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INTRODUCTION |
Equimolar amounts of
- and
-globin chains of human
hemoglobin self assemble to form
2
2
tetramer. In addition, isolated
chains also assemble to form
4 homo-tetramers (1). Extensive previous studies using
naturally occurring variants and our recent studies using recombinant
chain variants showed that affinity between
and
chains is
promoted by negatively charged
chains and is independent of charge
location on the surface except at the
1
1
interaction site (2-5). Our previous studies showed that
affinity is promoted by negatively charged
chains up to a maximum
of two additional net negative charges (5). In addition, we showed that
112 Cys located at an
1
1 interaction
site on the G helix is critical for facilitating formation of stable

dimers which then form functional hemoglobin tetramers. We also demonstrated that
112Cys
Asp inhibits formation of
stable
1
1 and
1
2 interactions in
2
2 and
4 tetramers,
respectively (5).
X-ray diffraction analysis of
4 and
2
2 showed that
112 Cys on the G helix
is involved in
1
2 and
1
1 subunit interactions, respectively,
and that there is similarity between the quaternary structure of
carbonmonoxy
4 and carbonmonoxy
2
2 tetramers (6). The detailed role of
1
1 interaction sites involving
112 Cys (G 14) in
2
2 and
4
tetramer formation is, however, not clear. Assembly of hemoglobin
tetramers at least in vitro occurs as shown in Reactions
I. (7).
The
chains are in monomer-dimer equilibrium and
dissociation into monomers is favored, whereas the
chains are in
monomer-tetramer equilibrium and association into tetramers is favored.
It is generally assumed that dissociation of these oligomeric subunits
(Reactions I and II) into monomers must occur before they can combine
to form the 
dimers (Reaction III). Two 
dimers then
associate to form tetrameric hemoglobin (Reaction IV). The dissociation of oligomeric
subunits is a first-order reaction, while assembly of

dimers from
and
monomers (Reaction III) is a
second-order reaction (2, 4, 7). Formation of functional hemoglobin tetramers is dependent on these reactions (7). In vitro
assembly of the liganded forms of
- and
-globin chains show that
the rate of dissociation of
4 homo-tetramers is a
rate-limiting step in formation of 
hetero-tetramers (2).
Therefore, clarification of the role of
112 Cys at the
1
1 and
1
2
interaction site on 
and
4 formation,
respectively, is critical in order to understand the mechanism of
hemoglobin assembly. In this report, we expressed four
112 variants
and characterized effects of these changes on
2
2 and
4 tetramer
formation in vitro in order to further assess the role of
112 Cys on assembly of hemoglobin.
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MATERIALS AND METHODS |
Expression of Soluble Recombinant Human
-Globin
Chain Variants in Escherichia coli--
Four
112 globin chain
variants (e.g.
112 Ser,
112 Val,
112 Thr, and
112 Asp) were constructed and expressed using the pHE2
plasmid
vector that contains cDNAs coding for each
chain variant and
methionine aminopeptidase which was originally developed to express
and
chains at the same time (8, 9). The basic strategy for
generation of these variants by site-specific mutagenesis of the normal
chain involves recombination/polymerase chain reaction as described
previously (10). Clones were subjected to DNA sequence analysis of the
entire
-globin cDNA region using site-specific primers and
fluorescently tagged terminators in a cycle sequencing reaction in
which extension products were analyzed on an automated DNA sequencer.
Plasmids were transfected into E. coli (JM 109) (Promega
Co.), bacteria were grown at 30 °C, and soluble
-globin chain
variants were isolated as described (5, 8). Expression and purification
of
-globin variants were basically as described previously (5, 8).
However, cation-exchange chromatography on a Source 15 S column
(Amersham Pharmacia Biotech) instead of Superose 12 gel filtration was
used for purification of the
chain variants. Authentic human
hemoglobin,
-,
A (
6 Glu)- and
S
(
6 Val)-globin chains were purified from erythrocyte lysates from
normal controls and patients with sickle cell disease, respectively, according to previously described methods (11). Removal of
p-mercuribenzoate from
chains was accomplished using 20 mM dithiothreitol
(DTT),1 and globin chains
were isolated using gel filtration on a Superose 12 column for the
final purification step.
Biochemical Characterization of Purified
-Globin
Chains--
Molecular mass and sample purity were assessed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described (12). Mutations in each purified
-globin chain variant were confirmed using mass spectral analysis. Electrospray ionization mass spectrometry was performed on a VG BioQ triple quadrapole mass
spectrometer (Micromass, Altrincham, UK) using the multiple charged ion
peaks from the
-globin chain (Mr = 15,126.4) as reference for mass scale calibrations (13). Data analysis
employed the MassLynx® software package (Micromass).
