From the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
Received for publication, February 27, 2001, and in revised form, March 17, 2001
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ABSTRACT |
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A cluster of amino acid residues located in the
AB-GH region of the Sickle cell anemia is a consequence of a point mutation
(Glu-6 The polymerization process is triggered by lateral interactions
of the donor Val-6 We have chosen three sites, namely We have adopted a chemo-enzymatic strategy for the construction of
CM-52 and DE-52 were purchased from Whatman. V8 protease was
obtained from Pierce. The chemicals used in peptide synthesis were from Novabiochem. All other chemicals and reagents were of analytical purity and procured from standard commercial sources. The
hemoglobins from sickle cell patients and marmosets were purified form
respective red cell hemolysates by established procedures employing
successive anion (DE-52) and cation (CM-52) exchange chromatography.
The hydroxymercuribenzoate Preparation of Synthesis of Construction of Mutant Reconstitution of Spectroscopic Studies--
The spectra were recorded on a Lambda
Bio20 spectrophotometer (PerkinElmer Life Sciences). The UV
region-derivative spectra were recorded in the first derivative mode of
the spectrophotometer. The hemoglobin concentration used for the
spectral measurements was ~50 µM on heme basis.
Circular dichroism spectra were recorded on a J710 spectropolarimeter
(Jasco) fitted with a Peltier-type constant temperature cell
holder (PTC-348W). The calibration of the equipment was done with
(+)-10-camphorsulfonic acid.
Analytical Procedures--
The synthetic peptides were purified
by RPHPLC on an aquapore RP300 column (250 × 7 mm) using a
4-72% linear gradient of solvent B (acetonitrile containing 0.1%
trifluoroacetic acid) in 130 min at a flow rate of 2 ml/min. Globin
chains from respective hemoglobins were separated on a similar column
of a smaller dimension (250 × 4.6 mm) under identical conditions
but at a flow rate of 0.7 ml/min.
Analytical anion-exchange chromatography of HbS constructs was
performed by FPLC (AKTA, Amersham Pharmacia Biotech) on a Mono Q
HR5/5 column. The respective protein samples were prepared in Tris
acetate buffer (50 mM, pH 8.5) and loaded on the column
that was pre-equilibrated with the same buffer. The samples were
chromatographed using a linear pH gradient of 50 mM Tris
acetate buffer, pH 8.5 to 7.0 over 20 min with a 1 ml/min flow rate.
The elution profile was monitored at 540 nm.
Electrospray mass spectrometric analysis was carried out on a VG
Platform (Fisons) mass spectrometer. The instrument was usually calibrated with standard myoglobin solution. Appropriate amounts of
globin chains isolated from each HbS sample by RPHPLC were taken in
50% acetonitrile containing 1% formic acid and analyzed under the
positive ion mode. The spectra of globins produced a series of
protonated species typically ranging from 13 to 22 positive charges.
The average molecular mass of each globin from the respective spectra
was obtained by using the software provided by the manufacturer.
Oxygen Affinity Measurements--
The oxygen affinity of
hemoglobins was measured by a Hemox-Analyzer (TCS Medical Products, New
Hope, PA) at 29 °C in 0.1 M sodium phosphate
buffer, pH 7.4. The hemoglobin concentration was ~0.1 mM
based on heme. The P50 value (partial oxygen
pressure at 50% saturation) and the Hill coefficients
(nmax), a measure of cooperativity, were
determined from each dissociation curve.
Measurement of Gelation Concentration, Csat--
The
gelation concentration of HbS constructs was determined by the
dextran-Csat method of
Bookchin et al. (30). This method allows
measurement of Csat under near-physiological
conditions and at a much lower concentration of HbS (about 5-fold or
less) than that required in standard Csat assays
but essentially provides the same information. Briefly, a suitable
aliquot of a concentrated solution of hemoglobin in potassium phosphate
buffer (0.05 M, pH 7.5) was taken in a 1.5-ml
microcentrifuge tube. A concentrated dextran (70 KDa) solution
prepared in the same buffer was added to the aliqout and mixed
well. This mixture was overlaid with 0.5 ml of mineral oil, chilled on
an ice bath, and deoxygenated with an anaerobically prepared dithionite
solution through an airtight Hamilton syringe. The final concentrations
of dextran and dithionite in the mixture were 120 mg/ml and 0.05 M, respectively. The above deoxygenated sample was allowed
to polymerize at 37 °C for 30 min, after which the gel under the oil
layer was disrupted with the plunger of a Hamilton syringe. The tube
was centrifuged at room temperature at 14,000 rpm for 30 min. The above
process of gel disruption and centrifugation was repeated twice,
subsequent to which the oil layer was aspirated, and suitable aliquots
from the supernatant were taken for estimation of
Csat by Drabkin's reagent.
