(Received for publication, September 13, 1994; and in revised form, April 14, 1995)
From the
Hemoglobin S (HbS) Hoshida and three substituted forms of HbS
Hoshida (the substituents being on the amide nitrogen of Gln-43())
have been prepared by the amidation of Glu-43(
) of HbS with
ammonia, methylamine, glycine ethyl ester, and galactosamine. The
O
affinity of HbS is increased slightly on amidation of
Glu-43(
). All the four amidated derivatives exhibited nearly the
same oxygen affinity. On the other hand, the influence of amidation on
the solubility exhibits some sensitivity to the chemical nature of the
substituent on the Gln-43(
). The solubility of HbS Hoshida (a case
with no substitution on Gln-43(
)), and the methyl-substituted
derivatives are about 33 and 36% higher than that of HbS. The
solubility of the HbS modified with the glycine ethyl ester or
galactosamine is increased to 41 and 47%, respectively. The first
derivative UV spectra of HbS Hoshida and its methyl derivative reflect
very little perturbations in their
interface as compared with that of HbS, whereas the amidated
derivatives with larger substituents on Gln-43(
) reflected
noticeable differences. Thus, the increase in the solubility and the
oxygen affinity of HbS on the amidation of Glu-43(
) is primarily a
consequence of the loss of the negative charge at 43(
), a residue
proximal to the
interface. The
copolymerization studies of amidated HbS with HbA, and HbS with
amidated HbA demonstrate that cis Glu-43(
) is the
``active'' residue. This assignment is discrepant with the
earlier implication of a trans configuration for this residue in the
polymer (Edelstein, S. J. (1981) J. Mol. Biol. 150,
557-575). However, it is consistent with the solution studies of
Nagel et al. (Nagel, R. L., Bookchin, R. M., Johnson, J.,
Labie, D., Wajcman, H., Isaac-Sodeye, W. A., Honig, G. R., Schiliro,
G., Crookstan, J. H., and Matsutomo, K.(1979) Proc. Nat. Acad. Sci.
U. S. A. 76, 670-672) and McCurdy et al. (McCurdy,
P. R., Lorkin, P. A., Casey, R., Lehmann, H., Uddin, D. E., and
Dickson, L. G.(1974) Am. J. Med. 57, 665-760).
The mutation of Glu to Val that is located at the sixth position
of -chain in sickle cell hemoglobin molecule is responsible for
its polymerization in the deoxy conformation. The assembly of
deoxyhemoglobin S (HbS) (
)tetramers into fiber involves the
cooperative participation of number of residues of
- as well as
-chains of the molecule, in addition to Val-6(
). X-ray
diffraction and electron microscopic studies have revealed that the
structural motif of deoxy HbS fibers as well as deoxy HbS crystals is
the Wishner-Love double strand (Wishner et al., 1975; Padlan
and Love, 1985). The
-chain segment containing Val-6(
),
namely A helix, of one tetramer from one strand of the double strand
interacts laterally with the hydrophobic pocket formed between the E
and F helices (including Phe-85 and Leu-88) of
-chain of another
tetramer located in the second strand of the double strand. However,
only one of the two Val-6(
) residues of the tetramer participates
in the polymerization reaction, and this also appears to be true of
most of the other contact sites. Consequently, the inhibition of
polymerization by a given mutation or chemical modification of an amino
acid residue depends on its position relative to the active
Val-6(
). When the inhibitory mutation/modification is located on
the
- and
-chains of
dimer that provides the
Val-6(
) for the polymerization reaction, it is referred to as a
``cis'' residue. When the mutation/modification is located on
the other
dimer of the HbS, the contact residue position is
referred to as ``trans.'' The information on the cis/trans
configuration of the intermolecular contact residues of HbS derived by
the copolymerization studies has served a unique role in the
cross-correlation of the structural concepts of the fiber from
biophysical studies (Watowich et al., 1989, 1993; Cretegny and
Edelstein, 1993; Nagel et al., 1980) with the solution
studies, particularly in evaluating the current models of deoxy HbS
fiber. However, the cross-correlation between the results of
biophysical studies and the solution studies is not a perfect one. A
correct definition of the contact sites is essential to describe the
quintic structure of HbS polymer unequivocally. The molecular model of
deoxy HbS fiber should serve as the blueprint for the rational design
of either the antisickling agents or antisickling hemoglobins for gene
therapy of sickle cell disease.