Purified
-globin chains were also analyzed by cellulose acetate
electrophoresis, and mobilities were compared with those of authentic
human globin chains. Isoelectric focusing of purified
-globin
variants, Hb A and Hb S, was performed on Ampholine PAG plates
(Amersham Pharmacia Biotech), pH 5.5-8.5, using a Multiphor II system
(Amersham Pharmacia Biotech). After focusing for 2 h at a constant
25 watts at 4 °C, the gel plate was cut in half, one portion was
stained using the JB-2 staining system (Isolab inc., Akron, Ohio) to
detect heme proteins, and the other half was stained with Coomassie
Brilliant Blue R-250 (Sigma) to detect protein. Isoelectric point of
each
-globin variant was estimated using a calibration curve
prepared with isoelectric focusing standards (Bio-Rad).
Absorption spectra of purified
-globins in the CO form were recorded
using a Hitachi U-2000 spectrophotometer (Hitachi Instruments, Inc.
Danbury, CT). Globin concentration was determined
spectrophotometrically using a millimolar extinction coefficient of
13.4 at 540 nm for carbonmonoxy hemoglobin (14). Circular dichroism
(CD) spectra of
-globin variants were recorded using an Aviv model
62 DS instrument (Varian Analytical Instruments, San Fernando, CA)
employing a 0.1-cm light path cuvette at 10 µM globin
concentration. CD ellipticity of
-globin variants compared with
normal
A was monitored between 190 and 260 nm. Oxygen
dissociation curves of hemoglobin tetramers were determined in 50 mM Bis-Tris buffer containing 0.1 M NaCl and 5 mM EDTA, pH 7.2, at 20 °C using a Hemox Analyzer (TCS
Medical Products, Huntingdon Valley, PA) (15).
Dissociation of
-globin homo-tetramers was studied by fast protein
liquid chromatography (FPLC) on a Superose 12 gel filtration column.
-Globin was mixed with blue dextran and vitamin B12, internal markers for determination of void volume
(V0) and total column volume
(Vt), respectively. The final
-globin
solution (1.5 to 75 µM) was injected into a Superose 12 column, and gel filtration was done using 0.1 M potassium
phosphate buffer, pH 7.0 at 4 °C. Elution coefficients were
calculated using the following equation, where
Ve represents elution volume of each sample.
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(Eq. 1)
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2
2 tetramer formation was assessed
by mixing purified
chain variants (75 µ M) with
varying amounts of
-globin chain in the CO form in 0.1 M
potassium phosphate buffer, pH 7.0 at 25 °C (2, 5), and tetramer
formation was monitored by FPLC using a Mono-S column. Assembled
tetramers were characterized after separation from excess, free globin
chains by FPLC using a Source 15 S column. Dissociation of in
vitro assembled
2
2 tetramer into
subunits was assessed at a flow rate of 0.5 ml/min at 4 °C by large
zone chromatography using a Superose 12 gel filtration column (1 × 31 cm) as described previously (16, 17). Sample volumes of
2
2 variant tetramers (25 ml in 0.1 M potassium phosphate buffer, pH 7.0) were equal to the
column bed volumes. Elution was monitored at 405 nm using a 5-mm flow
cell. Elution volume (Ve) was measured at the
centroid of the ascending leading boundary (16, 17). Dissociation
constants of
2
2 tetramers to 
dimers were estimated by evaluation of the change in
Ve as a function of hemoglobin concentration
(17, 18).
 |
RESULTS |
Expression and Purification of Soluble
-Globin Chain
Variants--
We previously reported expression of
112Cys
Asp chains in bacteria (5). In order to further
define the role of
112 Cys in
2
2 and
4 tetramer formation, three additional
-globin chain
variants (e.g.
112Cys
Ser,
112Cys
Thr, and
112Cys
Val) were
expressed and characterized. Ser is similar in size to Cys, whereas Thr
and Val are slightly larger. In addition, Ser and Thr are hydrophilic
like Cys, whereas Val is highly hydrophobic. After DNA sequence
confirmation, the three
-globin chain variants were expressed in
bacteria and purified by a combination of two anion-exchange
chromatography steps using DEAE-cellulose and Mono-Q columns (9).
Further purification was achieved using cation-exchange chromatography
on a Source 15 S column. Purified
A chains migrate
predominantly as 32-kDa dimers (9), whereas the four
112 variants
migrate mainly as 16-kDa monomers with small traces of dimers on
SDS-PAGE (Fig. 1). Addition of 20 mM DTT to the chains in solution prior to SDS-PAGE converts
the dimers to monomers, indicating that small amounts of
93 Cys
oxidize and form disulfide-linked dimers.

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Fig. 1.