Kinetics of Polymerization--
The delay time kinetics of
deoxyhemoglobin were studied in 1.8 M phosphate buffer (pH
7.25) as described by Adachi and Asakura (31, 32) using a Cary 400 spectrophotometer equipped with a Peltier temperature controller. The
polymerization of deoxyhemoglobin samples was initiated by a
temperature jump from 4 to 30 °C within 10 s, and the progress
of the reaction was followed by monitoring turbidity changes at 700 nm.
The delay time was calculated from the kinetic traces.
Assembly and Chemical Characterization of HbS Twin Peaks
(
The semisynthetic Twin Peaks
The purified HbS Twin Peaks was analyzed by reverse-phase HPLC to
establish the stoichiometry and chemical integrity of the globin
chains. The Structural Characterization of HbS Twin Peaks--
The CD spectrum
(Fig. 2) in the soret region for HbS Twin
Peaks was similar to that of the HbS, suggesting that interactions of
heme with the relevant aromatic residues in the mutant protein are
maintained and that the Leu-113
UV spectroscopy was employed to further probe the quaternary structural
status of HbS Twin Peaks. The ligand-dependent fine spectral changes around 290 nm are considered as diagnostic of the
quaternary structure of the Hb molecule (33-35). The first derivative
UV spectra of oxy (liganded) and deoxy (unliganded) forms of HbS Twin
peaks and native HbS were compared to assess the presence of gross
quaternary structural changes, if any, in HbS Twin Peaks (Fig.
3). Both HbS and HbS Twin Peaks displayed fine structural characteristics with a peak at 289 nm and a double minimum at 285 and 293 nm, respectively. In both Hbs, the magnitude of
the double minimum was reduced to about half upon deoxygenation. These
results suggest that quaternary structural features of native HbS are
preserved in HbS Twin Peaks.
Oxygen Affinity of HbS Twin Peaks--
The functional aspect of
HbS Twin Peaks was assessed by measuring the oxygen affinity at pH 7.4 in 0.1 M sodium phosphate buffer at 29 °C using a Hemox
Analyzer. P50 of HbS Twin Peaks was found to be
8, against 8.5 for a control sample of native HbS. The Hill coefficient
(nmax) value for HbS Twin Peaks (2.4) was
comparable with that of native HbS (2.5). Thus the mutant Hb exhibited
normal oxygen binding and cooperativity, suggesting that the
Polymer Solubility of HbS Twin Peaks--
The gelation
concentration (Csat) of HbS Twin Peaks was
measured in the presence of high concentrations of dextran developed by
Bookchin et al. (30) and as described under "Materials and Methods." We carried out the polymerization of deoxygenated
tetramers, HbS Twin peaks and native HbS, under identical conditions
and subsequently measured the concentration of the respective
hemoglobins in the supernatants to obtain their polymer solubility
(Fig. 4). The native HbS yielded a
Csat of 29-31 mg/ml in the initial
concentration range of 50-80 mg/ml HbS. In contrast, the
Csat values for HbS Twin Peaks at the initial
concentrations of 70 and 80 mg/ml were 53 and 55 mg/ml, respectively.
Thus the polymer solubility of HbS Twin Peaks was considerably higher
(about 1.8-fold) as compared with that of HbS. To further confirm that
the observed Csat was specific to HbS Twin
Peaks, we constructed an HbA counterpart of Twin Peaks. This Hb was
assembled from the Twin Peaks Kinetics of Polymerization--
To further authenticate the
participation of the Assembly of HbS Tetramers from the
The role of the
The desired substitutions in
The separation of chains by reverse-phase HPLC showed the presence of a
1:1 stoichiometry of Functional Characterization--
The oxygen
affinities of all of the HbS mutants (Table
II) were in the normal range
(P50 = 7.0-7.5), albeit slightly higher than that of native HbS (P50 = 8.5). Hill
coefficients of all of the mutants were also normal, indicating
preservation of HbS-like quaternary structure. HbS I
( Polymer Solubility of the Mutants--
The
Csat values of all HbS constructs were obtained
in the presence of dextran under similar conditions as those used for HbS Twin Peaks. The Csat values of the single as
well as double mutants were significantly higher than the
Csat of native HbS, albeit to different extents
(Table II). The innate Csat of HbS (30 mg/ml)
increased by nearly 30% (39 mg/ml) and 49% (44 mg/ml) with point
mutations of A 14-strand model of the HbS fiber composed of seven Wishner-Love
double strands and stabilized by intra- and inter-double strand
interactions is now well accepted, although ambiguities persist
regarding the arrangement of the crystal double strands within the
fiber (9). Whereas intra-double strand contacts of the fiber are
similar to those found in the single crystals of HbS, inter-double
strand contacts are somewhat different. This is due to the fact that
double strands in the crystal are straight, whereas packing
requirements entail twisted strands. The map of contact sites displayed
by different fiber models varies considerably depending on the distance
of separation used between Wishner-Love double strands to achieve a
reasonable packing (6-8). Most of the interactions within the double
strand consist of residues from the We chose to replace the Leu residue at the The participation of the The evaluation of the individual strength of contact sites in
quantitative terms has been a current subject of intense focus. In
recent studies, site-directed mutagenesis has been employed to generate
perturbation in both We are now in a position to compare the individual strength of -chain are shown in intra-double strand axial
interactions of the hemoglobin S (HbS) polymer. However,
Leu-113 (GH1) located in the periphery is not implicated in
any interactions by either crystal structure or models of the fiber,