Glu-43(), CD2 residue of
-chain, is located near the
interface of Hb. It has been implicated as an inter-double strand
contact residue based on the biophysical studies. Crystallographic
evidence suggests that the side chain of Glu-43(
) interacts with
Ala-53(
) and Gln-54(
) (Wishner et al., 1975; Padlan
and Love, 1985) and with His-45(
) (Cretegny and Edelstein, 1993;
Watowich et al., 1993) in the linear double strand of the
crystal. Based on the model building studies of the fiber structures,
Edelstein(1981) had suggested a trans configuration for the
``active'' Glu-43(
).
The results of our earlier
amidation studies (Seetharam et al., 1983; Acharya and
Seetharam, 1985) have established the unusually high reactivity of the
-carboxyl group of Glu-43(
) for modification with glycine
ethyl ester in the presence of carbodiimide. An increase in the
solubility (C
) of deoxy HbS also occurred as a
consequence of the modification of Glu-43(
). This observation is
consistent with the implication of Glu-43(
) as the contact site by
the biophysical studies discussed above. However, the contributions of
the loss of the negative charge and that of the bulky substituents
placed on the
-carboxyl group of Glu-43(
) could not be
segregated from the results of these studies. Interestingly, the
solubility of the amidated HbS did not exhibit a direct correlation
with the extent of the amidation of Glu-43(
). Increasing the
average modification of Glu-43(
) beyond 1 residue/tetramer had
minimal additional influence on the solubility of HbS. However, the
molecular explanation for this phenomenon could not be elucidated from
the results of these studies (Acharya et al., 1991). An
explanation for this observation is the differential roles of
Glu-43(
) residue placed in the cis and the trans dimers of the HbS
tetramer in the fiber.
In order to obtain a clear insight into the
role of negative charge of -carboxyl group of Glu-43(
) on the
polymerization of deoxy HbS, we have now carried out the amidation of
HbS with ammonia, methylamine (MA), glycine ethyl ester (GEE), and
galactosamine (GA) and have isolated the corresponding derivative of
HbS in a homogeneous form (Fig. 1). The copolymerization studies
have also been now undertaken in an attempt to define the configuration
of the active Glu-43(
) in the HbS tetramer, and the results are
presented here.
Figure 1:
Chemical
structures of the four amidated species of HbS used in the present
studies. A, HbS Hoshida; B, HbS amidated with
methylamine; C, amidated with glycine ethyl ester; D,
amidated with galactosamine. Hb-CO, shown in bold in
the figure, represents the part of -carboxyl group of
Glu-43(
) of HbS that gets converted into amide or substituted
amide.
Preparation of stripped hemoglobins (HbA from normal adult human donors and HbS from SS patients), their purification, and the amidation of Hb have been described previously (Rao and Acharya, 1991, 1994). Characterization of the amidated site by HPLC of the tryptic digest and amino acid sequencing of the modified peptide have been described previously (Rao and Acharya, 1991). Procedures for the oxygen affinity determinations, equilibrium solubility measurements, oxygen affinity method for estimating the solubility of HbS, and kinetics of the polymerization have also been described previously (Rao and Acharya, 1992; Acharya et al., 1991).
Figure 2:
Chromatography of HbS amidated with
galactosamine on CM-52 cellulose column. Sickle hemoglobin (0.5
mM) was treated with
1-ethyl-3-[3`-(dimethylamino)propyl]carbodiimide (10
mM), N-hydroxysulfosuccinimide (2 mM), and
galactosamine (100 mM) in MES buffer, pH 6.0, for 30 min at
room temperature. After this reaction period, the protein was desalted
on a Sephadex G-25 column equilibrated with phosphate-buffered saline,
pH 7.4. The desalted protein sample was further dialyzed against with
10 mM phosphate buffer, pH 6.0, containing 1 mM EDTA.
This amidated protein was chromatographed on a CM-52 cellulose column
(0.9 30 cm). The column was pre-equilibrated with 10 mM phosphate buffer, pH 6.0, containing 1 mM EDTA. A linear
pH gradient generated from 10 mM phosphate buffer, pH 6.0, to
25 mM phosphate buffer, pH 8.5 (250 ml each) was employed for
elution. Both buffers contained 1 mM EDTA. The protein elution
from column was monitored by measuring the absorbance at 540 nm. Inset represents isoelectric focusing of HbA, HbS, HbC, and
disubstituted derivatives of HbS. Agarose IEF gels with a blend of pH
6-8 Resolve ampholytes were used. LaneA, HbA; laneB, HbS; laneC, diGA-HbS; laneD, diGEE-HbS; laneE,
diMA-HbS; laneF, HbC.
