(Received for publication, June 1, 1995; and in revised form, August 2, 1995)
From the
In order to investigate the role of the R (relaxed) to T (tense)
structural transition in facilitating polymerization of deoxy-Hb S, we
have engineered and expressed two Hb S variants which destabilize
either T state (Hb S-Kempsey,
) or R state
structures (Hb S-Kansas,
). Polymerization of deoxy-Hb S-Kempsey, which shows high
oxygen affinity and increased dimer dissociation, required about 2- and
6-fold higher hemoglobin concentrations than deoxy-Hb S for
polymerization in low and high phosphate concentrations, and its
kinetic pattern of polymerization was biphasic. In contrast, oxy- or CO
Hb S-Kansas, which shows low oxygen affinity and increased dimer
dissociation, polymerized at a slightly higher critical concentration
than that required for polymerization of deoxy-Hb S in both low and
high phosphate buffers. Polymerization of oxy- and CO Hb S-Kansas was
linear and showed no delay time, which is similar to oversaturated oxy-
or CO Hb S. These results suggest that nuclei formation, which occurs
during the delay time prior to deoxy-Hb S polymerization, does not
occur in T state oxy-Hb S-Kansas, even though the critical
concentration for polymerization of T state oxy-Hb S-Kansas is similar
to that of T state deoxy-Hb S.
Oxygen binding has a dramatic effect on Hb S polymerization.
Deoxygenated Hb S forms gels, while oxygenated Hb S does
not(1) . The reason for this difference between oxy and deoxy
forms of Hb S is the marked change in quaternary conformation of the
hemoglobin molecule upon deoxygenation(2, 3) . When
normal hemoglobin is fully deoxygenated, most of the molecules assume
the T ()structure, which has a relatively low affinity for
oxygen and other heme ligands. Conversely, normal oxyhemoglobin exists
almost exclusively in the R conformation and has a relatively high
affinity for heme ligands such as oxygen. Structural alterations that
affect the equilibrium between T and R states are expected to have a
marked effect on hemoglobin function as well as Hb S polymerization.
X-ray crystallographic studies show that hemoglobin can assume two
different quaternary structures: one characteristic of unliganded (T)
and the other of liganded (R) hemoglobin(2) .
The key to Hb
S polymerization is the presence of Val-6 and the formation of a
hydrophobic acceptor pocket between E and F helices that occurs in the
T state but is absent in the R state(3, 4) . However,
if a specific amino acid substitution decreases stability of the T
structure, then transition to the R state is favored and the molecule
exhibits increased oxygen affinity and decreased heme-heme interaction.
This has been demonstrated for a number of chemically modified
hemoglobins as well as for many hemoglobin variants(3) . Hb
Kempsey (
) and Hb
Kansas (
) are well
studied
examples(3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) .
Hb Kempsey is a
-chain variant in which Asp-
99 is replaced by
Asn(5) . In deoxy-Hb A, Asp-
99 normally forms an important
hydrogen bond with Tyr-
42 at the
interface(11) . Upon oxygenation, the two subunits shift
in a dovetail fashion, so that the
99-
42 hydrogen bond is
broken and another one forms between Asp-
94 and Asn-
102. In
Hb Kempsey, substitution of Asp with Asn at
99 prevents formation
of the former hydrogen bond with Tyr-
42 and therefore decreases
stability of ``deoxy'' or T state structures. Thus, when this
tetramer is fully deoxygenated, it remains partially in the R state.
In contrast, Hb Kansas is a -chain variant in which
Asn-
102 is replaced by Thr(14) . This position is also an
important site at the
interface,
which is involved in the transition between the oxy and deoxy
conformation of hemoglobin. Hb Kansas has a low oxygen affinity because
the hydrogen bond that usually occurs between Asn-
102 and
Asp-
94 in normal Hb A, which functions to stabilize the oxy
conformation, is absent, thus shifting the equilibrium toward the deoxy
(T) conformation(3, 12, 13, 18) .
Asp-99 and Asn-
102 in Hb S are located internally at the
interface and are not exposed to the
surface of tetrameric hemoglobin. These positions are involved in
interactions, which lead to formation of tetramers, and are
not defined as interaction sites in Hb S polymers. These amino acids do
affect the R to T quaternary structural transition of hemoglobin
tetramers. In this report, we have studied polymerization of R state
deoxy-Hb S and T state oxy or CO Hb S by preparing Hb S-Kempsey
(
) and Hb
S-Kansas (
)
using a yeast expression system (19) in an attempt to clarify
the role that quaternary structural changes associated with T to R
state transition play in polymerization of Hb S.
