D1, 4C), exhib-
its the individual properties of the a and b subunits
intermediate between those of the protein solution
before the dilution at pH 6.8 (Figs 3C1, 4B) and
pH 8.5 (Fig. 3C3, 4D). However, the absence of any
detectable changes in oxygenation at the dilution at
pH 8.5 (Table 2; Fig. 3C3, D3) suggests that, under
these conditions, oxyhemoglobin is completely in the
form of dimer. Thus, the dimer oxygenation properties
can be determined at pH 8.5 (2.0 m NaCl) at a hemo-
globin concentration of 100 or 20 lm in heme. Taking
into account the almost identical ligand-binding prop-
erties of the subunits within liganded tetrameric and
dimeric HbA under salt-free conditions [13,14], the

tendency for the increase by a factor of 1.35 ± 0.37 in
the total dimer affinity to oxygen, K
t
, can be found
with increasing the salt concentration at pH 8.5
(Fig. 3A3, B3, C3). The value agrees reasonably well
with that ($ 1.4) obtained for the liganded dimer in a
variety of salt solutions at pH 7.4 and quoted previ-
ously [31]. The observed tendency for an increase in
the total dimer affinity is caused mainly by the increase
by a factor of 2.2 ± 0.9 in the O
2
affinity of the a sub-
units within oxygenated dimer (Fig. 4D3). In turn, this
a subunit affinity increase results from the remarkable
decrease by 1.8 ± 0.6 times in the dissociation rate
constant at an insignificant change in the association
rate constant (Fig. 4, D2 and D1, respectively). Also,
at pH 8.5, the rebinding study reveals an increase in
the association and dissociation rate constant for the
b subunit within dimer by a factor of 1.62 ± 0.09 and
1.5 ± 0.4, respectively (Fig. 4, D4 and D5). At such
rate constant variation, b subunit affinity does not
change noticeably (Fig. 4, D6). As a result, at the
highest salt level the b subunit within the liganded
dimer exhibited a threefold lower affinity than that for
the a subunit.
Table 1. Kinetic parameters for oxygen rebinding to the oxygenated forms of human hemoglobin after laser photolysis. Protein concentra-
tions are 100 l
M on a per heme basis. Conditions: 10 mM Tris ⁄ HCl buffer, at 21 °C.

pH
k¢
a
lM
)1
Æs
)1
k¢
b
lM
)1
Æs
)1
a
a
%
a
b
%
c
a
, d
a
a
·10
)2
, ·10
)2
c
b

, d
b
a
·10
)2
, ·10
)2
c, d
a
·10
)2
, ·10
)2
8.5 31±2 66±3 24±3 76±3 1.1±0.2
[4.8 ± 1.0]
3.4 ± 0.4
[15 ± 3]
2.2 ± 0.3
[9.7 ± 1.6]
7.4 27.9 ± 1.3 63.2 ± 0.9 22 ± 2 78 ± 2 1.01 ± 0.16
[4.4 ± 0.9]
3.6 ± 0.4
[16 ± 3]
2.3 ± 0.3
[10.0 ± 1.7]
6.8 29.0 ± 1.5 65.0 ± 1.8 26 ± 3 74 ± 3 1.3 ± 0.2
[5.5 ± 1.1]
3.7 ± 0.5
[16 ± 3]
2.5 ± 0.3

[10.7 ± 1.8]
Average
b
<30 ± 3> <66 ± 3> <24.5 ± 4.5> <75.5 ± 4.5> <1.2 ± 0.3>
<[5.1 ± 1.6]>
<3.6 ± 0.6>
[16 ± 4]
<2.4 ± 0.5>
[10 ± 2]
a
The efficiency of O
2
escape from the protein matrix, d , is presented in square brackets. For the kinetic parameters the uncertainties are
presented as 95% confidence intervals.
b
The average bimolecular oxygenation parameters are given in the row ‘Average’ in the angled
brackets.
S. V. Lepeshkevich and B. M. Dzhagarov pH and NaCl effects on HbA subunits oxygenation
FEBS Journal 272 (2005) 6109–6119 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6113
Effect of NaCl concentration on the rates of O
2
binding to the a and b subunits within liganded
tetramer
As evident from the experiment at pH 8.5 and the pre-
vious one at pH 7.4 [31], the effect of NaCl concentra-
tion on dimer oxygenation is manifested as a slight
increase in the total protein affinity to oxygen but not
as an affinity decrease. Therefore, the reduction in pro-
tein affinity at 0.5 m NaCl at pH 6.8 and 7.4 cannot
be attributable solely to dimerization at the increased