SDS-PAGE of 112 variants. Purified
preparations of each 112 variant (~2 µg) were subjected to
SDS-PAGE following incubation with (+) or without (-) 200 mM DTT for 30 min at 25 °C. Samples were heated in 3%
(w/v) SDS in a boiling water bath for 3 min and applied to a 15% (w/v)
polyacrylamide gel, and electrophoresis was performed at a constant
voltage of 100 V. The gel was stained with Coomassie Brilliant Blue
R-250 to detect proteins. A, molecular weight standards;
B, A chains (expressed in bacteria (5));
C, 112Cys Asp chains; D,
112Cys Ser chains; E,
112Cys Thr chains; and F,
112Cys Val chains.
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Mass spectral analysis of the four
chain variants using
electrospray ionization mass spectrometry resulted in values of 15,878.9, 15,850.3, 15,865.8, and 15,863.5 for
112Asp,
112Ser,
112Thr, and
112Val, respectively, which were in agreement with
expected masses for the variant
-globin chains. Carbonmonoxy forms
of all four variants showed typical absorption spectral characteristics
of human hemoglobin chains with peaks at 568, 540, 419, 344, and 276 nm
(14), indicating correct heme insertion into the variant
-globin
chains. CD spectra were also measured to examine structures of each
-globin variant compared with
A-globin chains (Fig.
2). Ellipticities between 190 and 260 nm for the
-globin variants were almost identical to that of normal human and recombinant
-globin, but there are small differences in
the region of the peak around 213 nm that are likely to be characteristic of these different
chains. Interestingly, the region
of the peak of the spectrum for
112Val chains around 213 nm was slightly left shifted compared with those of
A,
112Asp, and
112Ser chains (Fig. 2). These
CD results indicate that
-globin chain variants made in the E. coli were properly folded and had secondary structures similar to
authentic
-globin. In addition, the differences in the spectrum for
the
112Val chains may be a reflection of increased
conformational changes.

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Fig. 2.
Circular dichroism spectra of purified
-globin chain variants. CD spectra of A globin
purified from human red cells (4) compared with those of the four
112 variants ( 112Cys Asp (3),
112Cys Ser (5), 112Cys Thr (2), and
112Cys Val (1)). All chains were in the CO form in
0.01 M phosphate buffer, pH 7.0, at 10 °C.
Inset represents CD spectra between 205 and 230 nm of each
chain normalized to values at 222 nm after subtraction of the buffer
spectrum.
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Characterization of Purified
-Globin Chain
Variants--
We previously reported that electrophoretic mobility on
cellulose acetate of
112Cys
Asp chains was identical
to
s chains (5), indicating that these chains migrate as
monomers (charge of -2) rather than tetramers (charge of -4) like
4A chains (Fig. 3). In
contrast, mobility of
112Cys
Val and
112Cys
Thr chains was identical to authentic human
A. These results indicate that conversion from Cys to
Val or Thr at
112 does not cause significant changes in surface
charge while conversion to Asp appears to increase net positive charge
of the
-globin molecule because formation of monomers is favored
rather than tetramers. Effect of
112 amino acid on
-globin charge
was studied further by isoelectric focusing on polyacrylamide gel plates.
112Cys
Ser and
112Cys
Asp
chains focused as sharp bands with pI values of 6.68 and 6.35, respectively, whereas
112Cys
Val and
112Cys
Thr chains had pI values of 6.30 and 6.68, respectively, but focused as diffuse bands possibly because they exist
as mixtures of
chain monomer, dimer, and tetramer during
electrophoresis (19).

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Fig. 3.
Cellulose acetate electrophoresis of purified
-globin chain variants and their corresponding
2 2 tetramers. Purified -globin
variants (A) and their corresponding in vitro
assembled 2 2 tetramers (B)
were analyzed by electrophoresis on cellulose acetate membranes.
A, lanes 1 and 2, A and
S globins, respectively (purified from human red blood
cells); lane 3, 112Cys Val chains;
lane 4, 112Cys Thr chains; lane
5, 112Cys Ser chains; lane 6,
112Cys Asp chains. B, lane 1, Hb
A; lane 2, Hb S; lane 3, Hb C112V; lane
4, Hb C112T; lane 5, Hb C112S; and, lane
6, Hb C112D.
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4 tetramer and
-monomer levels for each of the
chain variants were assessed by size-exclusion chromatography (17, 20, 21).
A-Globin chains in solution exist as homo-tetramers
(
4) rather than monomers in the absence of
-globin
chains (1). In contrast, we found previously that
112Cys
Asp chains exist as monomers rather than
4 tetramers (5). In order to further characterize
effects of the different
112 amino acids on tetramer formation, each
-globin variant was chromatographed on a Superose 12 gel-filtration
column equilibrated with 0.1 M phosphate buffer, pH 7.0, at
4 OC in order to separate tetrameric and monomeric
species. The gel-filtration pattern of
A globin, which
contains
112 Cys, depended on concentration and temperature. At 75 µM,
A chains eluted mainly as tetramers
(86%) with a minor shoulder of monomers (14%) (Fig.
