From the Dipartimento di Scienze e Tecnologie
Biomediche, Università di Milano and § Istituto
Tecnologie Biomediche Avanzate del Consiglio Nazionale delle
Ricerche, Laboratori Interdisciplinari Tecnologie Avanzante
20090 Segrate, Italy
Received for publication, November 2, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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Understanding mechanisms in cooperative proteins
requires the analysis of the intermediate ligation states. The release
of hydrogen ions at the intermediate states of native and chemically modified hemoglobin, known as the Bohr effect, is an indicator of the
protein tertiary/quaternary transitions, useful for testing models of
cooperativity. The Bohr effects due to ligation of one subunit of a
dimer and two subunits across the dimer interface are not additive. The
reductions of the Bohr effect due to the chemical modification of a
Bohr group of one and two A vast amount of data on the structure/function of human
hemoglobin in solution apparently supports the mechanism of a concerted transition between two quaternary structural states in the course of
ligand binding, in agreement with the Monod-Wyman-Changeaux model (1).
Due to cooperativity, the end states largely prevail on species in a
partial state of ligation under equilibrium conditions, masking the
functional properties of the intermediate species. This is demonstrated
by the close agreement between the isotherms of CO binding calculated
from the experimental distributions of the CO ligation intermediates
according to the Monod-Wyman-Changeaux model and the alternative
Koshland-Nemethy-Filmer model, which assumes transitions through
intermediate structural/functional states (2), as shown by Perrella and
Di Cera (3). The functional/structural studies of the intermediates
still provide the most critical test for any model of cooperativity.
Such studies are difficult because of the high rates of the
dissociation and association reactions of the physiological ligand, the
complexity of the intermediate ligation states Fig.
1, and the instability of tetrameric
hemoglobin. The partially liganded hemoglobin tetramers reversibly
dissociate into dimers faster than the rate of resolution of the
separation techniques (4), and dimer rearrangement reactions occur
under nonequilibrium conditions, as depicted in Fig.
2. In a previous study of the Bohr effect
of the intermediate ligation states (5), the problem of the ligand
mobility was circumvented by using cyanide bound to the ferric subunits
to mimic ligation and a cryogenic technique to determine the proportion
of any asymmetrical hybrid species in equilibrium with the respective
symmetrical parental species (6). This information was needed to
calculate the contribution of each species from the total Bohr effect
of a mixture of hybrid and parental species. The study of the pH
dependence of the Bohr effects of the mono- and diliganded
intermediates revealed the absence of additivity of the effects, an
important clue to the mechanism of tertiary/quaternary transitions in
ligand binding to hemoglobin. However, the discovery by Shibayama
et al. (7) that the cyanomet intermediates undergo valency
exchange has made such studies questionable. We have now repeated the
measurement of the Bohr effect of the mono- and some diliganded species
under conditions of slight or negligible valency exchange, confirming the results of the previous study.
or
subunits are additive. The Bohr
effects of monoliganded chemically modified hemoglobins indicate the
additivity of the effects of ligation and chemical modification with
the possible exception of ligation and chemical modification of the
subunits. These observations suggest that ligation of a subunit brings
about a tertiary structure change of hemoglobin in the T quaternary
structure, which breaks some salt bridges, releases hydrogen ions, and
is signaled across the dimer interface in such a way that ligation of a
second subunit in the adjacent dimer promotes the switch from the T to
the R quaternary structure. The rupture of the salt bridges per
se does not drive the transition.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
The 10 ligation states of
hemoglobin. The non-dissociating dimers
( 1
1) and
(
2
2) are shown in brackets.
The states are labeled as [ij], indicating the ith ligation state and
the corresponding degeneracy j. Since the
1
1 and
1
2
contacts are different, species 21 is structurally nonequivalent to
species 22.
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Fig. 2.
Dimer rearrangement reactions of tetrameric
hemoglobin. The ligated or chemically modified subunits are
labeled by superscript X. The dashed lines
indicate the 1
2 and
2
1 contacts that dissociate under
physiological conditions. Symmetrical tetramers dissociate into
identical dimers that re-associate to yield the original tetramer.
These species can be studied in a pure form. Asymmetrical tetramers
dissociate into different dimers that re-associate, yielding two
symmetrical parental species in addition to the asymmetrical
species.