and its role in HbS polymerization has not been explored by solution
experiments. We have constructed HbS Twin Peaks (
Glu-6
Val,
Leu-113
His) to ascertain the hitherto unknown role of the
113
site in the polymerization process. The structural and functional
behavior of HbS Twin Peaks was comparable with HbS. HbS Twin Peaks
polymerized with a slower rate compared with HbS, and its polymer
solubility (Csat) was found to be about
1.8-fold higher than HbS. To further authenticate the participation of
the
113 site in the polymerization process as well as to evaluate
its relative inhibitory strength, we constructed HbS tetramers in which
the
113 mutation was coupled individually with two established fiber
contact sites (
16 and
23) located in the AB region of the
-chain: HbS(
Lys-16
Gln,
Leu-113
His), HbS(
Glu-23
Gln,
Leu-113
His). The single mutants at
16/
23 sites were also
engineered as controls. The Csat values of the
HbS point mutants involving sites
16 or
23 were higher than HbS
but markedly lower as compared with HbS Twin Peaks. In contrast,
Csat values of both double mutants were
comparable with or higher than that of HbS Twin Peaks. The
demonstration of the inhibitory effect of
113 mutation alone or in
combination with other sites, in quantitative terms, unequivocally
establishes a role for this site in HbS gelation. These results have
implications for development of a more accurate model of the fiber that
could serve as a blueprint for therapeutic intervention.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Val) at the sixth position in the
-chain of the hemoglobin molecule (1). The replacement of a charged residue with a hydrophobic one on the surface of the protein drastically reduces the solubility of
the deoxygenated sickle hemoglobin
(HbS)1, leading to its
polymerization into long helical fibers that are responsible for the
clinical manifestations of sickle cell disease. Electron microscopy and
crystallographic studies have suggested that both the deoxy HbS crystal
and fiber are constructed from the same "Wishner-Love"
double strands (2-5). The model of the fiber structure derived from
these analyses consists of seven Wishner-Love double strands
(6-9).
of a tetramer of one strand of the double strand
with the acceptor pocket at the EF corner (elicited mainly by
Phe-85
and
Leu-88) of the
-chain of an adjacent molecule present in the
second strand of the double strand. Subsequent intra-double strand and
inter-double strand interactions involving several amino acid residues
from both
- and
-chains contribute to the stabilization of the
fiber structure. The polymerization-impairing or -enhancing propensity
of mutant hemoglobins, in a binary mixture of mutant hemoglobins and
HbS, has facilitated the mapping of several contact residues of the HbS
polymer (10-12). The list of contact sites has been expanded by
subsequent studies involving chemical modifications of HbS (13, 14) and
site-directed mutagenesis (15-19). However, the identities of all the
fiber contacts that are predicted by model studies have not yet been
tested in solution experiments. The fiber models themselves are not
perfect, because they include several polymerization-insensitive sites,
exclude polymerization-sensitive sites, and possibly underestimate or overestimate the number of contact residues (6-9). Thus, it is prudent
to identify and authenticate all of the participating residues by
solution polymerization studies. More importantly, knowledge of the
inhibitory strength of each contact site and the combinatorial effects
of two or more contact sites (interaction linkage) is imperative for
designing effective antisickling agents or vectors for gene therapy
that could bring about optimum inhibition of fiber formation needed for
clinical amelioration of sickling. The relative strength of contact
sites, as well as their "interaction linkage" relationships in
terms of synergy and/or additivity, is largely unknown and is beginning
to be addressed only now by solution polymerization studies (20).
16,
23, and
113, for
further delineation of their contributions to the polymerization of
HbS. Whereas sites
Lys-16 and
Glu-23, located in the AB region, are established contact points in the HbS polymer, the participation of
Leu-113 (GH1) in the polymerization process is unknown. The
113
site is of interest because of its unique structural location. First,
113 is in sequence contiguity with a cluster of GH corner residues,
114,
115, and
116, which are established or implicated intra-double strand contact sites of the HbS fiber (7, 11, 16). Second,
the three-dimensional structure of the hemoglobin brings the AB region
of the
-chain in close proximity to the GH corner. The involvement
of several residues of the AB corner (
16,
20,
23) in HbS
polymerization has been deduced from crystal structures and also been
validated in solution experiments (21-24). However,
Leu-113, which
is located in the periphery of several physiologically relevant axial
contacts in the AB-GH region, is not implicated in any contacts by
crystal or model studies. Interestingly, a natural Hb variant at this
site, Hb Twin Peaks (
Leu-113
His), is reported (25), but the
functional properties of Hb Twin Peaks or its participation in deoxy
HbS polymerization has not been examined. Here, we constructed HbS Twin
Peaks (
Leu-113
His,
Glu-6
Val) to establish the role of the
113 site in HbS fiber generation. Furthermore, we have combined the
113 mutation at the GH corner with mutations of contact sites
involving residues
16 and
23 of the AB region to see whether the
inhibitory sites in the "contact-rich" AB-GH domain have additive
or synergistic influence on the Val-6
-dependent polymerization of HbS.