4a). The patterns of the
variants depended on the
112 amino acid. When the same concentration
as
A globin for each
chain variant was applied to
the column, results showed that the
112Cys
Ser variant
contained 51% tetramer and 49% monomer (Fig. 4c), whereas the
112Cys
Thr variant contained a slightly higher
percentage of tetramer than that of
A (Fig.
4d). The
112Cys
Val chains eluted only as
tetramers when the concentration was 75 µM. Dilution of
samples of the other
chain variants and
A chains
decreased the amounts of tetramer while monomer amounts increased. In
contrast,
112Cys
Asp chains eluted only as monomers at
all hemoglobin concentrations used. The ratio of tetramer/monomer
decreased as a function of
112 amino acid in the following order:
112Cys
Val >
112Cys
Thr >
A(112Cys) >
112Cys
Ser >
112Cys
Asp. Values for
112Cys
Thr and
112Cys
Val were 2- and 600-fold, respectively, higher
than that for
A(112Cys) chains. Association constants
for
4 homo-tetramer formation for the
112 variants
was calculated using results from gel-filtration chromatography and are
summarized in Table I. The
A-globin chain value (6 × 1016
M
3) was similar to results reported
previously (22). The association constant for the
112Cys
Asp variant was 0 since this variant existed
only as a monomer. It is interesting to note that dissociation of
S chains to monomers with decreasing concentrations was
significantly less compared with that of
A. The
tetramer association constant for
S chain monomers was
about 30-fold higher than that of
A, indicating that the
6 Val mutation in
S also affects tetramer-monomer
equilibrium and promotes homo-tetramer assembly.

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Fig. 4.
Superose 12 gel-filtration chromatogram of
purified -globin chains. Purified -globin variants were
mixed with internal markers (blue dextran for identification of void
volume (V0) and vitamin B12 for
total bed volume (Vt)) in 150 µl (final globin
concentration = 75 µM) and applied to a Superose 12 column (1 × 30 cm). Gel filtration was accomplished at a flow
rate of 0.5 ml/min using 100 mM potassium phosphate buffer,
pH 7.0. a, A chains; b,
S chains; c, 112Cys Ser
chains; d, 112Cys Thr chains; e,
112Cys Asp chains; and f,
112Cys Val chains.
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Table I
Homo-tetramer/monomer association constants of purified -globin
variants
Association constants were calculated from monomer/tetramer ratios
according to the equation, K = [ 4]/[ ]4 (20). Values were calculated from
three to five independent experiments and represent the mean ± S.E.
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Hetero-tetramer Formation
(
2
2) in Vitro
Using
-Globin Variants and Native
-Globin--
-Globin readily forms hetero-tetramers
(
2
2) in vitro in the presence
of
-globin. Tetramers were formed in vitro, purified and
separated from unreacted globins by chromatography, and then were
analyzed by cellulose acetate electrophoresis (Fig. 3B). We
previously reported that
112 Asp chains did not form
4 tetramers but did form
2
2112Asp tetramers. All three
new recombinant
-globin variants formed
2
2 tetramers in vitro.
Cellulose acetate electrophoresis showed cathodic shifts in migration
of tetramers compared with
chain variants upon binding of
-globin chains. It is interesting to note that, although individual
-globin chain variants migrated differently, all
2
2 tetramers containing the different
112 variants co-migrated to the same position as that of Hb A (Fig. 3B).
We also performed functional studies of the variant tetramers in 50 mM BisTris buffer, pH 7.2, containing 0.1 M
NaCl at 20 °C in the presence and absence of 2,3-biphosphoglycerate
and compared results with those of Hb A. Results of oxygen-binding
properties for the
112 variant
2
2
tetramers are summarized in Table II. Hb
C112S, Hb
C112T and Hb
C112V exhibited similar oxygen
affinities and n values to those of Hb A tetramers. Oxygen
affinity of recombinant Hb A was previously reported to be the same as
that of normal human Hb A (5, 8), while oxygen affinity of Hb
C112D
(P50 = 2.2) was higher than that of Hb A with a
similar cooperativity.
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Table II
Oxygen-binding properties of 112-variant hemoglobin tetramers
Oxygen equilibrium curves of hemoglobins were determined using 35 µM Hb concentration in 50 mM BisTris/HCl
buffer, pH 7.2, containing 100 mM NaCl and 5 mM
EDTA at 20 °C. P50 is partial oxygen pressure
required to give 50% oxygen saturation of hemoglobin.
nmax values were calculated from the Hill plot of
oxygen-equilibrium curves. Concentration of 2,3-biphosphoglycerate
(BPG) when present (+) is 2 mM. Mean P50
values (n = 5) for Hb A with and without BPG are shown
with a mean ± S.E. of 0.1 and 0.3, respectively.