Using the same technical approach we have measured the decrease in Bohr
hydrogen ions in hemoglobin derivatives in which either one or both
Bohr groups of the and
subunits of deoxy hemoglobin and of the
deoxy/cyanomet intermediates were chemically modified by carbamoylation
(8) and by the NEM1 reaction
of cysteine F
93 (9). We found that the functional effects of the
single and double chemical modifications were additive, as were the
combined effects of ligation and chemical modification, with just one
possible exception. These findings help define the role of the salt
bridges with regard to the stabilization of the hemoglobin T quaternary
structure, which was described by Perutz in his stereochemical
mechanism of cooperativity (10).
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MATERIALS AND METHODS |
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Hemoglobin Purification-- HbA0 was obtained from normal adult blood and HbS from heterozygous donors. The hemoglobins were purified by ion exchange chromatography on CM-52 cellulose, as previously described (5), equilibrated with 0.2 M KCl, and stored in liquid nitrogen at a concentration of 6 mM in heme.
Preparation of NES Hemoglobin-- Samples (4.5 g) of HbO2 were reacted with a 5-fold excess of NEM at 4° C and pH 7.3 for 2 h (11). The reactants were gel-filtered on Sephadex G-25 equilibrated with 5 mM potassium phosphate, 0.5 mM Na2EDTA, pH 6.8, and loaded onto a column (8 × 27 cm) of CM-52-cellulose equilibrated with the same buffer. Elution with 7.5 mM potassium phosphate, 0.5 mM Na2EDTA, pH 7.3, at 300 ml/h was continued until a good resolution of NES Hb was achieved. The resin-bound derivative was collected and eluted in a batch procedure with 20 mM Tris-HCl, 50 mM KCl, pH 7.5. Titration with p-chloromercurybenzoate was carried out to check for the absence of free thiol groups in the product (11).
Preparation of Hemoglobin Carbamoylated at the -Amino Groups
of the
Subunits,
(
C
)(
C
)--
Hb samples (4.5 g)
were reacted anaerobically at 20° for 1 h with a 50-fold molar
excess of KCNO in the presence of a 10 mM excess of
inositol hexaphosphate at pH 6.5. The carbamoylation reaction was
stopped by anaerobic gel filtration of the protein at pH 6.5, and the
excess inositol hexaphosphate was removed by aerobic gel filtration
using 50 mM potassium phosphate, pH 8 (12). Most of the
protein carbamoylated at the
-amino groups of the lysines was
removed by ion exchange chromatography on a DEAE-cellulose column
(8 × 30 cm) equilibrated with 50 mM Tris-HCl, pH 8.1, using a pH gradient of the same buffer (pH 8.1
7.6). Finally
(
C
)(
C
) was purified by
chromatography on a CM-52 column as for the purification of
HbA0. The product obtained by this method had the same
chromatographic properties as the carbamoylated hemoglobin prepared by
the procedure of Williams et al. (13) and the same isoelectric point as the
chain carbamoylated hemoglobin prepared by
the chain separation and recombination method (8, 14).
Hemoglobin Incubations-- HbO2 solutions were deoxygenated by N2 tonometry and transferred into thermostatted vials for the anaerobic incubation at 20° C using a flow of humidified N2. The Hb stability over 60-h periods was checked by measuring the spectra of undiluted oxygenated samples using a 0.2-mm optical path flow cell. When the amount of Hb+ raised above the initial value of 2-3% because of the presence of O2 traces in the gas flow, N2 was purged through an alkaline solution of sodium pyrogallate. The same procedure was used for the anaerobic incubation of solutions containing CNHb. The excess of cyanide (5 mM) added to the solutions was enough to compensate for the cyanide loss due to evaporation (5). The absence of free Hb+ due to cyanide evaporation was checked routinely by measuring the spectra of the oxygenated solutions after incubation. Alternatively, to keep the cyanide concentration during long incubations of the solutions constant, N2 was flown over a 5 mM solution of cyanide before reaching the incubation vial. Oxygen scavenging enzymes, catalase and superoxide dismutase, were not used.
Measurement of the BE-- Samples (1 ml) of 6 mM Hb were transferred into an anaerobic vessel thermostatted at 20° for pH measurement. The solution was exposed to O2 and the pH was titrated back to the value of the anaerobic sample using carbonate-free 20 mM NaOH (15).