The plasmid pGS389 Hb S contains the full-length human -
and
-globin cDNAs under transcriptional control of
dual GGAP promoters as well as a partially functional yeast LEU2d gene and the URA3 gene for selection in
yeast(19, 20) . The plasmid pGS189
contains a single GGAP promoter and
-globin cDNA
and was constructed by mutagenesis and subcloning as described
previously(19) . The basic strategy for site-specific
mutagenesis at
99 or
102 involves recombination polymerase
chain reaction as described previously(19) . Asn-
99 and
Thr-
102
-chain variants were subjected to DNA
sequence analysis of the entire
-globin cDNA using site-specific
primers and fluorescently tagged terminators in a cycle sequencing
reaction in which extension products were analyzed on an automated DNA
sequencer(19) . The mutated
-globin cDNA region was then
excised by XhoI digestion and subcloned back into the XhoI site of the expression vector pGS389(19) .
Yeast growth, plasmid transformation, induction, and purification of
recombinant hemoglobin tetramers were described
previously(19, 20, 21) . Abnormal forms of
the recombinant hemoglobins, which include sulfhaem-containing
hemoglobin and/or misfolded hemoglobin, were eliminated by
chromatographic purification(21) . The purified - and
-chains of the Hb S variants containing Asn-
99 or
Thr-
102 were subjected to electrospray mass analysis (Fisons
Instruments, VG Biotech, Altricham, UK) using the multiply charged ion
peaks from the
-globin chain (M
=
15,126.4) as an external reference for mass scale
calibrations(22) . Val-
6 and N-terminal amino acid
sequences of purified
- and
-chains were directly confirmed
by Edman degradation employing a pulsed liquid, protein sequencer (ABI
477A, Applied Biosystems, Inc., Foster City, CA). Absorption spectra of
the purified hemoglobin variants were recorded using a Hitachi U-2000
spectrophotometer. Circular dichroism spectra of the variants were
recorded using an Aviv model 62 DS instrument employing a 0.1-cm light
path cuvette at
10 µM Hb concentrations. Methods for
determination of oxygen dissociation curves and electrophoretic
analysis of hemoglobins and their kinetics of polymerization in high
phosphate buffers were performed as reported
previously(19, 23) . Kinetics of polymerization and
solubility of hemoglobins in 0.1 M phosphate buffer, pH 7.0,
as well as measurements of minimum concentrations required for gel
formation were performed as described previously with slight
modification(24, 25) . The time course for the
gelation of deoxy-Hb S in 0.1 M phosphate buffer, pH 7.0, at
30 °C was done by the temperature jump method by monitoring change
in optical density at 800 nm using an anaerobic cylindrical cuvette
with a 2-mm diameter. Solubility of deoxy-Hb S was measured by the
determination of soluble deoxy-Hb S concentration after centrifugation
at 30 °C for 3 h at 130,000
g. Tetramer-dimer
dissociation properties of hemoglobins were evaluated by monitoring
fluorescence changes of haptoglobin using a Hitachi F-2000 fluorescence
spectrophotometer (26) .
Figure 1:
Electrophoretic mobilities of Hb S
D99N and Hb S N
102T. Electrophoretic mobilities of Hb S
D
99N (lane 4) and Hb S N
102T (lane 5) were
compared with native Hb A (lane 1), Hb S (lane 2),
and Hb C (lane 3) following cellulose acetate
electrophoresis.
Mass spectral analysis of Hb S variants containing Asn-99 or
Thr-
102 showed the expected
-globin chain molecular masses,
15,835.1 and 15,823.8 Da, respectively, which corresponded to expected
masses of 15,836 and 15,824 Da, respectively. Absorption spectra of
these two Hb S variants were the same as those of native and
recombinant Hb S(19) . Circular dichroism spectra in the region
from 190 to 290 nm for the Hb S variants were also similar to that of
native Hb S (Fig. 2), indicating that these substitutions do not
significantly affect globin folding and/or overall secondary structure
of hemoglobin tetramers.
Figure 2:
Circular dichroism spectra of Hb S
D99N and Hb S N
102T. Spectra were recorded for the CO form of
Hb S D
99N and Hb S N
102T (
10 µM) in 0.1 M phosphate buffer, pH 7.0, at 5 °C, and results were
corrected for small differences in protein concentration. A, B, and C represent Hb S, Hb S D
99N, and Hb S
N
102T, respectively.