K4,2 values for Hb A, Hb C112T, and Hb C112V
were calculated as shown in Fig. 6. However, the
K4,2 values for Hb C112T and Hb C112V were not
calculated since these two tetramers dissociated directly to monomers
instead of dimers upon dilution as shown in Fig. 6.
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Tetramer Formation in Vitro with Limiting Amounts of
Chains--
Competition for
2
2
hetero-tetramer formation between two different
-globin chains can
be assessed by monitoring tetramer assembly in the presence of limiting
amounts of
chains using 1:1 mixtures of the two different
-globin chains (3, 5). Previous studies in vitro showed
that variant hemoglobin percentages were higher when using more
negatively charged
chains like J-Baltimore (
16Gly
Asp) and N-Baltimore
(
95Lys
Glu) (4, 5). These studies suggested that more
negatively charged
chains bind positively charged
chains more
readily than
A chains (2-5). Furthermore, Hb A
tetramers formed twice as readily as Hb S, suggesting that
A chains interact with
chain more readily than
S under limiting
-globin conditions and that this was
due to the positive net charge of Val instead of Glu at the
6
position (3-5). In addition, our previous subunit competition studies
with equimolar mixtures of
S and
112Cys
Asp chains in the presence of limiting
chains suggested that
112Cys (G14) is a key amino acid
in formation of stable 
dimers (5). The
112 position is known
to be localized at the 
subunit interface after hetero-tetramers
assemble (6).
In order to further clarify the effect of
112 amino acid side chain
on assembly, tetramer formation in vitro was evaluated for
each of the purified
-globin variants after addition of
-globin chain isolated from human red blood cells. Competition experiments were
done in which varying amounts of
chains were added to equimolar mixtures of
S and either
112Cys
Val,
112Cys
Thr,
112Cys
Ser, or
A chains, and assembled tetramers were separated from
free chains by ion-exchange chromatography (Fig.
5). Our previous competition experiments
in vitro using mixtures of purified
and
chains showed that 
A dimers form about twice as readily as

s dimers when the concentration of
chains becomes
limiting (5). This results in assembly of less Hb S relative to Hb A
when equimolar amounts of
A and
S chains
compete for limiting amounts of
-globin (Fig. 5). Similar amounts of
variant tetramers (Hb
C112T and Hb
C112V) and Hb S were formed in
mixtures at all
-globin concentrations tested. These results
indicate that levels of Hb
C112T and Hb
C112V would be less
than that of Hb A in competition studies with the
A
chain. Total amounts of Hb
C112S were always more than that of Hb S,
with the Hb
C112S/Hb S ratio approaching 3.5 when
chain/total
chain approaches 0.1. On the other hand, as shown previously, much
less Hb
C112D formed in mixtures of
112Cys
Asp and
S, and almost all
-globin chains assembled with
S chains to form Hb S, when the ratio of
/total
chains was < 0.5 (5).

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Fig. 5.
Effect of 112 amino acid on relative
amounts of in vitro assembled tetramers as a function of
varying amounts of input -globin. Equimolar mixtures (75 µM) of the chain variant ( X) and
S were added to varying amounts of -globin chain in
0.1 M potassium phosphate buffer, pH 7.0, at 25 °C, and
assembled tetramers were analyzed by FPLC. The relative ratio
(y axis) is shown for Hb A to Hb S ( ) in
( A + S)/ mixtures, Hb C112D to Hb S
( ) in ( S + 112Cys Asp)/ mixtures,
Hb C112S to Hb S ( ) in ( S + 112Cys Ser)/ mixtures, Hb C112T to Hb S ( ) in
( S + 112Cys Thr)/ mixtures, and Hb
C112V to Hb S ( ) in ( S + 112Cys Val)/ mixtures. Each value represents the
mean ± S.E. from three-five independent experiments.
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Effect of
112 Variants on Dimer (
)
and Tetramer (
2
2)
Equilibrium--
In order to evaluate effects of
112 amino acid on
dimer-tetramer equilibrium, large-zone gel-filtration chromatography of
112-variant
2
2 tetramers was performed
using a Superose 12 HR column at various hemoglobin concentrations
(17). Decreasing concentrations of
2
2
variant tetramers in the CO form (25 ml in 0.1 M potassium
phosphate buffer, pH 7.0), whose volumes were equal to the column bed
volume, were loaded on the column in order to prevent dilution of
samples during chromatography. Elution profiles of hemoglobin variants
and Hb A exhibited sharp leading boundaries which then plateaued at
different levels depending on the concentration applied. The elution
volume (Ve), assessed by the centride of the
leading boundary, depends on hemoglobin concentration below 1.3 µM; the lower the concentration then the higher the
Ve. Ve values were
constant above 1.3 µM hemoglobin, indicating most
molecules exist as tetramers (Fig. 6).
