(Received for publication, July 10, 1996, and in revised form, November 13, 1996)
From the Department of Biochemistry and Molecular
Biology, Medical School, University of Maryland, Baltimore,
Maryland 21201, the ¶ Laboratory of Molecular Carcinogenesis, NCI,
National Institutes of Health, Bethesda, Maryland, 20892, the
Department of Molecular Biology and Biochemistry,
Hall-Atwater-Shanklin Laboratories, Wesleyan University,
Middletown, Connecticut 06459, and the ** Department of Chemistry,
Temple University, Philadelphia, Pennsylvania 19122
The -globin of human hemoglobin was expressed
in Escherichia coli and was refolded with heme in the
presence and in the absence of native
-chains. The functional and
structural properties of the expressed
-chains were assessed in the
isolated state and after assembly into a functional hemoglobin
tetramer. The recombinant and native hemoglobins were essentially
identical on the basis of sensitivity to effectors (Cl
and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the
heme region, and nuclear magnetic resonance of the residues in the
1
1 and
1
2
interfaces. However, the nuclear magnetic resonance revealed subtle
differences in the heme region of the expressed
-chain, and the
recombinant human normal adult hemoglobin (HbA) exhibited a slightly
decreased cooperativity relative to native HbA. These results indicate
that subtle conformational changes in the heme pocket can alter
hemoglobin cooperativity in the absence of modifications of quaternary
interface contacts or protein dynamics. In addition to incorporation
into a HbA tetramer, the
-globin refolds and incorporates heme in
the absence of the partner
-chain. Although the CO binding kinetics
of recombinant
-chains were the same as that of native
-chains,
the ellipticity of the Soret circular dichroism spectrum was decreased
and CO binding kinetics revealed an additional faster component. These results show that recombinant
-chain assumes alternating
conformations in the absence of
-chain and indicate that the
isolated
-chain exhibits a higher degree of conformational
flexibility than the
-chain incorporated into the hemoglobin
tetramer. These findings demonstrate the utility of the expressed
-globin as a tool for elucidating the role of this chain in
hemoglobin structure-function relationships.
The basis of cooperative oxygen binding by human hemoglobin is an
old problem that is being elucidated with the aid of modern technologies. Of particular importance is the elucidation of the properties of - and
-chains, both in the isolated state and incorporated into the hemoglobin tetramer. Such studies should provide
fine details on the linkage between tertiary and quaternary structure
that forms the basis of cooperative oxygen binding. The recent
development of hemoglobin expression systems offers great promise for
facilitating the study of such structure-function relationships. This
approach has been particularly useful for investigating the
physicochemical properties of hemoglobin and myoglobin and has been
employed in the development of hemoglobin variants with properties
suitable for clinical use as red cell substitutes (1).
Although Escherichia coli expression systems for
HbA1 have been developed, the product
exhibits structural and functional modifications relative to native HbA
(2-4). Alternatively, tetrameric hemoglobins in which only the - or
-subunits are recombinant (
rHbA and
rHbA) can be obtained by
in vitro refolding of the expressed globin.
-Globin has
thus been expressed (5, 6) and refolded in the presence of native
-chains to yield recombinant hemoglobin whose conformational and
functional properties resemble those of natural HbA (5, 6). In
contrast, development of an analogous
-globin expression system was
not as successful (7), although it is highly desirable as a tool for
elucidating the tetrameric assembly and cooperativity of HbA.
In this paper we report the construction of a system for the synthesis
of high levels of an -globin fusion protein in E. coli.
The conformational properties of the isolated chain are presented along
with the functional and conformational characterization of
reconstituted HbA tetramer following refolding of the expressed
-globin with heme in the presence of native
-chains. Spectral measurements and oxygen binding of these proteins reveal new insights into the linkage between the conformation of the heme pocket, ligand
access channel, and quaternary interactions.