BE of the Singly Modified Hemoglobins--
Hemoglobin species
chemically modified at a Bohr group of one or one
subunit
cannot be studied in a pure form. Because of the tetramer dissociation
reaction, they disproportionate into the parental species,
i.e. unmodified and doubly modified hemoglobin, as shown in
Fig. 2. The BE of the singly modified species was measured by the same
approach used to study the asymmetrical deoxy/cyanomet analogs of the
intermediates (5). Since HbS differs from HbA0 for the
surface charge but is otherwise functionally equivalent, a one to one
mixture of two parent species, e.g. HbS and NES
HbA0, was incubated anaerobically until the equilibrium
with the asymmetrical species modified by NEM at just one
subunit
was reached. The total BE of the mixture was measured, and the
contributions to the BE of the fractions of HbS and NES
HbA0 at equilibrium were subtracted from the total. The
fractions of the three species at equilibrium were measured using a
cryogenic separation method, as follows. A sample of the anaerobic
mixture was quenched into a hydro-organic solvent at
30 °C to stop
the tetramer dissociation reactions, the mixture was resolved by
cryofocusing at
25 °C, and the three fractions were assayed by the
pyridine hemochromogen method (16). The data on the rate of
equilibration at 20 °C in 0.2 M KCl, pH 7, of an
equimolar mixture of HbS and (
C
)(
C
)
plotted in Fig. 3 indicate that
equilibrium was reached after a 20-h incubation.
|
Valency Exchange Controls--
Valency exchange was measured by
a procedure similar to that used by Shibayama et al. (7). At
the end of each anaerobic incubation of mixtures of deoxy-HbS and
cyanomet HbA0,
(+CN
)(
+CN
) or (
+CN
)(
+CN
) valency exchange was
stopped by exposure to O2, and the two hemoglobin species,
oxy/cyanomet HbS and oxy/cyanomet HbA0, were separated by
ion exchange chromatography using small CM-52 columns equilibrated with
buffer containing cyanide. The proportion of oxy versus
cyanomet hemoglobin in each separated fraction, determined by a
spectral analysis of the samples in the 450-600-nm range, yielded the
amount of valency exchange. The proportions of each component were
obtained by fitting the spectra of the mixtures with the spectra of
pure HbO2, CNHb, and Hb+ using a Matlab 5.3 program. The error was 1-2% of the total. Similar spectral analyses
of the undiluted oxygenated samples before chromatography allowed a
check for the absence of free Hb+ due to cyanide
evaporation and for any increase in the total CNHb concentration due to
Hb oxidation by O2 leaking in during the anaerobic incubations.
BE of the Deoxy/Cyanomet Analogs of the Intermediates--
In
the absence of valency exchange, the anaerobic incubation of
HbA0 and species
(+CN
)(
+CN
) or (
+CN
)(
+CN
) should yield the
monoliganded intermediates 11 or 12, respectively (Fig. 1). As shown in
Fig. 3, the incubation for 3 h at neutral pH of a 1 to 1 mixture
of HbS and species (
+CN
)(
+CN
)
or (
+CN
)(
+CN
) yielded an
amount of hybrid comparable with that observed by incubating under the
same conditions HbS and (
C
)(
C
). The
proportion of hybrid was approximately that predicted by the kinetics
of the Hb tetramer-dimer reactions (4). A higher proportion of hybrid
was obtained when the mixture was pre-incubated under aerobic
conditions before deoxygenation (1/2 h) and anaerobic incubation (2 h).
Under these conditions the valency exchange was negligible at neutral
or alkaline pH and slight (
5%) at the most acidic pH
values.2 The valency
exchange, proportion of hybrid, and BE were measured using the same
anaerobic mixtures.
Data Analysis-- The concentration of hydrogen ions released per tetramer of asymmetrical hybrid in mixture with the parental species was calculated as follows.
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(Eq. 1) |
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RESULTS AND DISCUSSION |
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BE of the Deoxy/Cyanomet-diliganded Intermediates 23 and 24 and
Monoliganded Intermediates 11 and 12--
The BE of species 23 and 24 measured after 3 h of anaerobic incubation are compared in Fig.
4, a-b, with the values
previously obtained immediately after deoxygenation of the oxygenated
species (5). Valency exchange controls were not carried out, but the close agreement with the previous data suggests that the exchange had
only minor effects during a 3-h incubation.
|
The data on the BE of the hybrid monoliganded intermediates, compared
in Fig. 4, c-d, with the previously published data (5), were calculated from the titration data of the ternary mixtures of
parental species, HbS plus species 23 or 24, and hybrid species incubated under anaerobic conditions for 2 h after deoxygenation (data not shown) and the fractional values of the concentration of
hybrid in the mixtures, shown in the inset of Fig. 3.