-globin mutants. The propensity of V8 protease to catalyze the
ligation of complementary fragments,
1-30 and
31-141, to generate a full-length
-globin (
1-141) has been utilized for this purpose (26). Appropriate synthetic
1-30 segments were employed to incorporate desired mutations at sites
16 and
23. The
Hb Twin Peaks mutation was introduced through the
31-141 segment of
the marmoset (Callithrix argentata)
-chain, which contains a single amino acid substitution,
Leu-113
His, with respect to the human
31-141 segment (27). HbS tetramers were assembled from
s-chain and respective single and double
mutant
-chains. The structural/conformational, functional, and
polymerization behavior of mutants was studied with a view to examining
the hitherto unknown role of the
113 (GH1) site in the HbS gelation
process as well as to quantifying its inhibitory strength relative to
selected AB region
-chain contact residues.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- and
-chains were prepared as
described previously (28). The chains were freed from heme by
acid-acetone precipitation.
1-30 and
31-141 Segments of Human or
Marmoset
-Globin--
The complementary segments of
-globin
needed for the semisynthesis of mutant chains were prepared by V8
protease digestion (26). The respective globins were dissolved in 10 mM ammonium acetate buffer (pH 4) at a concentration of 0.5 mg/ml and digested at 37 °C with V8 protease (1:200, w/w) for 3 h. The completion of digestion was ascertained by RPHPLC. The
complementary segments,
1-30 and
31-141, from the respective
digestion mixtures were isolated in pure form by gel permeation
chromatography on a Sephadex G50 column. The column was equilibrated
and run in 0.1% trifluoroacetic acid. The lyophilized sample of
the digest was dissolved in the above solvent and loaded on to the
column. The column was run at a flow rate of 45 ml/h, and the elution
profile was monitored at 280 nm. The individual chromatographic profile
of the
-globin digest (human or marmoset) showed only two peaks,
31-141 and
1-30, as expected from a single cleavage at
the 30-31 peptide bond. The peak fractions from each digest were
pooled separately and lyophilized.
1-30 Analogs:
(Lys-16
Gln) and
(Glu-23
Gln)--
Peptides were synthesized by a standard solid
phase N-(9-fluorenyl)methoxycarbonyl (Fmoc) strategy
on a peptide synthesizer (model 90, Advanced Chemtech). For this, Wang
resin pre-loaded with N-
-Fmoc-Glu was used as the
starting material. The stepwise coupling of Fmoc amino acids was
performed with the
N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole activation procedure. The coupling of each step was monitored by a
Kaiser test (29), and wherever necessary, a double coupling was used to
increase the yield. On completion of the synthesis, the N-terminal Fmoc
group was removed using piperidine. The peptides were cleaved from the
resin and deprotected with an appropriate volume of a mixture
containing trifluoroacetic acid, ethanedithiol, phenol, thioanisole,
and water (80:5:5:5:5, v/v). The resin was removed by filtration, and
the crude cleaved peptides were precipitated using cold diethyl ether.
The peptides were purified by RPHPLC, and their chemical identity was
checked by electrospray mass spectrometry. The experimental masses of
the peptides were in agreement with their theoretical masses:
(Lys-16
Gln), observed mass of 3040.73 Da (theoretical mass,
3040.73);
(Glu-23
Gln), observed mass of 3040.50 (theoretical
mass, 3039.39).
-Globins--
V8 protease-mediated
semisynthesis of
-globin was carried out at 4 °C in 50 mM ammonium acetate buffer (pH 6) containing 30%
1-propanol. For this, the lyophilized samples of natural or synthetic
analogs of
1-30 and human or marmoset
31-141 were individually
prepared in water. Suitable volumes of the complementary fragments were
mixed to obtain a 1:1 molar ratio and lyophilized. The lyophilized
material (150 mg) was dissolved in 6 ml of 84 mM ammonium
acetate buffer (pH 6). To this solution, 3 ml of 1-propanol was added.
The mixture was cooled on ice, subsequent to which 1 ml of V8
protease solution (1.5 mg/ml prepared in water) was added. The ligation
reaction mixture was incubated at 4 °C for 24 h. The reaction
was stopped by addition of 2 ml of 5% trifluoroacetic acid and
lyophilized. The semisynthetic
-globin was isolated from the mixture
by CM52-urea chromatography, extensively dialyzed against 0.1%
trifluoroacetic acid, and lyophilized (26). The semisynthetic yield of
the protein varied between 35 and 45%. The identity of the
-globin
constructs were checked by mass spectrometry and tryptic peptide mapping.