The -globin expression plasmid
pNF
is structurally analogous to pJKO5, from which
-globin has
been routinely produced as a fusion protein, and comprises 83 residues
of a flu virus protein, NS1, a Factor X recognition sequence followed
by
-globin. The construction of pNF
is similar to that of pJKO5
(6). Human
-globin cDNA was provided by B. G. Forget (Yale
University) as a 700-base pair PstI insert in pKT218 (8). In
an initial effort to express
-globin in E. coli, the
700-base pair PstI fragment was inserted into the
PstI site of pKK-233-2 (Pharmacia Biotech Inc.), and the
short region between the NcoI sites at the initiation ATG
site in the globin gene was subsequently removed by digestion with
NcoI followed by religation. The resulting plasmid, pB
2, in JM101 induced with
isopropyl-1-thio-
-D-galactopyranoside was found to
produce very low levels of
-globin. Although unsatisfactory as an
expression system, the 750-base pair NcoI to XmnI
fragment from this plasmid was used as a source of the
-globin gene
for the construction of pNF
. pNF
was constructed in two steps.
The initial construct, pN
, was formed by inserting the
NcoI to XmnI fragment from pB
2 into pJKO4 (6),
from which the
-globin gene had been removed by digestion with
NcoI and EcoRV. (Note that both EcoRV
and XmnI produce blunt ended DNA.) pJKO4 codes for a fusion
protein composed of 81 residues of NS1 followed by
-globin but does
not have an intervening Factor X recognition sequence. pN
in
E. coli strain AR120 (9), induced with nalidixic acid,
produced high levels of the fusion protein NS1-
-globin (data not
shown). A Factor X recognition sequence was inserted between the NS1
segment and
-globin by first digesting pN
with NcoI
and making the DNA blunt ended with mung bean nuclease. Subsequent digestion with BglII removed the NS1 portion of the fused
gene. This segment was replaced by the BglII to
StuI fragment from pJKO1 (6), which contains the same NS1
portion followed by a Factor X recognition sequence. The resulting
plasmid, pNF
, is structurally identical to pJKO5 except that
-globin cDNA replaces the
-globin cDNA of pJKO5. This
plasmid in strain AR120, induced with nalidixic acid, yielded large
amounts of the NS1-FX-
-globin fusion protein as the principal
component of the insoluble inclusion bodies.
Growth, expression, and purification of the fusion protein followed a previously described protocol (6). Enzymatic cleavage was monitored by reverse phase HPLC using a Vydac C4 column. The solvents were: A, 20% CH3CN, 0.1% trifluoroacetic acid; and B, 60% CH3CN, 0.1% trifluoroacetic acid. The gradient was 44% B to 75% B over 120 min. Peaks were identified by eight cycles of Edman sequencing.
Reconstitution ofUpon cleavage with Factor X, the
-globin was recovered as a precipitate, dissolved in a minimum
volume of cold 0.1 M NaOH, and diluted to 2 mg/ml
(A280 = 1.0 is taken as 1 mg/ml) with 0.04 M borate buffer, pH 9.0, 0.002 M EDTA. In order
to solubilize the aggregates, the solution was deoxygenated by stirring
under a constant stream of N2 followed by addition of
dithiothreitol to a final concentration of 0.001 M. After
24 h at 4 °C the protein was reconstituted with cyanoheme and
native
-chains, prepared from carboxy-HbA by reaction with
p-mercuribenzoate (10). p-Mercuribenzoate was
removed from the isolated
-chains as described by Geraci and Li
(11). After 1 week the protein was concentrated and the heme converted
to the CO derivative by addition of sodium dithionite under a stream of
CO. The hemoglobin was purified on an affinity column of immobilized
hemoglobin (12, 13). The collected fractions were analyzed on paragon
gel electrophoresis (Beckman Instruments).
The -globin was
purified and reconstituted with cyanoheme as described. After 1 week at
4 °C the protein was concentrated and the heme converted to the CO
form followed by dialysis against 0.001 M NaCl saturated
with CO. The
r were purified by preparative isoelectrofocusing
(Bio-Rad) according to the manufacturer's instructions. The pH range
of the ampholyte was 3-10. Screening of the fractions was carried out
on paragon electrophoresis (Beckman). The ampholyte was removed by
repetitive washing with water using a Centriprep concentrator
(Amicon).