With decreasing concentrations of Hb A, the Ve
values approached 15.5 ml, which corresponds to the value for 
dimers. A plot of Ve as a function of hemoglobin
concentration showed similar curves for Hb
C112T and Hb A, whereas
the curve for Hb
C112V was slightly shifted to the left. These
findings suggest that
112 Cys
Val inhibits tetramer dissociation.
The tetramer-dimer dissociation constants for Hb A, Hb
C112T and Hb
C112V were calculated as described previously (23) and were 0.11, 0.12, and 0.07 µM, respectively (Table I). In contrast,
the plots of elution volume as a function of concentration for Hb
C112S and Hb
C112D were right shifted from that of Hb A
(Fig. 6). Furthermore, Ve values for Hb
C112S and Hb
C112D below 0.1 µM were greater
than that for 
dimers and approached 16.25 ml at lower
concentrations, which corresponds to the Ve
value for monomeric forms determined using
112Cys
Asp
chains. The plot for Hb
C112D was more right shifted than that for
Hb
C112S (Fig. 6). These results indicate that tetrameric forms of
Hb
C112S and Hb
C112D continue to dissociate to
and
monomers upon dilution rather than remaining as 
dimers and that
Hb
C112D dissociates more than Hb
C112S.

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Fig. 6.
Effect of 112 amino acid on dissociation
of 2 2 tetramers. Varying
concentrations ranging from 0.01 to 5 µM hemoglobin
tetramer in 25 ml of 0.1 M potassium phosphate buffer, pH
7.0, were loaded onto a Superose 12 column, and gel-filtration
chromatography was performed at a flow rate of 0.5 ml/min in the same
buffer at 4 °C. A, elution position
(Ve) was plotted against hemoglobin
concentration, and Ve of globin monomers was
determined using 112Cys Asp chains. Samples included
Hb A from human red blood cells ( ) and in vitro assembled
112 variant 2 2 tetramers (Hb C112D
( ), Hb C112S ( ), Hb 112T ( ), and Hb C112V ( )).
B, K4,2 values of Hb A
(a), Hb 112V (b), and Hb C112T
(c) were calculated from the results of Fig. 6A.
K4,2 = [D]2/[T] is the value when log (1 - ) = 2 log , where is the fraction of dimers. D
(dimer) and T (tetramer) fractions were estimated from the
results in Fig. 6A. K4,2 values of Hb
C112D and Hb C112S could not be calculated since these tetramers
dissociate directly to monomers upon dilution.
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 |
DISCUSSION |
Effects of
112 Amino Acids on Homo-tetramer
Assembly--
X-ray analysis of
4 homo-tetramers at 1.8 Å resolution showed that
112 Cys (G14) is located at the
1 and
2 chain interface and that the side chains of
1112
Cys and
2112 Cys in the
4 tetramer are
very close to the molecular dyad at this interface (6). These two
residues exist on the surface of the
chains and may be involved in
weak interactions with other residues. Our present results showing
absence or trace amounts of disulfide dimer formation for all four of
the
112 chain variants in contrast to
A and other
chain variants, suggest that
1112 Cys and
2112 Cys are physically close together and that
disulfide
chain dimer formation is governed in part by these two
cysteine residues. Our results also show that
112 Val chains exist
mainly as
4 tetramers in solution, which is in contrast
to the previous results with
1112 Asp chains that exist
as monomers (5). In addition,
112 Thr and
112 Ser chains can form
4 homo-tetramers, whose levels are intermediate between
those of
112 Val and
112 Asp. Relative order of tetramer
formation among the different
112 variants is a direct function of
hydrophobicity of the
112 amino acid, the higher the hydrophobicity
then the more tetramers are formed. These results suggest that
1 and
2 interactions in homo-tetramers are governed to a large extent by
hydrophobic interactions involving the
112 amino acid and that
hydrophobic interactions between
1112 Val and
2112 Val are strongest followed by
1112
Thr-
2112 Thr,
1112
Cys-
2112 Cys, and then
1112
Ser-
2112 Ser. In contrast,
112 Asp chains exist as
monomers since
1112 Asp and
1112 Asp cannot participate in hydrophobic interactions, which results in lack
of formation of homo-tetramers. The ellipticity values for the CD
spectra in the far ultraviolet region (190-280 nm) are expected not to
be dependent on the heme moiety but on overall protein conformation
(24). The spectral difference in
112 Val compared with those of
A- and other
chain variants may be related to the
high homo-tetramer formation of the
112 Val variant. Detailed
studies of the conformational change differences between homo-tetramer
and monomer forms are now in progress.