salt concentration or to the moderate change in the
dimer oxygenation.
From this reasoning, the ligand-binding properties
of the completely liganded tetrameric HbA should be
considered as sensitive to proton and NaCl concentra-
tions. Therefore, our study indicates that the hemo-
globin solution is comprised of dimers and tetramers,
whose ligand-binding properties are dependent on the
buffer conditions. As the chloride concentration
increases at pH 8.5, complete dissociation of tetrameric
hemoglobin to dimer without a detectable change in
the O
2
-binding properties of the tetramer may be
inferred to take place. By contrast, at pH values of 6.8
and 7.4, addition of NaCl to a concentration of 0.5 m
results not only in an increase in the dimer fraction
[33], but also in a change in tetramer oxygenation.
Subsequent increases in salt concentration at these pH
values leads, for the most part, to the further tetramer
dissociation.
Referring to Fig. 3, the largest decrease in the total
protein affinity to oxygen and, consequently, the lar-
gest change in the O
2
-binding properties of the ligan-
ded tetramer are observed at a NaCl concentration of
0.5 m at pH 6.8. Here, the ligand-binding properties of
the tetramer can be found if two initial conditions are
imposed: (a) $ 30% of the hemoglobin is in the dimer

form under these buffer conditions [33]; and (b) total
dimer affinity does not vary practically above 0.5 m
NaCl [31], so the ligand-binding properties of the
Table 2. Mutual effects of proton and NaCl on hemoglobin oxygenation. Conditions: 10 mM Tris ⁄ HCl buffer, at 21 °C.
pH
Protein
conc. l
M
(in heme)
NaCl
conc.
M
k¢
a
lM
)1
Æs
)1
k¢
b
lM
)1
Æs
)1
a
a
%
a
b
%

c
a
, d
a
a
·10
)2
, ·10
)2
c
b
, d
b
a
·10
)2
, ·10
)2
c, d
a
·10
)2
, ·10
)2
8.5 100 None 31±2 66±3 24±3 76±3 1.1±0.2
[4.8 ± 1.0]
3.4 ± 0.4
[15 ± 3]
2.2 ± 0.3
[9.7 ± 1.6]

0.5 31 ± 4 76 ± 3 18 ± 4 82 ± 4 1.0 ± 0.3
[4.3 ± 1.3]
4.4 ± 0.6
[19 ± 3]
2.7 ± 0.3
[12 ± 2]
1.0 32 ± 3 84 ± 2 15 ± 2 85 ± 2 0.84 ± 0.15
[3.7 ± 0.8]
4.7 ± 0.6
[20 ± 4]
2.8 ± 0.3
[12 ± 2]
20
2.0
2.0
35 ± 3
38 ± 5
106.4 ± 1.3
100 ± 5
10.8 ± 1.6
11 ± 2
89.2 ± 1.6
89 ± 2
0.6 ± 0.2
[2.8 ± 1.0]
0.66 ± 0.14
[2.9 ± 0.7]
5.4 ± 0.7
[23 ± 4]
5.3 ± 0.7

[23 ± 4]
3.0 ± 0.4
[13 ± 2],
3.0 ± 0.4
[13 ± 2]
7.4 100 None 27.9 ± 1.3 63.2 ± 0.9 22 ± 2 78 ± 2 1.01 ± 0.16
[4.4 ± 0.9]
3.6 ± 0.4
[16 ± 3]
2.3 ± 0.3
[10.0 ± 1.7]
0.5 16.6 ± 1.1 64.6 ± 1.5 13.9 ± 1.2 86.1 ± 1.2 0.85 ± 0.13
[3.7 ± 0.7]
5.3 ± 0.6
[23 ± 4]
3.1 ± 0.4
[13 ± 2]
1.0 11.9 ± 1.1 70 ± 2 11.3 ± 0.4 88.7 ± 0.4 0.69 ± 0.09
[3.0 ± 0.5]
5.4 ± 0.7
[24 ± 4]
3.1 ± 0.4
[13 ± 2]
2.0 16.1 ± 1.1 94 ± 2 10.0 ± 0.3 90.0 ± 0.3 0.66 ± 0.08
[2.9 ± 0.5]
5.9 ± 0.7
[26 ± 4]
3.3 ± 0.4
[14 ± 2]
6.8 100 None 29.0 ± 1.5 65.0 ± 1.8 26 ± 3 74 ± 3 1.3 ± 0.2