The natural and recombinant -globins
were isolated from the respective hemoglobin using reverse phase HPLC
and a Vydac C4 column (14). Prior to tryptic digestion, the
isolated
-globins were S-pyridylethylated and desalted
(15). TPCK-treated trypsin (Sigma) was added to a
ratio of 1:50 (w/w) and the solution let stand at room temperature for
20 h. The resulting clear solution was then injected into a
4.6 × 250 mm Vydac C18 column previously equilibrated
with Buffer A (0.1% trifluoroacetic acid). The peptides were eluted
using a gradient of 0-50% Buffer B (0.1% trifluoroacetic acid in 9:1
(v/v) CH3CN:H2O) over 60 min. The absorbance
was monitored at 220 nm. Sequences were determined using a
Hewlett-Packard model G1005A protein sequencing system following the
procedures standardized by the manufacturer.
Oxygen equilibrium measurements were performed using the thin layer dilution technique of Dolman and Gill (16) with an Aviv 14DS spectrophotometer. In this technique the oxyhemoglobin is deoxygenated by stepwise dilution of the equilibrating gas with constant volumes of N2. Formation of methemoglobin as judged by spectral deconvolution of the initial and final spectra was less than 10%. Protein concentration was 1.5-2.0 mM in heme in 0.05 M HEPES buffer at 25 °C. The experimental data were fitted to the Adair equation (17) using an iterative procedure incorporating the Marquardt algorithm,
![]() |
(Eq. 1) |
Flash photolysis was carried out on solutions containing 5 µM heme and 50 µM CO at 23 °C in 0.1 M Bis-Tris (pH 7.0) containing 0.1 M KCl. Approximately 0.5 mg of sodium dithionite was added to reduce any ferric heme to the ferrous state. The instrumentation and experimental details for laser flash photolysis were essentially as described previously (19). A pulse (0.6 µs) from a dye laser disrupted the photolabile heme Fe-CO bond, and the recombination of CO with the heme protein was then monitored by following the absorbance change at 436 nm. Data were transmitted to a microcomputer for processing and analysis.
Standard multiexponential analysis of the kinetic data was performed according to,
![]() |
(Eq. 2) |
The CD spectra of the carboxyl derivatives were recorded in 0.05 M phosphate buffer at pH 7.0 using an Aviv CD 60 spectropolarimeter. Before recording the spectra hemoglobin was treated with dithionite under an atmosphere of CO and rapidly filtered through Sephadex G-25 resin. The optical spectra were deconvolved to verify that the heme was present only in the carboxyl form and to determine the exact protein concentration. The CD spectra, which are the average of three scans, were recorded every 0.3 nm using a bandwidth of 0.5 nm and a time constant of 1 s in a cuvette of 0.5 cm path length. Protein concentration was 0.2 mg/ml.
NMR MeasurementsThe NMR experiments were carried out on a
VXR-400/54 spectrometer operating at 9.4 tesla. All NMR spectra
reported here were obtained at 29 °C using the jump-and-return pulse
sequence 90°()-
-90°(-
) (20). The delay
was adjusted
for each experiment such that maximum excitation was obtained for the
spectral region of interest. The relaxation delay between successive
transients was 2.5 s. The proton chemical shifts are referenced to
2,2-dimethyl-2-silapentane-5-sulfonate.2
The samples of native HbA and rHbA used for the NMR measurements
were in 0.1 M Bis-Tris buffer, pH 6.85, in 90%
H2O, 10% D2O. Hemoglobin concentration was
between 7 and 9%, and methemoglobin content was 5% or less.
Fig.
1 shows SDS-polyacrylamide gel electrophoresis of the
total cell proteins before and after induction as well as the
detergent-insoluble fusion protein. This figure illustrates the high
expression level of NS1-FX--globin and shows that treatment of the
insoluble fraction with detergent eliminates a large part of the
contaminant proteins.
The procedures for purification, cleavage, and reconstitution of the
tetrameric hemoglobin were essentially the same as those used for
rHbA (6). The pH values of the various reactions were increased due
to the higher isoelectric point of the
-globin, which renders it
less soluble than
-globin at pH 8.5. The yield of
rHbA was 15-20
mg/liter of cell culture. Cleavage of the fusion protein with Factor Xa
was followed by reverse phase HPLC. Fig. 2A
shows the elution pattern of the detergent-purified fusion protein, and
Fig. 2B shows the elution pattern of the fusion protein following 20 h of digestion with Factor Xa. The major peak
corresponds to
-globin with the correct amino-terminal end, whereas
the smaller peak is a product of overdigestion, which occurs at
Arg
31(B12).