Figure 3: Tryptic peptide map of diGA-HbS. The tryptic digest of globin of diGA-HbS (double-purified on CM-52 chromatography) was analyzed by RP-HPLC. The tryptic digest of 2 mg of globin was taken in 1.5 ml of 0.1% trifluoroacetic acid and loaded onto an preparative Aquapore RP-300 column. The peptides were eluted with a gradient of 5-50% acetonitrile over a period of 160 min, each containing 0.1% trifluoroacetic acid. The peptides were eluted at a flow rate of 1 ml/min. 1-ml fractions were collected, and a 50-µl aliquot from each fraction was used to measure the radioactivity. All the peptides were monitored at 230 nm.
Furthermore, as shown
in Fig. 4, the modification of Glu-43() to Gln also
increased the solubility of HbS. The solubility of deoxy HbS Hoshida is
about 25.5 g/dl as compared with a value of 19.0 g/dl for HbS (these
determinations were carried out at 37 °C). The relative increase in
the solubility of HbS on modification of its Glu-43(
) to a Gln
residue is 33% and is comparable to the solubility increase of 36% that
occurs on amidation of HbS with methylamine (Table 1). Thus, from
these results it is concluded that the methyl group placed on the amide
nitrogen of Gln-43(
) of HbS Hoshida influences neither the oxygen
affinity nor the solubility by itself. The increase in the solubility
seen on amidation of HbS with methylamine is a consequence of the loss
of the negative charge of this residue.
Figure 4:
Relation between the oxygen affinity and
hemoglobin concentration of HbS Hoshida and HbS. P was
obtained from the complete oxygenation curves recorded using an Aminco
Hem-O-Scan at 37 °C and pH 6.8 at each concentration
shown.
Figure 5:
Comparison of first derivative UV spectra
of HbS and its disubstituted derivatives. UV spectra (260-340 nm)
of HbS, diMA-HbS, diGEE-HbS, and diGA-HbS in oxy and deoxy conformation
was recorded in 50 mM BisTris, pH 7.4, buffer at 25 °C.
Protein concentration was maintained as 55 µM on heme
basis. HbS, HbS-Hoshida, diMA-HbS, diGEE-HbS, diGA-HbS in the oxy
conformation (-), deoxy HbS(- -), deoxy HbS Hoshida
and deoxy diMA-HbS(- - -), deoxy diGEE-HbS (
), and
deoxy diGA-HbS (-
-).
Figure 6: Kinetics of polymerization of HbS and diGEE-HbS in high phosphate buffer. Polymerization of HbS and diGEE-HbS was carried out in 1.8 M potassium phosphate buffer, pH 7.2. The polymerization of deoxy protein was initiated by the temperature jump from 0 to 30 °C, and the extent of polymerization was monitored by measuring the absorbance of turbidity at 700 nm. The protein concentration was maintained as 0.11 g/dl. Inset represents correlation between the initial hemoglobin concentration and delay time for polymerization in high phosphate buffer.
The solubility of an
equimolar mixture of diGEE-HbS and HbA is approximately 26% higher as
compared to that of an 1:1 mixture of HbS and HbA (Table 2). The
amidation of the cis Glu-43() has a strong inhibitory effect on
polymerization. The equilibrium solubility of 1:1 mixture of HbS and
diGEE-HbA is only about 8% more than that of the solubility of the 1:1
mixture of HbA and HbS. This reflects the fact that the polymerization
inhibitory potential of amidated Glu-43(
) is small when it is on
the
chain, i.e. when it is in the trans
chain. It may be further noted that the solubility of an equimolar
mixture of HbS and diGEE-HbS is very close to that of diGEE-HbS itself
(10% lower).