It is known that Hb Kempsey and Hb Kansas
have higher and lower oxygen affinity than normal Hb A,
respectively(6, 12) . Oxygen affinities of Hb S
D99N and Hb S N
102T differed from Hb S as anticipated; the
former had higher and the latter lower oxygen affinity than Hb S or Hb
A (Fig. 3). P
values in 0.1 M phosphate buffer, pH 7.0, at 20 °C for the Asn-
99 and
Thr-
102 Hb S variants were 0.8 and 18, respectively, compared with
6.5 for recombinant Hb S(19) . Hill coefficients for both
variants were reduced to 1.2 and 1.5 for Hb S D
99N and Hb S
N
102T, respectively, compared with 2.6 for Hb S (Table 1).
Figure 3:
Oxygen equilibrium curves of Hb S
D99N and Hb S N
102T. Oxygen equilibrium curves of Hb S
D
99N (
) and Hb S N
102T (
) were determined in 0.1 M phosphate buffer, pH 7.0, at 20 °C and compared with
native human Hb S (
).
Differences in oxygen affinity for Hb S D99N and Hb S
N
102T compared with Hb S should be correlated with differences in
kinetics of dimer formation as has been shown for deoxy-Hb Kempsey and
oxy-Hb Kansas(6, 12) . Tetramer-dimer dissociation
properties of the two Hb S variants were measured by monitoring
quenching of haptoglobin fluorescence caused by existing hemoglobin
dimers (Table 2). Deoxy-Hb S N
102T showed similar dimer
formation properties to that of deoxy-Hb S, while deoxy-Hb S D
99N
exhibited increased dimer formation. In contrast, oxy-Hb S N
102T
showed a slightly increased tendency, while oxy-Hb S D
99N showed a
similar tendency for dimer formation as oxy-Hb S.
Figure 4:
Kinetics of polymerization for deoxy-Hb S
D99N. The time course of polymerization for deoxy-Hb S D
99N
was defined using 1.8 M phosphate buffer, pH 7.4, at 30 °C
by the temperature jump method. a, b, and c represent 506, 432, and 332 mg/dl deoxy-Hb S D
99N,
respectively.
Figure 5:
Relationship between log of reciprocal
delay time and hemoglobin concentration. Polymerization studies of
hemoglobins at different concentrations were performed in 1.8 M phosphate buffer, pH 7.4, at 30 °C. ,
, and
refer to native deoxy-Hb S, deoxy-Hb S D
99N, and deoxy-Hb
S N
102T, respectively.
In contrast to Hb Kempsey, the deoxy form of Hb S
N102T (Hb S-Kansas) polymerized like deoxy-Hb S (Fig. 6);
however, a small amount of detectable polymers formed prior to the
major phase of polymerization. Logarithmic plots of delay time versus hemoglobin concentration for the major phase of
deoxy-Hb S N
102T polymerization showed a straight line slightly
shifted to the right of the line for deoxy-Hb S (Fig. 5). It is
known that the oxy structure is destabilized in Hb Kansas, which favors
the low oxygen affinity T (deoxy) state(3, 12) .
Polymerization of liganded oxy- and CO-Hb S-Kansas occurred at a
slightly higher concentration than that required for deoxy-Hb S
polymerization; however, polymer formation was not accompanied by a
delay time prior to polymerization, and oxy-Hb S N
102T polymerized
linearly like oxy- and CO-Hb S (27) (Fig. 7). A
hemoglobin concentration 20 times less than that for oxy-Hb S was,
however, required to initiate polymerization. The CO form of Hb S
N
102T also polymerized linearly like the oxy form(27) .
Polymerization of oxy, CO, and deoxy-Hb S N
102T was reversed by
decreasing the temperature to 0 °C; however, when deoxy-Hb S
N
102T polymers were exposed to CO, about 60% of the polymers
depolymerized (Fig. 8) in contrast to complete depolymerization
for deoxy-Hb S polymers(19) . It is noteworthy that
polymerization of oxy-Hb S N
102T in 1.8 M phosphate
buffer was not affected by addition of inositol hexaphosphate, even
though organic phosphates are known to stabilize the T state of
liganded Hb Kansas(18) .
Figure 6:
Kinetics of polymerization for deoxy-Hb S
N102T. The time course of polymerization for the deoxy form of Hb
S N
102T was defined using 1.8 M phosphate buffer, pH 7.4,
at 30 °C by the temperature jump method as described previously. a and b represent 97.2 and 81 mg/dl deoxy-Hb S
N
102T, respectively.