-Globin and the
s-Chain into
HbS Tetramers--
The semisynthetic
-globin was reconstituted with
heme and the
s-chain into tetrameric hemoglobin through
the "Alloplex pathway" as described previously (26). The
reconstituted tetramers were purified by CM-52 chromatography. The heme
stoichiometry in purified tetramers was ascertained by 280:540 nm
absorbance ratios. The A280:A540 ratio for
native HbS was 2.54. For reconstituted tetramers, this ratio varied
between 2.49 and 2.55. The tetramers were checked for the correct
stoichiometry of chains by RPHPLC. The
- and
-chains from each Hb
were isolated and subjected to electrospray mass spectrometry and
tryptic peptide mapping to ensure that the reconstitution procedure did
not alter the chemical integrity of the chains.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Leu-113
His,
Glu-6
Val)--
The construction of the
-chain of Hb Twin Peaks (
Leu-113
His) through the
-globin
semisynthetic strategy involves the stitching of human
1-30 with an
31-141 segment containing the Twin Peaks mutation. The two peaks
isolated from G-50 chromatography of the V8 protease digest of the
marmoset
-globin were subjected to electrospray mass spectrometry.
The reported sequence of marmoset
-globin (27) contains amino acid
substitutions at four sites compared with the human (T8S, A19S, E23D,
and L113H). The experimental masses obtained for the two peaks, 3029.23 and 12126.05 Da, respectively, were in agreement with the calculated
masses of the complementary fragments of marmoset
-globin (
1-30,
3028.32 Da;
31-141, 12127.99 Da).
-globin was obtained by ligation of
human
1-30 and marmoset
31-141 fragments through the V8 protease-catalyzed reaction as described under "Materials and Methods." The purified material was reconstituted with the
s-chain and heme to obtain the tetramer. HbS Twin Peaks
was isolated in pure form CM-52 chromatography. The purity of the
protein was further established by FPLC. Under identical
chromatographic conditions, HbS Twin Peaks eluted slightly earlier than
the native HbS from the Mono Q anion-exchange column (Fig.
1). This elution behavior of HbS Twin
Peaks is consistent with the replacement of Leu by His at the
113
site in HbS.
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Fig. 1.
FPLC of HbS Twin Peaks
( Leu-113
His). A sample of the mutant
HbS was chromatographed on a Mono Q (HR5/5) column using the AKTA
system of Amersham Pharmacia Biotech. The protein sample was prepared
in 50 mM Tris acetate buffer (pH 8.5) and loaded on to the
column. The elution of Hb was carried out using a linear pH gradient of
the above buffer (pH 8.5-7.0) over a period of 20 min at a
flow rate of 1 ml/min. The elution profile was monitored at 540 nm.
Broken line, HbS; continuous line, HbS Twin
Peaks. The inset shows the RPHPLC separation of the globin
chains of HbS Twin Peaks and native HbS. The purified samples of each
hemoglobin were analyzed on an RP300 column (250 × 4.6 mm) using
a 4-72% gradient of acetonitrile containing 0.1% trifluoroacetic
acid in 130 min at a flow rate of 0.7 ml/min.
- and
-chains of HbS Twin Peaks were separated on a
C8 column (RP300) using an acetonitrile- trifluoroacetic acid-water
solvent system and compared with native HbS. The chromatographic profile showed identical retention times for
s-chains
from both samples and indicated correct stoichiometry of the
- and
-chains in HbS Twin Peaks (Fig. 1, inset). Interestingly, the order of elution of chains of HbS Twin Peaks was reversed as
compared with natural HbS. The
-chain of HbS Twin Peaks eluted earlier than the
s-chain, suggesting that Leu to His
substitution exerted considerable influence on the chromatographic
behavior of the
-chain. To rule out the possibility that the above
elution behavior was a consequence of chemical modifications
during semisynthesis or tetramer assembly,
- and
s-chains of HbS Twin Peaks were isolated by
reverse-phase HPLC and subjected to electrospray mass spectrometry. The
molecular mass of the isolated
-chain (15150.03 Da) obtained by
electrospray mass spectrometry agreed very well with the
calculated value of 15150.36 Da for the
-chain of Hb Twin Peaks.
Likewise, the experimental mass of the
-chain (15837.99 Da) was in
accord with the calculated mass of the
s-chain (15837.25 Da). Taken together, the results unambiguously established the chemical
integrity of HbS Twin Peaks.
His mutation does not have a
deleterious effect on the folding of the heme pocket.
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Fig. 2.