HbS Hoshida and the three substituted forms of HbS Hoshida
(the substituents being on the amide nitrogen of Gln-43()) exhibit
nearly the same oxygen affinity, which is slightly higher than that of
HbS. Thus, it could be concluded that the increased oxygen affinity of
the amidated derivatives is a consequence of the loss of the negative
charge of Glu-43(
). This conclusion is consistent with the
observation that the oxygen affinities of naturally occurring mutants
of Glu-43(
), Hb-Galvaston (
43 Glu
Ala) (Bowman et
al., 1964), and Hb Hoshida (
43 Glu
Gln) (Iuchi et
al., 1978) are comparable to that of the amidated derivatives of
HbS prepared in the present study. Furthermore, even though the
solubility of deoxy HbS exhibits some degree of sensitivity to the size
and/or the chemical nature of the alkyl groups on the Gln-43(
),
most of the solubility increase of HbS is achieved by converting the
-carboxyl group of Glu-43(
) to an amide.
The UV derivative
spectroscopic investigations of the amidated derivatives have
demonstrated the presence of some amount of conformational
perturbations of the interface
region in the deoxy structure of the protein resulting as a consequence
of amidation of Glu-43(
). Such perturbations are minimal in HbS
Hoshida and its methyl derivative (Fig. 5). On the other hand,
the first derivative UV spectra of the diGEE-HbS and diGA-HbS are
distinct from HbS Hoshida and suggest greater degrees of perturbations
of the oxy-deoxy conformational transition-mediated local
conformational aspects of the deoxy protein. Thus, the size and/or the
chemical nature of the alkyl chain on the Gln-43(
) appears to
dictate the degree to which the oxy-like conformational aspects of the
region could be conserved in the molecule even on deoxygenation.
Thus, the microenvironment of Glu-43() appears to have a degree
of flexibility to accommodate alkyl chains of differing molecular sizes
(one- to six-carbon chain lengths) and still retain the same level of
polymerization inhibitory potential seen with an unsubstituted amide
function at 43(
). This aspect can be considered as the reflection
of the conformational (stereochemical) freedom that exists at the inter
molecular contact regions of the deoxy HbS fiber. This aspect is
reminiscent of the distinct propensities of the side chains of various
amino acid residues placed at Val-6(
) site (Bihoreau et
al., 1992; Adachi et al., 1993) to induce polymerization.
Polymerization potentiating influence is not unique to the side chain
of Val placed at 6(
) position of HbA. Although the original
molecular modeling studies implicated a perfect fit between the side
chain of Val-6(
) and the acceptor pocket (Dean and Schechter,
1978), protein engineering studies have demonstrated that a higher
degree of flexibility is present for the side chain stereochemistry for
the residues placed at 6(
) site. The results of the present study
suggests that such a flexibility is probably a general phenomenon of
most of the intermolecular contact sites.
The copolymerization
studies of the amidated HbS has demonstrated that the polymerization
inhibition seen on amidation of Glu-43() is primarily a cis
effect. This result is consistent with the observation that the lysate
of the double heterozygous patient with HbS and mutant Hb Galvaston
(
43 Glu
Ala) exhibits an equilibrium solubility comparable
to that of the lysate of sickle cell trait (McCurdy et al.,
1974). Mutation of trans Glu-43(
) to Ala thus appears to have very
little influence on the copolymerization. Glu-43(
) is located in
the CD region of the
chain. It should be noted here that Nagel et al.(1979, 1980) have investigated the influence of the
mutation of the amino acid residues of the CD region (47(
)) and
the D helix (58(
) and 59(
)) of trans
-chain through
copolymerization studies. These mutations appear to have very little
influence on the polymerization (Table 3) in the trans position,
even though these are the implicated as contact domains in the current
molecular models of the fiber. The present results on the role of
Glu-43(
) in the cis configuration is discrepant with the results
of the crystallographic and the molecular modeling studies, which have
implicated a trans configuration for the active Glu-43(
) in the
polymerization of HbS (Edelstein, 1981; Watowich et al.,
1989). The Glu-43(
) is the only known amino acid residue of CD
region of the
-chain, the mutation/modification of which has
resulted in the inhibition of the polymerization. Interestingly,
polymerization inhibitory potential of its amidation is primarily from
the cis position. Accordingly, further studies of implicated contact
residues of this CD region in the cis configuration will have to be
delineated to facilitate the resolution of the discrepancy between the
present results and the current molecular models. Furthermore, studies
of chemical mapping of this region through photo affinity functional
labeling coupled with the electronmicrographic studies of the fiber of
HbS Hoshida should help in establishing the stereochemical aspects of
this inter molecular contact region in the HbS polymer, which appears
to play a pivotal role in triggering the polymerization reaction.
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