[5.5 ± 1.1]
3.7 ± 0.5
[16 ± 3]
2.5 ± 0.3
[10.7 ± 1.8]
0.5 8.5 ± 0.2 59.1 ± 0.5 14.5 ± 0.3 85.5 ± 0.3 0.92 ± 0.11
[4.0 ± 0.7]
5.4 ± 0.7
[24 ± 4]
3.2 ± 0.4
[14 ± 2]
1.0 10.2 ± 0.7 72.3 ± 1.4 11.8 ± 0.2 88.2 ± 0.2 0.77 ± 0.09
[3.3 ± 0.6]
5.7 ± 0.7
[25 ± 4]
3.3 ± 0.4
[14 ± 2]
2.0 12.9 ± 0.4 91.2 ± 1.0 11.8 ± 0.3 88.2 ± 0.3 0.78 ± 0.10
[3.4 ± 0.6]
5.8 ± 0.7
[25 ± 4]
3.3 ± 0.4
[14 ± 2]
20 2.0 19 ± 4 90 ± 2 9 ± 2 91 ± 2 0.54 ± 0.14
[2.4 ± 0.7]
5.5 ± 0.7
[24 ± 4]
3.0 ± 0.4
[13 ± 2]
a

The efficiency of O
2
escape from the protein matrix, d, is presented in square brackets. For the kinetic parameters the uncertainties are
presented as 95% confidence intervals.
pH and NaCl effects on HbA subunits oxygenation S. V. Lepeshkevich and B. M. Dzhagarov
6114 FEBS Journal 272 (2005) 6109–6119 ª 2005 The Authors Journal Compilation ª 2005 FEBS
dimer in the presence of 0.5 m NaCl can be considered
to be equal to those found at the highest salt level at
pH 8.5. Thus, the quantum yield of BR for the a and
b subunits within completely oxygenated tetrameric
HbA (c
a
and c
b
) are found to lie in the range of
0.011 ± 0.002 and 0.061 ± 0.013, respectively. The
rate constant of BR for the a and b subunits within
triliganded HbA are k¢
a
¼ 6.9 ± 0.3 lm
)1
Æs
)1
and
k¢
b
¼ 47 ± 3 lm
)1
Æs
)1

, respectively.
Using Eqns (4) and (6), total tetramer affinity is
found to be reduced by a factor of three in the pres-
ence of NaCl. This decrease is seen to be due to the
a and b subunit affinity reduction of 4.0 ± 1.6 and
2.4 ± 0.9 times, respectively. The association rate con-
stant for the a subunits is decreased in 4.4 ± 0.5 times
in the presence of NaCl, whereas the dissociation rate
constant does not vary virtually. In contrast, the b
subunits exhibit a larger 1.7 ± 0.6 times dissociation
rate constant and a lower 1.40 ± 0.11 times associ-
ation rate constant in the presence of NaCl with
respect to the absence of NaCl.
Discussion
It has long been known that the binding of various
heterotropic effectors including chloride ions modu-
lates the O
2
affinity and cooperative function of HbA
[34–36]. Previous measurements [35] have suggested at
least two classes of chloride-binding sites. Over the
range 0.1–2.5 m NaCl, oxygenated hemoglobin binds
chloride ions at high-affinity sites with an intrinsic
binding constant of $ 10 m
)1
. The data [35,37] have
Fig. 3. Total oxygen affinity (K
t
) of liganded hemoglobin as a func-
tion of NaCl concentration. Bars 1, 2 and 3 are the relative changes