Valency exchange controls carried out at the end of each anaerobic
incubation showed negligible exchange at neutral or alkaline pH and a
slight exchange (5%) at acidic pH.2 Such controls
carried out after longer periods of incubation (up to 48 h), as in
our previous studies (5), indicated a high proportion of valency
exchange.2 The data in Fig. 3 indicate that a high
proportion of hybrid was attained after 2-3 h, although not yet at
equilibrium. In contrast, the rate of hybridization between HbS and
CNHbA0, yielding intermediate 21 is slower (17), and
valency exchange controls under these conditions indicated a high
proportion of exchange, in agreement with the measurements of Shibayama
et al. (7). Therefore we could not confirm the data
previously published on the BE of species 21 (5). The apparent
agreement between the data on the BE of the monoliganded species
obtained in the present study under conditions of low valency exchange
and the previous data obtained under conditions of extensive valency
exchange (5) has two possible explanations. It could be an artifact of
the calculation of the BE of the monoliganded species from the total BE
of the mixture of monoliganded and parental species. In the previous
work, ignoring the valency exchange, it was assumed that only three
species were present. Alternatively, it could be due to the mechanism
of the exchange reactions. The method of Shibayama et al.
(7) measures the global valency exchange but does not provide
information on which species are generated in the exchange process.
The curves of the BE in Fig. 4, a-b, and Fig. 4,
c-d, are the functional responses of different structures.
The bell-shaped curve of the hemoglobin alkaline BE yields the hydrogen
ions released in the transition from the T to the R structure due to
oxygenation. The bell-shaped curves of the monoliganded intermediates
(Fig. 4, c-d) are indicative of the functional effect of a
tertiary structural change occurring in the T quaternary structure due to ligation of a subunit. In Fig. 4, c-d, the BE of the
vacant sites of the intermediates is measured by the difference between the total BE of Hb and the BE of the monoliganded intermediate. It is
clear that these vacant sites released hydrogen ions on oxygenation at
all pH values except where the alkaline BE of Hb itself vanishes. The
sigmoidal shape of the curves of the BE of the diliganded intermediates
(Fig. 4, a-b) are not equal to the sum of the bell-shaped
curves of the two monoliganded intermediates, indicating a profound
interaction between the ligation sites. The vacant sites of the
diliganded intermediates did not release hydrogen ions upon oxygenation
at pH values at which the BE of Hb is still significant, Fig. 4,
a-b. This is the response of a molecule in the R quaternary
structure in which all salt bridges are broken, as described by Perutz
(10) in his stereochemical mechanism. At neutral and acidic pH values,
the two vacant sites in diliganded species 23 and 24 released on
oxygenation an amount of hydrogen ions comparable or even less than the
amount released by the three vacant sites in the monoliganded species.
If the quaternary structures of these diliganded species were in T/R equilibrium, the molecules in T structure should have an additive BE
twice as large as the BE of the monoliganded species, and the molecules
in R structure should have a BE similar to that observed at alkaline
pH. Such a hypothesis is not consistent with the experimental data.
Instead the sigmoidal curve of the diliganded intermediates is
consistent with the hypothesis that the quaternary structures of
diliganded species 23 and 24 have switched to the R conformation at all
pH values. The hydrogen ions released on oxygenation by the unliganded
subunits of these species would then be the functional effects of
tertiary structure changes modulated by pH. It is not known whether
such effects are associated with the R2 structure discovered in
studying the crystals of carboxy hemoglobin crystallized under low salt
(0.1 M Cl) and acidic (pH 5.8) conditions
(18). However, the crystallographic studies indicate the possible
existence of alternative R quaternary structures.