Effects of
112 Amino Acid on Hetero-tetramer
Assembly--
112 Cys is located at the interface of
1
1 in
2
2
hemoglobin tetramers and interacts with Val
107 (G14) and Ala
110
(G11), which are critical for stabilization of the 
interface
(6). Amino acid changes at
112 also affected in vitro
assembly of
2
2 variant tetramers. Order
of
2
2 formation under limiting
-globin
chain conditions showed Hb
C112S > Hb A > Hb S = Hb
C112T = Hb
C112V >>> Hb
C112D. In
addition, apohemoglobin dimers were reported previously to dissociate
into monomers 170-fold faster than normal 
dimers because no
interactions occur between
112Cys and
104
Cys as a result of loss of the heme moiety (25). These findings and our results indicate that relative affinity of
for
chains is highly dependent on direct
1 and
1
interaction sites even though surface charge of the chains also affects
interactions during assembly (2-5). Amino acids at
1
1 interaction sites such as
112 Cys
may be critical for 
assembly.
Although the precise steps in
2
2 tetramer
assembly in vivo are not completely understood, functionally
intact
and
chains can be isolated and readily reconstituted
into
2
2 tetramers by mixing equimolar
amounts of
and
chains. As noted previously, it is generally
assumed that isolated
and
chains exist in a monomer-dimer and
monomer-tetramer equilibrium at least in vitro, respectively
(see Reactions I and II in the Introduction). Dissociation of
2 dimers and
4 tetramers follows kinetics
of a first-order reaction, while the monomer and dimer combination
steps (Reaction III in the Introduction) are second-order processes
that depend on protein concentration (7). Interaction of
and
chain monomers in Reaction III is very rapid compared with dissociation of
4 tetramers to monomers (7). The second-order rate
constant of Reaction III for
and
A
(
k5) is about 105
M
1 s
1 (20, 26), which means
that
monomer binds monomeric
A chain very rapidly,
roughly following second-order reaction kinetics. In contrast,
dissociation of
4
4
is very slow exhibiting a
dissociation constant (
k4) of
about 10
3 s
1 (20). These results indicate
that isolated single
and
chains assemble quickly but that
formation of tetrameric hemoglobin depends on
4
stability and total amounts of monomer in solution. Substitution of
112 Cys with Val as well as
6 Glu with Val in
S
chains inhibits tetramer dissociation, whereas substitution of
112
Cys with Ser or Asp promotes dissociation. When these
chain variants competed with
S chain for assembly with
limiting
chains, the order of assembly of the
112 variant
hetero-tetramers (e.g. Hb
C112S > Hb A > Hb
C112V) shown in Fig. 5 implies that
112 Val forms a weaker
1
1 interface even though the hydrophobic
substitution to Val is expected to strengthen hydrophobic interactions
with
107 Val (G14) and
110 Ala (G11) (27). These results,
however, may be reconciled by the fact that
112 Val facilitates
increased tetramer stability compared with
A chains and
that dissociation of
112 Val homo-tetramers to monomers is much
slower than that of
112 Cys homo-tetramers. This slow dissociation
of
112 Val homo-tetramers leads to less monomer formation and
therefore less Hb C
112V compared with Hb A. Furthermore, homo-tetramers containing
112 Ser dissociate to monomers more readily than those containing
112 Thr. This results in
112 Ser promoting
2
2 tetrameric formation
compared with
112 Thr under limiting
chain conditions. In
contrast, even though
112 Asp promotes formation of monomers, the
results of the order of tetramer assembly (e.g. Hb
C112T = Hb
C112V >>> Hb
C112D)
indicates that stability of the interaction between
and
chains
plays a key role in assembly in addition to ability to form monomers. However, when
112 Val associates with
chains, this substitution of the strong hydrophobic amino acid Val for Cys at
112 could stabilize
1
1 interactions due to its
ability to exclude water. This would facilitate interactions with the
chain at
107 Val (G14) and
110 Ala (G11) (27). In contrast, a
112 Cys substitution to a hydrophilic amino acid like Asp would
inhibit this hydrophobic interaction and result in no homo-tetramer
formation and weaken 
interactions. This also would destabilize
formation of hetero-tetramers. In vivo, hemoglobin synthesis
occurs in erythroblasts and reticulocytes (28). Even though the
assembly scheme in vitro is clear, it is not clear whether
these results in vitro apply to assembly in vivo.
The newly synthesized
chains in the cytoplasm probably exist at low
concentrations (<10 µM), which would favor formation of
monomers rather than homo-tetramers (3). However, gel-filtration experiments using
chains at 1.5 µM indicate that more
than 80% exist as tetramers when analyzing
S and
112
Val chains. The combination of heme and apoglobin is so rapid that
newly translated subunits probably bind heme prior to assembly (3, 29).
Our present results on production of soluble
-globin chains in
bacteria in the presence of heme but in the absence of
-globin also
suggest that these conditions in vivo promote correct
-globin chain folding in the absence of
-globin chains. The
relevance of
4 tetramer formation and dissociation under
in vivo conditions in which
and
chain synthesis is
balanced is not clear. Hb H (
chain homo-tetramers) is formed in a
severe form of
thalassemia in which patients produce only about
one-fourth of the normal amount of
chains. As a result,
chain
homo-tetramers form in cells. The ease of dissociation of
chain
homo-tetramers to monomers in addition to direct
and
interactions may be rate-limiting for assembly in vivo.