Figure 7:
Kinetics of polymerization for Hb S
N102T in the oxy form. The time course of polymerization for the
oxy form of Hb S N
102T was defined using 1.8 M phosphate
buffer, pH 7.4, at 30 °C by the temperature jump method. a and b represent 243 and 162 mg/dl oxy-Hb S N
102T,
respectively.
Figure 8:
Depolymerization of deoxy-Hb S N102T.
Depolymerization of deoxy-Hb S N
102T (98 mg/dl) polymers in 1.8 M phosphate buffer, pH 7.4, at 30 °C was evaluated after
exposure to CO and cooling the temperature to 0
°C.
We also evaluated gelation and
polymerization of CO Hb S N102T at room temperature in low
phosphate concentration (0.1 M) buffer, pH 7.0, under more
physiological conditions. CO Hb S N
102T formed gels at a
concentration of 25.7 g/dl compared with 24 g/dl for deoxy-Hb S and
24.5 g/dl for deoxy-Hb S
(Table 2). CO Hb S
N
102T polymerized linearly without a delay time in 0.1 M phosphate buffer, pH 7.0, at 30 °C (Fig. 9), which is
similar to results in 1.8 M phosphate buffer (Fig. 7).
Solubility after polymerization of CO Hb S N
102T in 0.1 M
phosphate buffer, pH 7.0, at 30 °C was 19.2 versus 17 g/dl
for deoxy-Hb S. Depolymerization of CO Hb S N
102T in low phosphate
buffer was also observed upon lowering the temperature to 0 °C.
Figure 9:
Kinetics of polymerization for Hb S
N102T in the CO form in low phosphate buffer. Time course of
polymerization for the CO form of Hb S N
102T was defined using 0.1 M phosphate buffer, pH 7.0, at 30 °C by the temperature
jump method. a and b represent 26 and 22 g/dl CO Hb S
N
102T, respectively.
Total polymer formed in 1.8 M phosphate buffer as a
function of hemoglobin concentration was also determined in order to
evaluate effects of the Asn-99 and Thr-
102 substitutions on
the critical concentrations required for polymerization. Critical
concentration depends on deoxyhemoglobin solubility: the higher the
solubility, the higher the concentration required for polymerization.
Polymer formation for the two variants increased linearly with
increases in initial hemoglobin concentration (Fig. 10).
Critical concentration for polymer formation was then determined by
extrapolation of the lines to zero turbidity(19) . Values for
deoxy-Hb S
, CO Hb S
, and
deoxy-Hb S
were 1.2-, 1.6-, and 6.3-fold higher,
respectively, than that for deoxy-Hb S.
Figure 10:
Polymer formation as a function of
hemoglobin concentration. Turbidity at the plateau of the
polymerization curves in 1.8 M phosphate buffer at various
hemoglobin concentrations was measured at 700 nm. ,
,
, and
refer to native deoxy-Hb S, deoxy-Hb S D
99N,
deoxy-Hb S N
102T, and oxy-Hb S N
102T,
respectively.
The functional properties of normal Hb A and polymerization
of Hb S depend on transition of their three-dimensional conformation,
which accompanies the addition and removal of oxygen. X-ray
crystallographic studies show that hemoglobin tetramers exist in
equilibrium between two quaternary conformations: R and
T(2, 11) . The change from T to the R conformation
involves a well defined series of structural changes, including rupture
of the salt bridges that stabilize the T conformation and rotation of
the -chains relative to the
-chains(3, 11) .
Intramolecular ``movements'' also occur during conformational
isomerization at the
interface.
Structural alterations that affect the equilibrium between R and T
states are expected to have marked effects on hemoglobin function and
Hb S polymerization. Thus, if a specific amino acid substitution
decreases T structure stability, then transition to the R state would
be expected to occur at an earlier stage in ligation, and this
hemoglobin would exhibit increased oxygen affinity and decreased
heme-heme interaction. In contrast, substitutions that decrease R
structure stability result in tetramers with decreased oxygen affinity
and increased heme-heme interaction. These results have been observed
for a number of hemoglobin variants. Both Hb Kempsey and Hb Kansas,
which have amino acid substitutions at the
interface, are well studied
examples(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) .
In most respects, deoxy-Hb Kempsey with high oxygen affinity bears more
resemblance to oxyhemoglobin A than to deoxyhemoglobin A and is
partially in the R conformation(6, 7, 8) .