Peptide Maps of
Fig. 3, A and B,
shows the tryptic peptide patterns obtained from normal and recombinant
-globins, respectively. The numbers above the peaks correspond to
the tryptic fragments of the
-chain as listed in Table
I. The HPLC profiles of tryptic digestion of the two
-globins are identical. Furthermore, five cycles of Edman sequential
degradation on intact
-globins isolated from natural and recombinant
HbA gave comparable yields of the expected 5 residues at the amino
terminus. Sequence analysis of the peak eluting at 22 min identified an
unresolved mixture of fragments 1 (residues 1-7) and 3 (residues
12-16). The peak eluting at 24 min contains fragment 1+2, a partial
cleavage product (residues 1-11). The difference between peaks 12 and
13 eluting at 52 and 54 min is 1 Lys residue located at position
61(E10).
|
-The spectrum in the Soret region is
sensitive to the interaction of the heme with the surrounding aromatic
residues (21). Fig. 4A shows the CD spectra
of the carboxyl derivatives of HbA and rHbA. Although these exhibit
the same ellipticity at the peaks of their spectra, small differences
are evident at lower wavelengths. Fig. 4B shows the CD
spectra of native and recombinant
-chains. The recombinant
-chains have a lower ellipticity and a broader spectrum than the
native
-chains.
Nuclear Magnetic Resonance
Fig. 5 shows the
downfield region of the NMR spectra of rHbA and HbA in the deoxy
form. The resonances between 15 and 24 ppm originate from protons in
the heme groups and/or from protons in amino acid residues located in
the heme pockets. These resonances are shifted downfield by the
hyperfine interactions between the corresponding protons and the
unpaired electrons of the iron atoms. In deoxy-HbA, the hyperfine
shifted resonances at 22.2 and 18.9 ppm have been assigned to the
-subunits, and those at 20.3 and 16.8 ppm have been assigned to the
-subunits (22-24). As shown in the figure, in deoxy-
rHbA the
same hyperfine shifted resonances are observed, and their chemical
shifts are, within experimental error, the same as in deoxy-HbA. The
spectral region from 11 to 15 ppm in Fig. 5 contains several other
hyperfine shifted resonances and four exchangeable proton resonances.
The hyperfine shifted resonances are significantly broader than the
exchangeable proton resonances (i.e. 350-500
versus 50-75 Hz). Due to these differences in the line
widths and to the spectral overlap, only the exchangeable proton
resonances in the spectral region 11-15 ppm in Fig. 5 can be observed
accurately. In deoxy-HbA, these four exchangeable proton resonances
have been assigned to specific hydrogen bonds in the Hb molecule as
follows: the resonance at 14.1 ppm to the hydrogen bond between
Tyr
42(C7) and Asp
99(G1) at the
1
2 interface (25, 26); the resonance at
13.0 ppm to the hydrogen bond between Asp
126(H9) and
Tyr
35(C1) at the
1
1
interface; the resonance 12.2 ppm to the hydrogen bond between
His
103(G10) and Asn
108(G10) at the
1
1 interface (26); and the resonance at
11.1 ppm to the hydrogen bond between Asp
94(G1)
and Trp
37(C3) at the
1
2
interface (27).3 As shown in the figure, in
deoxy-
rHbA these four exchangeable proton resonances are very close
if not identical to those in deoxy-HbA.
Our NMR results for rHbA in the ligated state are shown in Figs.
6 and 7. Fig. 6 shows the spectral region
from 9.5-14.5 ppm. In carboxy-HbA, the resonances at 12.95, 12.1, and
10.2 ppm originate from exchangeable protons and have been assigned as follows: the resonance at 12.95 ppm to the hydrogen bond between Asp
126(H9) and Tyr
35(C1) at the
1
1 interface (26); the resonance at 12.1 ppm to the hydrogen bond between His
103(G10) and
Asn
108(G10) at the
1
1
interface (26); and the resonance at 10.23 ppm to the hydrogen bond
between Asp
94(G1) and Asn
102(G4) at the
1
2 interface (25). In carboxy-
rHbA,
these exchangeable proton resonances occur at the same spectral
positions as those in carboxy-HbA.