Effect of
112 Variants on Dimer-tetramer
Equilibrium--
Interactions between amino acid residues at the
1
1 and
1
2
interfaces are different (6, 28, 30). The
1
1 interface remains relatively fixed
during deoxygenation, and contacts between the
1 and
1 subunits are identical in oxyhemoglobin and
deoxyhemoglobin. In deoxyhemoglobin, there are about 40 contacts,
including 19 hydrogen bonds. In contrast, there is considerable
movement at the
1
2 interface during
oxygenation and deoxygenation. When hemoglobin is oxygenated, the total
number of contacts drops to about 23, including 12 hydrogen bonds. In
view of the important role that conformational isomerization plays in
hemoglobin function, it is not surprising that residues at the
1
2 interface are invariant and highly
conserved throughout vertebrate evolution (28, 30).
Under physiologic conditions, liganded tetrameric hemoglobins
dissociate reversibly into dimers:
2
2
2
. The
1
1 interface in 
dimers participates in formation of noncovalent intersubunit bonds that
do not dissociate easily in the oxy and deoxy forms. In contrast, the
1
2 contacts can dissociate upon
oxygenation and exhibit large oxygenation-induced subunit interaction
energies (28, 30). The reduction in contacts at the
1
2 interface following oxygenation
suggests that dissociation occurs there rather than at the
1
1 interface. Accordingly, human
hemoglobin variants with substitutions at the
1
2 interface usually exhibit dramatic
alterations in their functional properties, whereas those with
substitutions at the
1
1 interface do not.
In fact, oxygen affinities and cooperativity (n value) of Hb
C112T, Hb
C112V, and Hb
C112S were similar to those of Hb A,
whereas oxygen affinity was higher and cooperativity was lower for Hb
R40S (P50 = 2.2, n = 2.0), Hb
R40A (P50 = 2.2, n = 1.6), Hb
E101A (P50 = 3.4, n = 2.2),
and Hb
E101K (P50 = 2.5, n = 2.2),
which have substitutions at the
1
2
interface compared with Hb A (P50 = 5.1, n
= 2.7) (31). Furthermore, our results show that Hb
C112D
(P50 = 2.2, n = 2.6) has similar
cooperativity to Hb A and higher oxygen affinity compared with Hb A
(P50 = 4.2 ± 0.1, n = 2.7), whereas Hb
R40D (P50 = 1.2, n = 1.0) and Hb
E101D (P50 = 0.9, n = 1.9) (31) exhibited lower cooperativity and
much more increased oxygen affinities even though Hb
C112D favors
dissociation to dimer and monomer forms compared with Hb
R40D and Hb
E101D (31). These results reinforce the contention that allosteric
transition between oxy and deoxy forms of tetrameric hemoglobin is
influenced by amino acid residues located at the
1
2 interaction sites (28, 32, 33).
Interestingly, recent studies showed that
112Cys
Gly
chains in tetrameric hemoglobin unexpectedly stabilized
1
2 interactions (34). Our studies also
showed that
112Cys
Val stabilizes the
1
2 interface without significantly
changing oxygen affinity. Substitution of Cys at the
112 position
with Val appears to stabilize
1
2
interactions but may not affect local quaternary conformational changes
induced during the oxygenation-deoxygenation process. Furthermore, even
though the
112 Ser variant decreased 
stability by promoting
dissociation into monomers, the oxygen affinity of Hb
C112S was not
influenced by this mutation. These findings suggest that local
quaternary conformational changes induced during
oxygenation/deoxygenation are not influenced by this substitution at an
1
1 interaction site. In contrast, the
112 Asp variant destabilizes the
1
1
interaction sites and slightly reduced oxygen affinity. Furthermore,
this substitution also increased dimer dissociation from
hetero-tetramers compared with normal Hb A. These results indicate that
the
112 amino acid plays a critical role in tetramer-dimer stability
and suggest that structural changes at the
1
1 contact can be propagated through the
protein and lead to stability at the
1
2
interface. We are now investigating the relationship between mutation
at the
1
1 interface and dimer-tetramer stability of hemoglobin. These studies should aid in our understanding of hemoglobin assembly as well as in how changes at one site influence tertiary and quaternary structure at a distant location.
We thank Dr. Eric Rappaport and members of
the Nucleic Acid/Protein Core at the Children's Hospital of
Philadelphia for automated DNA sequence analysis. We are grateful to
Dr. H. E. Witkowska for mass spectral analysis of the
chain
variants performed at the Children's Hospital Mass Spectrometry
Facility in Oakland, CA. (Dr. C. Shackleton, director), which is
supported in part by NIH Grant HL20985 and a Shared Instrumentation
Grant RR06505.