Soret region CD spectra of HbS Twin
Peaks. The spectra were recorded at 10 °C in 10 mM
potassium phosphate buffer (pH 7.4) using a 1-mm path length cell. The
hemoglobin concentration was 0.64 mg/ml. Solid line, HbS;
broken line, HbS Twin Peaks. MRE, mean residue
ellipticity.
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Fig. 3.
First derivative UV spectra of liganded and
unliganded HbS Twin Peaks. The spectra of oxy and deoxy forms of
the protein were recorded at 25 °C in the first derivative mode of
the spectrophotometer. The hemoglobin concentration used was about 50 µM (on heme basis) in 10 mM potassium
phosphate buffer (pH 7.4) containing 0.1 M chloride.
Solid lines and broken lines represent the
spectra of deoxy and oxy forms of the hemoglobin, respectively.
1, native HbS; 2, HbS Twin Peaks.
Leu-113
His substitution did not cause any significant perturbation of the quaternary structure of the protein. This interpretation is consistent with the above spectroscopic studies of
HbS Twin Peaks.
-chain and
A-chain
(Glu-6
) through the same procedure as that employed for the assembly
of HbS Twin Peaks. Under similar assay conditions, HbA or HbA Twin
Peaks did not polymerize when tested at an initial concentration of 70 mg/ml, suggesting that the Csat values of HbS
Twin Peaks reflect the true potential of the
113 site to inhibit
s-dependent polymerization of HbS.
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Fig. 4.
Polymer solubility of HbS Twin Peaks.
The measurements were performed in the presence of dextran in 50 mM potassium phosphate buffer (pH 7.5) and at 37 °C as
described under "Materials and Methods." The hemoglobin
concentration in the supernatant (Csat) was
determined by Drabkin's method. , HbS;
, HbS Twin Peaks.
113 site in the HbS gelation process, kinetics
of polymerization of HbS Twin Peaks, HbS, HbA Twin Peaks, and HbA were
studied in 1.8 M phosphate buffer (Fig.
5). Two concentrations (0.5 and 0.78 mg/ml) of HbS Twin Peaks were tested. HbS Twin Peaks polymerized with a
longer delay time as compared with HbS at both concentrations. The
delay time for HbS Twin Peaks at an initial concentration of 0.5 mg/ml
was about 18 min compared with 12 min for HbS (Fig. 5a). At
a higher concentration (0.78 mg/ml), the delay time of HbS Twin Peaks
polymerization decreased to 8 min, which was still higher compared with
HbS (6 min) at the same concentration (Fig. 5b). In
contrast, HbA Twin Peaks and HbA did not polymerize at an initial Hb
concentration of 1 mg/ml (Fig. 5a).
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Fig. 5.
Kinetics of polymerization of HbS Twin
Peaks. Polymerization of hemoglobins was carried out in 1.8 M potassium phosphate buffer, pH 7.25. The polymerization
of the deoxy form of the protein was initiated by a temperature jump
from 4 to 30 °C within 10 s, and the progress of the reaction
was continuously monitored at 700 nm. a, x x, HbS (0.5 mg/ml);
- - -
, HbS Twin Peaks (0.5 mg/ml);
, HbA (1 mg/ml); x- - -x, HbA Twin Peaks (1 mg/ml). b, x
x, HbS
(0.78 mg/ml);
- - -
, HbS Twin Peaks (0.78 mg/ml).
s-Chain and
Mutant
-Chains (Lys-16
Gln; Glu-23
Gln; Lys-16
Gln,
Leu-113
His; Glu-23
Gln, Leu-113
His)--
To see whether the
inhibitory potential of the
113 site in the deoxy-HbS fiber could
exert a synergistic or additive effect when combined with surrounding
intermolecular contact residues of the AB region of the
-chain, we
generated HbS tetramers with double mutant
-chains that contained
amino acid substitutions at
16 (Lys
Gln) or
23 (Glu
Gln)
positions along with the Twin Peaks mutation,
113 (Leu
His). HbS
mutants with single amino acid replacements at the above selected AB
region sites (
16 and
23) were also constructed to evaluate the
quantitative strength of each of these sites vis à vis
their relative contributions to the polymerization reaction and the
extent of interaction linkage, if any, with the
113 site located at
the GH corner.
16 site in the polymerization of HbS has been
established by solution studies (21) with Hb variants, I (Lys-16
Glu). In our studies, we incorporated Gln rather than Glu
in the place of Lys at the
16 position, considering the
susceptibility of Glu residues to V8 protease hydrolysis during the
ligation reaction. The secondary hydrolysis at Glu-16 during the
proteo-synthesis of the 30-31 peptide bond between
1-30
(Lys-16
Glu) and
31-141 fragments would lower the yield of the
semisynthetic
-globin. Moreover, the presence of Gln is unlikely to
affect the overall structure and function of the tetramer, because Hb
Beijing, which contains a conservative residue (Lys-16
Asn) at this
site, exhibits normal stability and does not cause any hematological
complications in the heterozygotes (36). The Glu-23
Gln substitution
present in the
-chain of Hb Memphis was retained, because it was
compatible with the
-globin semisynthetic strategy.