The spectra shown in Fig. 6 also contain several resonances from the
heme groups. In carboxy-HbA these resonances are (28, 29): the
resonance at 10.45 ppm (mesoproton of the heme in
-chains); the
resonance at 10.1 ppm (mesoproton
of the heme in the
-chain);
and the resonance at 9.7 ppm (mesoproton
of the hemes in
- and
-chains and mesoproton
of the heme in the
-chain). In
carboxy-
rHbA, the latter two heme resonances occur at the same
positions as in carboxy-HbA. However, the resonance of the mesoproton
of the heme in
-chains (10.45 ppm) is missing from the
spectrum.
Fig. 7 shows the region from 2.2 to
0.4 ppm of the NMR spectra of
carboxy-
rHbA and HbA. This region contains resonances from protons
in the heme groups and/or amino acids in the heme pockets that are
shifted upfield by the ring-current effect of the heme groups. In
carboxy-HbA, the resonance at
1.77 ppm has been assigned to the
2-CH3 protons of the distal Val residues in
the
- and
-subunits, Val
62(E11) and
Val
67(E11) (28). As shown in Fig. 7, the position of
this resonance in carboxy-
rHbA is nearly identical to that in
carboxy-HbA.
The binding isotherms of HbA and rHbA
were measured using 1.5-2.0 mM heme, a concentration at
which dimers and their associated complications are negligible. For
these experiments, heme oxydation was below 5%, thus eliminating the
need for adding the reducing system. The curves were analyzed according
to the Adair equation (17) and yielded the thermodynamic parameters
listed in Table II. Although these parameters do not
differ significantly within the 66.7% confidence limit, the
cooperativity of
rHbA is decreased at all oxygen fractional
saturations as shown in Fig. 8.
|
The oxygen binding parameters of HbA and rHbA were measured in the
presence of Cl
and 2,3-diphosphoglycerate. The data
(Table III) indicate that the oxygen affinity and
cooperativity of natural HbA and
rHbA are similarly affected by
these effectors and that these exhibit the same Bohr effect.
|
The kinetics of CO binding to
native HbA and rHbA were measured (Fig. 9) and the
data analyzed using a multiexponential model. Each binding curve was
well represented by two components whose rates varied by about 30-fold
(Table IV). The slower phase originated from CO binding
to the tetramer, and the fast phase originated from CO binding to the
dimer as described previously (30). Comparison of the rate constants
shows that HbA and
rHbA exhibit similar rates of CO recombination
for both dimers and tetramers.
|
The kinetic analysis also yields absorbance values that reflect the
amounts of tetramer and dimer. These values were used to calculate the
dimer-tetramer association constant (Ka) and the
accompanying G value for this transition (Table IV). The
resulting Ka values for HbA (2.93 × 105 M
1) and
rHbA (2.0 × 105 M
1) and their respective
G values (
7.34 and
7.12 kcal/mol) were similar.
The kinetics of CO binding to native and recombinant -chains were
also measured. Each binding curve was well represented by two
components whose parameters are presented in Table V. The major components of the native and recombinant
-chains reacted at the same rate (k2 = 18.2 and 19.7 × 105 M
1 s
1,
respectively). However, the two samples differed significantly in that
the major component corresponded to virtually the entire (97%) native
-chain but to only 63% of the recombinant
-chain.
|
The long term objective of this work is elucidation of
the role of -chains on the structure and function of hemoglobin. Our strategy involved comparison of the expressed and native
-chains both as the isolated chain or incorporated into the HbA tetramer. Any
observed differences in conformational or functional properties can
then be attributed to the
-chain. Thus, although the
rHbA was
almost identical to native HbA, the subtle differences that were
observed provide new details on the role of proper
-chain folding in
maintaining the structure and cooperativity of the hemoglobin molecule.
The conformational and functional aspects will be considered
separately.