Our interpretation of the correlation between the observed functional
effects of mono- and diligation and the tertiary/quaternary structures
of the protein is consistent with the interpretation of the energetics
of the same species provided by Ackers et al. (19). The
cooperative free energy of ligand binding, GC, can be measured from the difference between the free energy changes for
the dimer-tetramer assembly of ligation intermediate ij and Hb, assumed
as the reference state,
GC(01
ij) =
Gij
G01 (20). At
neutral pH the
GC value for the first ligation
step in the deoxy/cyanomet analogs is 50% of the value for the
transition to CNHb (4). A similar value was calculated from the
distributions of the CO ligation intermediates reported by Perrella
et al. (6, 21) under similar conditions. At alkaline pH the
GC values remain intermediate (22). The
observation of an intermediate
GC value for the
first ligation step is consistent with a two state concerted model (23)
and has been interpreted as the energy of destabilization of the T
quaternary structure due to the binding of one ligand (19). In
contrast, the
GC values for the symmetrical
diliganded intermediates are the same as for the transition to CNHb in
the range from neutral to alkaline pH. This indicates that these
species are in the R quaternary structure under these conditions and
that
GC is not significantly modulated by the
effects of pH on the tertiary structure of the unliganded subunits.
Such effects were observed in our functional studies since the curve of
BE versus pH was sigmoidal in shape (Fig. 4). Sigmoidal
curves of the BE were also observed in the study of the triply liganded
intermediates (5). The present study is partly consistent with the
symmetry rule model for hemoglobin cooperativity proposed by Ackers
et al. (19). Important features of this model are the
energetic and other functional properties of the diliganded
intermediate 21 (Fig. 1), which have been recently confirmed to be
different from those of the diliganded species 22, 23, and 24 (24). As
discussed above, the low rate of formation of intermediate 21 from the
parental species Hb and CNHb together with the high rate of valency
exchange have precluded our study of the BE of this key intermediate.
BE of (C
)(
C
) and NES
HbA0--
The experimental values of the hydrogen ions
released on oxygenation per tetramer of HbA0 and its doubly
modified derivatives at 20 °C in 0.2 M KCl in the pH
range of the alkaline BE are shown in Fig.
5, a-c.
|
The NEM reaction of Cys93 causes a modification of the tertiary
structure of the
subunit that disrupts a network of salt bridges at
the
1
2 interface with the participation
of His
146, as observed in the Hb crystal structure (9, 10).
Carbamoylation of Val1
breaks a chloride ion-mediated salt bridge
within the structure of the
subunit (25, 26). The data in Fig. 5,
a-c, indicate that the chemical modification reduced
significantly the BE in each derivative, in qualitative agreement with
the values at physiological pH reported by several authors (8, 9). The
loss of Bohr hydrogen ions observed in NES HbA0,
i.e. the difference between the BE of native and chemically
modified hemoglobin, in a range of pH values is compared in Fig.
6a with the differential titration curve of His146
, assuming the values
pKdeoxy = 8.1 and pKoxy = 7.2 (27). The loss of Bohr hydrogen ions in
(
C
)(
C
) is compared in Fig.
6b with the differential titration curve of Val1
,
assuming the values pKdeoxy = 8.0 and
pKoxy = 7.25 (28). Also shown in Fig.
6b is the differential titration curve corrected on the
assumption that carbamoylation of Val1
perturbs His122
(26). The
differential titration curve of His122
required for the correction
was calculated assuming the values pKdeoxy = 6.1 and pKoxy = 6.6 (29). The simulations in Fig. 6,
a and b, indicate the strict correlation between
the rupture of the salt bridges inferred from the crystal structures of
deoxy and oxy hemoglobin and the functional effects we have measured in
the chemically modified protein.
|
BE of the Mixtures of Parental Species, HbS plus
(C
)(
C
) and HbS plus NES
HbA0, and Hybrid Species--
The experimental titration
data are shown in Fig. 7,
a-b. The fractions of BE due to the hybrid species (not
shown) were calculated from the data in Fig. 7, a-b, using
the values of the fraction of hybrid species in the mixture shown in
Fig. 7, c-d.
|
BE Loss in the Doubly and Singly Chemically Modified
Hemoglobins--
The Bohr hydrogen ions lost in NES HbA0
and (C
)(
C
) (Fig. 6,
a-b) are compared in Fig. 8,
a-b, with those lost in the singly modified derivatives
calculated from the data in Fig. 7. Within the experimental error, the
effects on function of both types of chemical modification were
additive. The additivity observed in this study of the NEM-modified
hemoglobins parallels the additivity observed in the study of the
energetics of the same species by Ackers and co-workers (30-32). The
modification by NEM of the Bohr group on 1
subunit results in a
1.4-kcal/mol increase in free energy for the dimer-tetramer
assembly, one-half the amount observed with a double modification (31,
32), indicating that the two sites are independent of one another with
regard to their effect on function despite the destabilization of the
quaternary structure brought about by the chemical modification. A
measure of such a destabilization is obtained by comparing the change
in free energy from Hb to NES Hb, 2.8 kcal/mol, with the free energy
change of 5.8 kcal/mol for the transition from Hb to NES
HbO2 (32).