-chains were introduced through the
semisynthetic strategy described under "Materials and Methods" and
as applied to the construction of the
-chain of Twin Peaks. The HbS
tetramers reconstituted from the respective semisynthetic
-chains
and
s-chain were purified by cation exchange CM-52
chromatography. The purity of the HbS constructs were further tested by
anion-exchange chromatography on a Mono Q column using FPLC (data not
shown). The FPLC profile displayed a single peak in all cases,
establishing the homogeneity of each preparation. The elution pattern
of the proteins was in accord with the charge difference created
because of amino acid replacement(s).
- and
s-chains in all the HbS
constructs. The order of elution of chains from HbS carrying single
mutations (
16 or
23) was similar to HbS;
-chains carrying
either point mutation eluted later than the
s-chain,
suggesting that the Lys-16
Gln or Glu-23
Gln substitution does not
cause a significant alteration of the behavior of
-chains on
reverse-phase columns. In contrast, the chromatographic pattern of
double mutants exhibited a reversal of elution order that was reminiscent of HbS Twin Peaks. In both HbS double mutants
(Lys-16
Gln, Leu-113
His or Glu-23
Gln, Leu-113
His), the
-chains eluted before the
s-chains. Thus the presence
of His at the 113 site alone seems to dictate the behavior of
-chains on reverse-phase columns. The experimental electrospray
masses of isolated chains from each HbS sample were in good agreement
with their respective theoretical masses (Table
I).
Electrospray mass of isolated globin chains from mutant HbS constructs
Lys-16
Glu) has been studied previously and found to exhibit the
usual functional properties (21). The normal oxygen binding behavior of
mutants, HbS(
K16Q) or HbS(
K16Q,L113H), suggests that abrogation
of the positive charge or reversal of the charge polarity at the
16
site does not appreciably affect the oxygen binding behavior of the
Hb.
Influence of -chain mutations on the oxygen affinity and polymer
solubility of HbS constructs
16 and
23 sites, respectively. This increase in
Csat for the above mutants was markedly less
than that observed for HbS Twin Peaks; Csat of
HbS Twin Peaks (53 mg/ml) was about 80% higher relative to native HbS.
The Csat of the double mutant comprising sites
16 and
113 (60 mg/ml) was twice that of HbS and significantly
higher than that of the mutation of the
113 site alone. In contrast,
the double mutant consisting of
23 and
113 sites produced a
Csat (55 mg/ml) that was very similar to that of
HbS Twin Peaks.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chain, and those involved
between the double strands emanate predominantly from the
-chains.
In this connection, it is noteworthy that the discrepancies between the
molecular modeling and solution experiments largely relate to the
contact sites involving
-chain residues. Therefore, further
delineation of interactions in the HbS fiber, particularly of those
surface residues of the
-chain that are not implicated in contacts
by fiber models or solution studies, appears to be necessary, because
it is likely to provide information on the inter-double strand contacts
specific to the fiber. The major aim of the present study was to
explore the role of one such residue,
Leu-113 (GH1), by solution
polymerization experiments.
113 site by His because
this point mutation is present in the
31-141 segment of the
marmoset
-chain and was compatible with our semisynthetic strategy.
Because this mutation (
Leu-113
His) is also naturally present in
Hb Twin Peaks (25), it would be expected to have no effect on
hemoglobin stability and function. Indeed, the environment of heme and
the
1
2 interface in HbS Twin Peaks was
found to be similar to that in HbS, indicating that HbS Twin Peaks
assumes an HbS-like quaternary structure. The
P50 and Hill coefficient of HbS Twin Peaks were
also similar to those of HbS. Thus, the inhibition of polymerization
seen with HbS Twin Peaks, both in high phosphate buffer and in the
presence of dextran, reflects the true inhibitory potential of the
113 residue.
113 site in the polymerization of HbS was
further corroborated by the polymer solubility data of double mutants
involving sites
113 and
23 or
16. The dextran Csat of point mutants at
16 or
23 (39 and
44 mg/ml, respectively) was much lower than the
Csat of HbS Twin Peaks (
113; 53 mg/ml), suggesting that the above AB region
-chain residues are relatively weak fiber contacts as compared with the
113 site. In contrast, the
Csat of the double mutants comprising sites
16 and
113 (60 mg/ml) or
23 and
113 (55 mg/ml) was higher
than or comparable with that of HbS Twin Peaks. Thus, the inhibitory
effect of
113 was additive with
16 but not with
23. This is in
accord with the crystal structure of deoxy HbS, which shows axial
interactions of
Lys-16 with
Pro-114 and
Ala-115 residues
located at the GH corner (37). The interactions of Lys-16 are likely to
be perturbed by Gln mutation. Indirect effects of Lys-16 replacements on the
113 mutation are also conceivable in view of the sequence proximity of
113 to the above GH corner sites. In contrast,
interactions of the
Glu-23 site do not involve the GH corner residues.