in K
t
at pH 6.8, 7.4 and 8.5, respectively. As a reference, the total
oxygen affinity for the liganded hemoglobin under salt-free condi-
tions was taken. The uncertainties are presented as 95% confid-
ence intervals. Conditions: 10 m
M Tris ⁄ HCl buffer, at 21 °C. (A)
Protein (100 l
M in heme) at 0.5 M NaCl. (B) Protein (100 lM in
heme) at 1.0
M NaCl. (C) Protein (100 lM in heme) at 2.0 M NaCl.
(D) Protein (20 l
M in heme) at 2.0 M NaCl.
Fig. 4. The parameters of O
2
binding to the a and b subunits within liganded hemoglobin as a function of NaCl concentration. Bars 1, 2 and
3 are the relative changes in the association (k¢), dissociation (k) rate constants, oxygen affinity (K) for the a subunits, respectively. Bars 4, 5
and 6 are the changes in k¢, k and K for the b subunits, respectively. As a reference, the averaged parameters of O
2
rebinding (Table 1, Aver-
age) under salt-free conditions were taken. Uncertainties are presented as 95% confidence intervals. Conditions: 10 m
M Tris ⁄ HCl buffer, at
21 °C. (A) Protein (100 l
M in heme) at pH 6.8 at 0.5 M NaCl. (B) Protein (100 lM in heme) at pH 6.8 at 2.0 M NaCl. (C) Protein (20 lM in
heme) at pH 6.8 at 2.0
M NaCl. (D) Protein (100 lM in heme) at pH 8.5 at 2.0 M NaCl.
S. V. Lepeshkevich and B. M. Dzhagarov pH and NaCl effects on HbA subunits oxygenation
FEBS Journal 272 (2005) 6109–6119 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6115
shown that Cl
–

interacts strongly with HbA but pro-
vide no evidence for binding of Na
+
up to concentra-
tions of 0.5 m. Furthermore, the chloride effect is
considered to arise indirectly from alterations in water
activity [38,39]. Dimer–dimer interactions (e.g. hydro-
gen bonds, salt bridges) within the interface might be,
to a certain degree, osmotic-pressure dependent. The
considered indirect effect arises when the high chloride
concentration alters the water activity and conse-
quently the hydration of hemoglobin.
Recent X-ray investigations have rekindled interest
in the links between oxygenation, salt binding and
dimer–dimer interactions. It has been shown that fully
liganded human HbA can be crystallized under low-
salt conditions with a ‘third quaternary structure’, des-
ignated R2 [40], whereas at high salt levels the protein
is found in the classical R quaternary structure [41].
Based on extensive structural analysis, it has been pro-
posed that the R2 state represents a crystallographi-
cally trapped intermediate in the transition between
the T- and R-states. Later modeling studies have
argued that the R2-state was actually the endpoint of
the transition from the T-state. The crystallization,
under different conditions, of liganded hemoglobin in
R, R2, and intermediate forms suggests that a family
of conformers (the R
e
ensemble) coexist in solution

[42]. Moreover, a recent NMR experiment [43] at near-
physiological conditions of pH, ionic strength and tem-
perature showed that the solution structure of HbCO
is a dynamic intermediate between two previously
solved R and R2 crystal structures. Most likely, this
intermediate structure is similar to the RR2 structure
reported previously [44]. On the basis of recent X-ray
studies [40–42,44], it has been concluded that the ligan-
ded HbA may undergo structural and functional chan-
ges in response to subtle changes in the ionic strength,
the concentration of allosteric effectors.
Summaries of our new and previous results [18] for
the a and b subunits within liganded tetrameric HbA
modified by the interactions with sodium chloride and
pyridoxal 5¢-phosphate are shown in Fig. 5. As evi-
dent, the rate constant (k¢) and the quantum yield of
BR (c) for the a and b subunits are modulated by the
interactions of the allosteric effectors with HbA in
quite different ways. The decrease in the association
rate constant of BR for the a subunits is seen at a
practically unchanged quantum yield of BR. By con-
trast, the decrease in the association rate constant for
the b subunits occurs with the increase in the quantum
yield of BR. The results for the b subunits show that
there is an inverse correspondence between k¢ and c.
The decreased association rate at increased quantum
yield may result from a low probability of binding to
the heme once the ligand has entered the protein [11].
This could arise from a decreased rate of bond forma-
tion between the ligand localized to the region of the

