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 subunit
Trang 1oxygenation 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 subsub-units, a2b2, 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 O2 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 O2 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: bmd@imaph.bas-net.by
(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 O2-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 O2-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.
Trang 2the 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 O2 association [14–16]
to both a and to b subunits within triliganded HbA
and the efficiency of O2 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 O2
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 O2 rebinding and the quantum yield
of BR, c The latter value is defined as the ratio of the
number of O2, 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 O2
molecules to that of the absorbed light quanta defines
the primary quantum yield of photodissociation, c0
[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
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 O2 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 O2 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 O2 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 O2was 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: ðaO2;bO2ÞðaO2;bO2Þ !hv ða; bO2ÞðaO2;bO2Þ þ O2
!k
0 a
ðaO2;bO2ÞðaO2;bO2Þ ðaO2;bO2ÞðaO2;bO2Þ !hv ðaO2;bÞðaO2;bO2Þ þ O2
!k
0 b
ðaO2;bO2ÞðaO2;bO2Þ
ð1Þ
where (aO2, bO2)(aO2, bO2) denotes the
oxyhemoglob-in molecule In Scheme 1, the oxygenated subunits are shown together with O2 The central terms in Scheme 1 represent the case of free O2 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 O2 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:
DAnorm¼ aa expðk0
a ½O2 tÞ þ abexpðk0
b ½O2tÞ ð2Þ whereDAnormis a normalized change in optical density
of the sample and aa, ab, k¢a and k¢b are the ampli-tudes and rate constants of BR The quantity [O2] is the concentration of molecular oxygen dissolved in the
Trang 3buffer 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
ca(b)¼ 2Æaa(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 c¼ 0.023 ± 0.003 was obtained [16] Also, the efficiency of O2 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, c0
Having the individual parameters of bimolecular oxygenation for each subunit type within HbA, the extent of subunit dissociation rate constant difference (k2/k1) and the magnitude of subunit affinity difference (K2/K1) [17] was calculated using the formulas:
k2
k1
¼ kth2
c02
kth1
c01
1
c2
and
K2
K1
¼ kth2
c02
kth1
c01
1
k
0 2
k0 1
c1
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, kth, 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, kdet¼ 430 nm Conditions: 10 m M 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 l M (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.
Trang 4kth, can be considered constant with an accuracy of
9% Dzhagarov et al [26] determined the value, c0, for
the a and b subunits within oxygenated HbA to be
equal to that for the isolated chains, c0¼ 0.23 ± 0.03
Therefore, the ratio of kth/c0 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, c2(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, Kt, for the last ligand binding step:
Kt ¼ K1 K2
K1þ K2
ð5Þ Here, K1 and K2correspond to the affinity of O2
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:
Ktðp1Þ
Ktðp2Þ¼
1þK1 ðp 2 Þ
K 2 ðp 2 Þ
K 1 ðp 2 Þ
K 1 ðp 1 ÞþK1 ðp 2 Þ
K 2 ðp 1 Þ
ð6Þ
Here, p1and p2correspond to two compared proteins
Subscripts 1 and 2 correspond to two different types
of subunits within considered proteins
Rates of O2binding 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 bindbind-ing to
dimer-ic HbA are almost identdimer-ical [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 ± 3 lm)1Æs)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 ± 3 lm)1Æs)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 O2 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, <da>), and in every six ligands leaves the b subunits (Table 1, <db>) Using Eqns (3) and (4), the dissociation rate con-stant, k, and the O2affinity, 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 O2 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 O2binding 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
Trang 5increase 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 O2
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 O2 binding to tetrameric HbA, the contribution of
O2 rebinding with dimer to the total protein
oxygen-ation must be taken into account and the effect of
NaCl concentration on the rates of O2 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 O2
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 subunexhib-its 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, Kt, 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 O2affinity 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 m M Tris ⁄ HCl buffer, at 21 C.
pH
k¢ a
l M )1Æs)1
k¢ b
l M )1Æs)1
aa
%
ab
%
ca, daa
·10)2, ·10)2
cb, dba
·10)2, ·10)2
c, d a
·10)2, ·10)2
[4.8 ± 1.0]
3.4 ± 0.4 [15 ± 3]
2.2 ± 0.3 [9.7 ± 1.6]
[4.4 ± 0.9]
3.6 ± 0.4 [16 ± 3]
2.3 ± 0.3 [10.0 ± 1.7]
[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 O2escape 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.
Trang 6Effect of NaCl concentration on the rates of O2
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 oxygenaconcentra-tion 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 O2-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 O2-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 m M Tris ⁄ HCl buffer, at 21 C.
pH
Protein
conc l M
(in heme)
NaCl conc.
M
k¢ a
l M )1Æs)1
k¢ b
l M )1Æs)1
aa
%
ab
%
ca, daa
·10)2, ·10)2
cb, dba
·10)2, ·10)2
c, d a
·10)2, ·10)2
[4.8 ± 1.0]
3.4 ± 0.4 [15 ± 3]
2.2 ± 0.3 [9.7 ± 1.6]
[4.3 ± 1.3]
4.4 ± 0.6 [19 ± 3]
2.7 ± 0.3 [12 ± 2]
[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]
[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]
[3.0 ± 0.5]
5.4 ± 0.7 [24 ± 4]
3.1 ± 0.4 [13 ± 2]
[2.9 ± 0.5]
5.9 ± 0.7 [26 ± 4]
3.3 ± 0.4 [14 ± 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]
[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.
Trang 7dimer 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 (ca and cb) 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¢a¼ 6.9 ± 0.3 lm)1Æs)1 and k¢b¼ 47 ± 3 lm)1Æs)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 O2 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 concentrafunc-tion Bars 1, 2 and 3 are the relative changes
in Ktat 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 l M in
heme) at 1.0 M NaCl (C) Protein (100 l M 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 l M in heme) at pH 6.8 at 2.0 M NaCl (C) Protein (20 l M in heme) at pH 6.8 at 2.0 M NaCl (D) Protein (100 l M in heme) at pH 8.5 at 2.0 M NaCl.
Trang 8shown that Cl– interacts strongly with HbA but
pro-vide no epro-vidence 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 Re 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 chanchan-ges 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 K2HPO4buffer, pH 7.4, at 20 C (4) 10 m M Tris ⁄ HCl buffer,
pH 6.8, 0.5 M NaCl, at 21 C.
Trang 9The 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 O2 in water depends strongly on
NaCl concentration Conversions from O2 partial pressures
to molarities of dissolved O2were 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 lmÆmmHg)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)5absorbance 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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