Recombinant Hb S-Kempsey exhibited high oxygen affinity with low
cooperativity. Polymerization of deoxy-Hb S-Kempsey occurred at higher
concentrations than deoxy-Hb S, and kinetic studies suggested
polymerization by linear as well as nucleation-controlled mechanisms (19, 27) . These results suggest that Hb S-Kempsey is
not completely converted to the R state upon deoxygenation. Asp-
99
is located in the interior of Hb S, but yet the change from Asp-
99
to Asn in Hb S-Kempsey inhibits polymerization, no doubt by affecting
the quaternary structure. These results indicate that inhibition and/or
acceleration of polymerization of Hb S is not always controlled by
residues at direct interaction sites of deoxy-Hb S polymers. We are now
attempting to define effects of this substitution at the allosteric
interface of Hb S on polymer and crystal structure.
Hb Kansas
contains Thr instead of Asp at 102, which is also a site at the
interface (3, 12) .
However, in contrast to Hb Kempsey, Hb Kansas has decreased oxygen
affinity, which is due in part to a relatively unstable R structure,
thereby shifting the equilibrium to that of the T structure. Our
results with Hb S-Kansas showed low oxygen affinity, and liganded forms
had decreased solubility, thereby facilitating polymerization like
deoxy-Hb S. The critical concentrations for polymerization of liganded
forms of Hb S-Kansas were similar to the deoxy form of Hb S-Kansas.
Facilitation of polymerization of liganded hemoglobin can be explained
by shifting conformational equilibrium to favor the T state like Hb
Kansas. However, the kinetics of polymerization of liganded Hb S-Kansas
were different from those of deoxy-Hb S and deoxy-Hb S-Kansas, which
were accompanied with a delay time prior to polymerization. These
results suggest that the surface structure of liganded Hb S-Kansas
resembles that of the T structure of Hb A or Hb S. The lack of
nucleation during polymerization of oxy or CO Hb S-Kansas, even though
the T-state is favored, may be caused by insufficient protein-protein
interactions that are required to form deoxy-Hb S or deoxy-Hb S-Kansas
nuclei.
There are seven known natural hemoglobin variants with
substitutions for
Asp-99(5, 6, 26, 28, 29, 30, 31, 32, 33, 34) .
Recently, recombinant Hb A containing Asp-
99
Lys and a
double mutant (Asp-
99
Asn and Tyr-
42
Asp),
which were produced in yeast and Escherichia coli expression
systems, respectively, were characterized(26, 35) .
All these variants show increased oxygen affinity with reduced
cooperativity. There are also three known natural hemoglobin variants
with substitutions at
102, and recombinant Hb A D
102A made in
yeast was also recently
reported(36, 37, 38) . These variants show
decreased oxygen affinity with reduced cooperativity. Functional
properties of hemoglobin variants with substitutions at
99 and
102, which involve the
contact,
have been attributed to increased dissociation of the variant
tetrameric hemoglobin dimers. Deoxy-Hb S-Kempsey and oxy-Hb S-Kansas
show increased dissociation to dimers like Hb Kempsey and Hb
Kansas(6, 12) . Recent studies of recombinant Hb A
D
99K and Hb A N
102A indicated that dimerization properties of
these variants depended on hemoglobin concentration: the higher the
concentration, the lower the tetramer-dimer dissociation constant of
hemoglobin(26, 38) . Although polymerization
properties of dimeric forms of Hb S are not known, the effect of
dimerization on polymerization of Hb S-Kansas and Hb S-Kempsey should
be negligible, since hemoglobin concentrations used for polymerization
in both high and low phosphate buffers (50-100 µM and 1.5-6 mM, respectively) are too high to favor
dimer formation. Polymerization properties of oxy- or CO Hb S-Kansas
and deoxy-Hb S-Kempsey in low and high phosphate buffers can therefore
be attributed to the quaternary and tertiary structural differences
from deoxy-Hb S rather than dimerization differences. It is interesting
to note that deoxy-Hb Kansas crystallizes like deoxy-Hb A and that
exposure of these crystals to CO results in formation of two new
crystal forms that are not identical to crystals formed upon
deoxygenation(15) . In contrast, deoxy-Hb A crystals dissolve
upon exposure to CO. These results are similar to those of exposure of
deoxy-Hb S-Kansas polymers to CO. Polymers of CO Hb S-Kansas may also
remain in the T state conformation, which may not favor CO-induced
depolymerization. Structural analyses of these Hb S variants are now
required to further our understanding of the relationship between
quaternary structure, hemoglobin function, and polymerization of Hb S.
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