heme pocket and the heme iron. This suggestion is
consistent with the NMR data [45,46]. By investigating
the ring-current shifted proton resonances in the NMR
spectra, it has been shown [45,46] that anions (both
phosphate and chloride) can affect the tertiary struc-
ture around the ligand-binding site of liganded hemo-
globin. The conformation of Val(E11) in the a and b
subunits relative to the heme plane is quite dependent
on the nature of the anions and the pD of the solution
as well as on the nature of the ligand. It has been
observed [45], that in the liganded hemoglobin under
different buffering conditions, Val(E11)b moves closer
to the iron atom in the presence of certain anions. It
leads to lowering the access of the dissociated ligand
to the heme. Consequently, it leads to increasing the
inner-most barrier controlling bond formation between
the ligand and the heme-iron. These appears to be a
direct relationship between the ability of the anions to
shift Val(E11)b closer to the iron atom and its ability
to lower the ligand affinity.
In this study, different NaCl effects on the associ-
ation rate constant and the quantum yield of BR (the
efficiency of the ligand escape) for the a and b sub-
units within the oxygenated tetramer and dimer of
human hemoglobin were revealed. As a consequence,
the regulation of the affinity for the a and b subunits
within the completely liganded tetrameric hemoglobin
is proposed to be achieved in two distinctly different
ways. The mechanism of the regulation can be unam-
biguously determined by the additional study of the

GR, i.e. the ligand rebinding from within the protein.
Fig. 5. Correlations between the values of the association rate con-
stant of BR (k¢) and the quantum yield of BR (c). The data for the a
and b subunits within liganded tetrameric HbA are shown in (A)
and (B), respectively. Conditions: (1)10 m
M Tris ⁄ HCl buffer, pH 6.8–
8.5, at 21 °C. (2) HbA modified with pyridoxal 5¢-phosphate (PLP-
HbA), 1.6 mol PLP per tetrameric HbA, 50 m
M K
2
HPO
4
buffer,
pH 7.4, at 20 °C. (3) PLP-HbA, 6.0 mol PLP per tetrameric HbA,
50 m
M K
2
HPO
4
buffer, pH 7.4, at 20 °C. (4) 10 mM Tris ⁄ HCl buffer,
pH 6.8, 0.5
M NaCl, at 21 °C.
pH and NaCl effects on HbA subunits oxygenation S. V. Lepeshkevich and B. M. Dzhagarov
6116 FEBS Journal 272 (2005) 6109–6119 ª 2005 The Authors Journal Compilation ª 2005 FEBS
The time course and the yield of the geminate phase
are both sensitive to the immediate environment of the
heme, and to the dynamics of structural changes in the
protein. Hence, the analysis of the GR parameters can
give a deep insight into modulation of the ligand bind-
ing properties of the hemoglobin subunits. The GR

study is in progress now.
Experimental procedures
Materials
Oxyhemoglobin was isolated from fresh donor blood using
the method described previously [47]. For experiments on
stripped HbA, it is necessary to use buffers that do not
affect the ligand affinity, for example, the phosphate buf-
fers. Therefore, the kinetic experiments were carried out in
10 mm Tris ⁄ HCl buffer, at 21 °C. Three pH conditions
were used as follows: 8.5, 7.4, and 6.8. The NaCl effects
were carried out at concentrations of 0.1, 0.5, 1.0, and
2.0 m. The solubility of O
2
in water depends strongly on
NaCl concentration. Conversions from O
2
partial pressures
to molarities of dissolved O
2
were made with the following
solubility coefficients [48,49]: 1.80 lmÆmmHg
)1
(salt-free
buffers), 1.74 lmÆmmHg
)1
(0.1 m NaCl), 1.50 l mÆmmHg
)1
(0.5 m NaCl), 1.26 lmÆmmHg
)1
(1.0 m NaCl), and 0.90

lmÆmmHg
)1
(2.0 m NaCl). The HbA concentration was 20
and 100 lm in heme.
Time-resolved spectroscopy
The bimolecular oxygenation parameters were measured
using a kinetic laser spectrometer described previously
[15,16]. The second harmonic (532 nm) of an Nd:YAG
laser was applied as an exciting light pulse. Transient
absorption measurements were performed in the spectral
region 430–435 nm. The sensitivity of the detection system
allowed us to measure photo-induced absorption changes
up to 1 · 10
)5
absorbance units per 2500 shots.
Acknowledgements
The authors are greatly indebted to Dr Vladimir S.
Starovoitov for fruitful discussion. The authors thank
Anna V. Chistyakova and Dr Nona V. Konovalova for
preparing protein solutions. This work was supported
by the Belarusian Republican Foundation for Funda-
mental Research (Grant B00-176) and the Belarus State
Program of Basic Research (Project ‘Spectr-06’).
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