- and
-chain contact points with sites in
question mutated individually or in combination. The
-chain contacts
have also been probed through construction of interspecies hybrids and
semisynthetic chimeric hemoglobins (38-40). Nonetheless, these
interspecies hybrids contain a large number of mutations, at contact
sites or otherwise, compared with the human
-chain, that interfere
with the unambiguous assignment of the role of each residue vis
à vis the synergistic and additive nature of their
interactions. Given the large number of participating residues and the
complexity of the polymerization process, precise delineation of the
individual or combinatorial strength of sites could be ideally achieved
by systematic variation of surface residues through DNA-based
site-directed mutagenesis (16, 18, 19, 41). The semisynthetic method,
although restricted in scope, could also be effectively applied in
appropriate situations as demonstrated in the present study.
113,
16, and
23 sites with other sites and also the interaction linkage of some fiber contacts in quantitative terms (Table
III), because the
Csat values of respective HbS mutants in all
these cases have been determined in the presence of dextran under
similar conditions, as described by Bookchin et al. (30).
The inhibitory strength of sites varies as follows:
95 >
88 >
113 ~
85 >
23 >
16. The
inhibitory strength of
113 is similar to that of
85 but
significantly lower than that of
95 or
88. The other two sites,
16 and
23, are rather weak. Among the above sites,
95 and
85 residues are implicated in inter-double strand contacts, whereas
88 is involved in crucial primary interactions with Val-6
. We
envision the
113 site as a potential inter-double strand contact point of the fiber, because it is not implicated in interactions by
crystal and model studies. The remaining two sites (
16 and
23)
with lowest inhibitory propensity are intra-double strand contacts.
Against this scenario, it appears that the inhibitory strength of
fiber-specific inter-double strand contact sites is likely to be
greater than those involved in intra-double strand contacts. It is
instructive to note that multiple mutations of the inter-double strand
contacts in the pig
-chain relative to human were posited as prime
sites that contributed to the total abrogation of polymerization in
interspecies hybrid HbS composed of pig
-chain and human
s-chain (38). The influence of simultaneous perturbation
of two or more contact sites has been analyzed in quantitative terms only for a few combinations of sites. Whereas a non-additive inhibitory effect was seen for
88/
95 residues, the potentiation of
polymerization by
6/
75/
121 appeared to be additive. Our data
show that the inhibitory effect of the
113 site was non-additive
with
23 but additive with
16. So far, a synergistic influence
between two contact sites has not been demonstrated in any definitive
fashion. The knowledge of the individual strength of all of the fiber
contact sites in quantitative terms appears to be an immediate need,
because it would help discriminate weak and strong sites, thereby
restricting the number of combinations that could be assessed as
potential therapeutic targets.
Quantitative strength of contact sites of HbS fiber measured by the
dextran Csat method
16,
23,
113,
16/113, and
23/113 (present study) were obtained by measuring the Hb
concentration in the supernatant by Drabkin's procedure, concentration
estimates in the case of other sites (Csat values
represented in parentheses) were made by quantitative amino acid
analysis. The apparent difference between Csat
values of HbS (Val-6
) obtained in the present report (30 mg/ml) and
the previous studies (34 mg/ml) is presumably related to the method of
Hb estimation. Nonetheless, this small difference may not have a
bearing on the comparative analyses of Csat data.
In conclusion, we unequivocally established a role for the 113 amino
acid residue in the polymerization of HbS. We not only demonstrated the
participation of this site in the gelation process but also quantified
its strength of interaction. The
113 amino acid residue is not
postulated in any interactions in the crystal structure of HbS and is
also excluded by the models of the fiber. These considerations lead us
to believe that the
113 site might be involved in fiber-specific
inter-double strand contacts. Further solution copolymerization studies
of HbS Twin Peaks coupled with electron micrographic analysis of the
HbS Twin Peaks fiber should help delineate the stereochemistry of the
113 residue in the polymer and facilitate efforts to develop an
accurate model of the HbS fiber.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. Sandip K. Basu for interest in this work and Dr. Debasisa Mohanty for helpful discussions. The SS blood sample was kindly provided by Dr. V. S. Dani of Indira Gandhi Medical College, Nagpur, India.
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FOOTNOTES |
---|
* This work was supported by a grant from the Department of Science and Technology (to R. P. R.) and grants to the National Institute of Immunology from the Department of Biotechnology, Government of India.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. Fax: 91-11-610-9433;
E-mail: rproy@nii.res.in.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M101788200
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ABBREVIATIONS |
---|
The abbreviations used are: HbS, sickle hemoglobin; HPLC, high pressure liquid chromatography; RPHPLC, reverse phase HPLC; Fmoc, N-(9-fluorenyl)methoxycarbonyl; FPLC, fast protein liquid chromatography.
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