The NMR spectra in Fig. 5 indicate
that the overall conformation of the heme pockets in deoxy-rHbA is
similar to that in deoxy-HbA. A subtle difference, however, is observed
for the relative intensities of the hyperfine shifted resonances at
22.2 and 16.8 ppm in
rHbA. In deoxy-HbA, the intensity of the
-chain hyperfine shifted resonance at 16.8 ppm is the same as that
of the
-chain hyperfine shifted resonance at 22.2 ppm (namely, six
protons/heme) (31).4 In contrast, in
deoxy-
rHbA the
-chain resonance at 16.8 ppm appears more intense
than the
-chain resonance at 22.2 ppm. In order to understand the
origin of this difference we have measured the absolute intensity of
the hyperfine shifted resonance of
rHbA using a reference standard
(tris(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionate) europium) complexed with t-butyl alcohol (31). The results
showed that the
-chain resonance at 22.2 ppm in
rHbA has the same
intensity as the corresponding resonance in HbA. In contrast, the
intensity of the
-chain resonance at 16.8 ppm in
rHbA is
increased by a factor of approximately 2 relative to that in HbA. The
origin of this difference is difficult to ascertain since, at present, it is not known whether the
-chain hyperfine shifted resonance at
16.8 ppm originates from protons in the heme groups, from protons in
the amino acids in the heme pockets, or from both. Future NMR experiments on HbA molecules containing fully deuterated heme groups
may provide an answer to this question.
Similarly, in carboxy-rHbA (Fig. 6) the resonance of the mesoproton
of the heme in the
-chain (10.45 ppm) is missing from the
spectrum, probably shifted to a different spectral position in which it
cannot be resolved. This result suggests that in the ligated form the
heme environment in the
-subunits of
rHbA may also differ from
that in HbA. Alterations in the heme pockets are also suggested by the
ring-current shifted resonances (Fig. 7). All these resonances are
broader in
rHbA than in native HbA, and in
rHbA the resonance at
0.64 ppm appears to be shifted from its position in HbA. Similar
differences in the ring-current shifted resonances have been previously
observed for a full recombinant HbA (4) and have been attributed to a
different mode of heme insertion. One possibility previously suggested
is the existence of two heme orientations that differ by a 180°
rotation about the
,
-mesoaxis of the porphyrin (32). Our results
for
rHbA do not support this suggestion, since a rotation about the
,
-mesoaxis would conserve the environment of the
-mesoproton.
In contrast, our results show that in
rHbA the
-mesoproton
resonance is shifted from its position in the spectrum of HbA (Fig. 6).
Moreover, our results show that the position of the distal (E11)Val
residues relative to the heme groups in ligated
rHbA is very close
to that in HbA. This is consistent with the observed similarity of the
CD spectra (Fig. 4A) and indicates that the small difference observed near 410 nm is not due to heme inversion. Further
investigations are necessary in order to characterize fully the
conformation of the
-chain heme pockets in
rHbA. Figs. 5 and 6
show that the exchangeable proton resonances of deoxy-
rHbA are very
similar if not identical to those in HbA in both deoxy and carboxyl
forms. These findings indicate that the hydrogen bonds at the
1
2 and
1
1
interfaces that can be monitored by NMR are identical in deoxy- and
carboxy-
rHbA and HbA.
The Soret region of the CD spectrum primarily reflects the interaction
of the heme chromophore with the surrounding aromatic residues (21).
Thus, it serves as a sensitive probe of correct heme pocket refolding.
In the heme pocket, in the absence of amino acid substitutions a
decrease in ellipticity has been attributed to the presence of inverted
heme (33). The CD spectra of HbA and rHbA have a similar ellipticity
(Fig. 4A), suggesting correct heme insertion. However, a
small difference in the shape of the spectrum is observed near 410 nm,
which suggests the presence of subtle conformational modifications.
Although no direct correlation between the NMR and the CD spectra can
be made, it is interesting to note that NMR data suggest the correct
refolding of the heme pocket, the absence of inverted heme, and the
presence of subtle modifications in the heme pocket resonaces.
The CD spectrum of r-chains (Fig. 4B) is broader than
that of native
-chains and has a decreased ellipticity, suggesting some heme disorder and heterogeneity. The absence of similar
heterogeneity in the
rHbA suggests that optimal refolding of the
-heme pocket occurs upon the tetrameric assembly of the protein.