|
BE of the Chemically Modified Deoxy/Cyanomet-monoliganded
Intermediates--
Fig. 9,
a-d, shows the BE of intermediates
(+CN
)(
C
),
(
+CN
)(
NEM),
(
+CN
)(
NEM), and
(
+CN
)(
C
). The fraction of hybrid
and parental species were determined by the cryogenic technique using
mixtures of HbS and species 23 and 24 carbamoylated at the
-amino
groups of the
subunits or NES HbS (data not shown). The putative
hydrogen ions released upon ligation of one
or
subunit in a
dimer and the Bohr hydrogen ions lost because of the chemical
modification of one
or
subunit in the adjacent dimer were in
most cases roughly additive, as shown in Fig. 9, a-c. This
indicates that the signal of ligand binding is not mediated by a
tertiary structural change in the adjacent subunits involving the
rupture of a salt bridge. A possible exception was species
(
+CN
)(
C
) (Fig. 9d).
The BE of this species, i.e. the difference between the
total BE of hemoglobin and the residual BE of its vacant, normal and
chemically modified sites (shaded area in Fig.
9d) was significantly greater than the sum of the BE of the
mono-liganded species and the hydrogen ions lost because of the
chemical modification of one
subunit (continuous line in
Fig. 9d).
|
Role of the Salt Bridges in the Ternary/Quaternary
Transitions--
From the above information we can draw the following
overall picture of the tertiary and quaternary transitions in the
process of hemoglobin ligation. Hydrogen ions are released upon
ligation of a subunit because ligation perturbs the tertiary structure of the ligated subunit in such a way as to break some salt bridge and/or hydrogen bond in agreement with the Perutz stereochemical mechanism (10). However, ligation is also signaled to the neighboring subunits, since a second ligation step promotes a dramatic change from
the T to the R structure, as monitored by the different characteristics and nonadditivity of the BE of the mono- and diliganded intermediates (Fig. 4, a and b and Fig. 4, c and
d) and by the energetics of the dimer-tetramer reaction of
these species (22). The structural basis for the inter-subunit
communication remains to be discovered. The data in Fig. 9d
suggest a mechanism by which ligation of an subunit brings about a
change in the tertiary structure of a neighboring
subunit involving
the perturbation of a Bohr group. Such a tertiary structural change may
also bring about an increase in the ligand affinity of the
subunit.
However, this mechanism does not appear to be the general case, as
shown in Fig. 9, a-c.
The rupture of a salt bridge, as it occurs in the chemically modified
hemoglobins, perturbs the tertiary structure of the subunit, causing a
destabilization of the T structure that is not signaled to the other
subunits via a T to R transition. This is demonstrated by the finding
that two different functional effects of hemoglobin chemically modified
by NEM, i.e. the loss of Bohr hydrogen ions (Fig. 8) and the
change in energy for the dimer-tetramer assembly reaction (31, 32),
have the additivity property. Our observations are also consistent with
the observation reported by Bettati and Mozzarelli (33) that silica
gel-entrapped Hb retains a T structure while binding oxygen with an
apparently normal BE.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. C. Ho and N. T. Ho for a generous gift of HbS and Drs. E. Di Cera and A. Mozzarelli for helpful comments on this paper.
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FOOTNOTES |
---|
* This work was supported by grants from Ministero Università Ricerca Scientifica Tecnologica and Consiglio Nazionale delle Ricerche, Rome.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: L.I.T.A., Via Flli Cervi 93, 20090 Segrate, Italy. Tel.: 02-26423-303; Fax 02-26423-302; E-mail: michele.perrella@unimi.it.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M010009200
2 L. Benazzi, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: NEM, N-ethylmaleimide; Hb, deoxyhemoglobin; HbO2, oxyhemoglobin; CNHb, cyanomet hemoglobin; HbA0, hemoglobin A0; HbS, hemoglobin S; NES HbA0, hemoglobin A0 modified by NEM; BE, Bohr effect.
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REFERENCES |
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