The thermodynamic analysis reported in
Table II shows that HbA and rHbA exhibit oxygen binding properties
that are indistinguishable except for a reduced cooperativity. Since
the NMR data indicate identical resonances for residues at the
1
1 and
1
2
interfaces, the reduced cooperativity cannot be attributed to altered
quaternary assembly. The free energy of cooperativity,
Gc, is the same for HbA and
rHbA (
1.8 ± 0.3 and
1.6 ± 0.2 kcal/heme). Thus, the decreased value of
nmax measured for
rHbA is probably due to
phenomena linked to the intermediate stages of oxygenation. At present
the molecular events associated with the intermediates of oxygenation
are still elusive, as these partially oxygenated forms are very
unstable and their fractional distribution is much smaller than
anticipated by a statistical distribution of ligands on tetrameric
hemoglobin (34). These results suggest that substitution of the
recombinant for the native
-globin is sufficient to alter these
oxygenation intermediates independently of the quaternary assembly and
propose a key role for nonquaternary interactions in the modulation of
oxygen cooperativity.
The Bohr effect and the oxygen affinity regulation by allosteric
effectors are sensitive probes of tertiary structure and quaternary
assembly. In rHbA the Bohr effect and the sensitivity to
Cl
and 2,3-diphosphoglycerate are equivalent to that in
native HbA (Table III). This indicates that the Bohr effect groups and
the effector binding sites are correctly aligned in the
half-recombinant protein. This is probably favored in the
half-recombinant hemoglobin by the native partner chains, which help
direct the correct refolding and reassembly of the recombinant chains
(35).
The CO binding experiments confirm the oxygen equilibrium data (Fig. 9
and Table IV) and reveal that HbA and rHbA are indistinguishable on
the basis of two additional criteria: rate of CO binding and the
energetics of dimer-tetramer association. Our observation that the
major component of both recombinant and native
-chains exhibited the
same rate (19.7 and 18.2 × 105
M
1 s
1) as that of the dimers of
native HbA (20.5 × 105 M
1
s
1) and
rHbA (19.5 × 105
M
1 s
1) is consistent with a
similar conformation of isolated
-chains, whether monomeric or
polymeric, and the
-chains within a hemoglobin dimer. The
differences observed between the binding kinetics of the expressed and
native
-chains thus are eliminated during tetramer formation, as the
energetics of quaternary assembly more than compensate for the
decreased conformational flexibility of
r upon its incorporation
into the tetramer.
In addition to characterizing HbA, it is also important to determine
the properties of the isolated chains. The ability of isolated
-globin to refold with heme may perhaps be explained by the results
from the CO binding kinetics and CD. The greater heterogeneity in CO
binding kinetics of the
r-chain suggests a correspondingly greater
degree of structural or dynamic heterogeneity relative to that of the
native
-chain. This result is consistent with the CD measurements,
which reflect the time-averaged structure of all
-chain components
and which show reduced ellipticity and a broader spectrum in the Soret
region for the
r-chains. Taken together, these approaches suggest
the presence of alternating conformations for the isolated
r-chain.
A dynamic access to a wide conformational space would enhance its
prospects for correctly refolding with the heme and ultimately
attaining a conformation suitable for pairing with
-chain and
formation of a functional HbA tetramer.
In conclusion, we have expressed -globin that can refold with
heme in the absence of the
-chain. The isolated
-globin exhibits a wide range of conformational flexibility. Furthermore, this
-globin can recombine with the heme and assemble with
-chain to
produce a recombinant HbA whose functional and conformational characteristics resemble those of natural HbA. Although subtle modifications in the heme pocket and a decreased cooperativity are
observed, the contacts across the
1
1 and
1
2 interfaces are retained. These
findings underscore the key role played by the tertiary structure of
the heme region, exclusive of the quaternary structure, in modulating
the functionality of the HbA molecule. Our results also suggest a key
role for nonquaternary interactions in processes involving the
relatively unstable oxygenation intermediates. The expressed
-globin
is thus suitable for construction of mutants to be used in future
studies of HbA structure-function relationships.
We thank Dr. Walter Kisiel for the gift of
Factor X and Daniel Youch for assistance in the construction of
pNF.