©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Polymerization of Hemoglobin S
QUINARY INTERACTIONS OF Glu-43(beta) (*)

(Received for publication, September 13, 1994; and in revised form, April 14, 1995)

M. Janardhan Rao K. Subramonia Iyer A. Seetharama Acharya (§)

From the Division of Hematology, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Hemoglobin S (HbS) Hoshida and three substituted forms of HbS Hoshida (the substituents being on the amide nitrogen of Gln-43(beta)) have been prepared by the amidation of Glu-43(beta) of HbS with ammonia, methylamine, glycine ethyl ester, and galactosamine. The O(2) affinity of HbS is increased slightly on amidation of Glu-43(beta). 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(beta). The solubility of HbS Hoshida (a case with no substitution on Gln-43(beta)), 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 alpha(1)beta(2) interface as compared with that of HbS, whereas the amidated derivatives with larger substituents on Gln-43(beta) reflected noticeable differences. Thus, the increase in the solubility and the oxygen affinity of HbS on the amidation of Glu-43(beta) is primarily a consequence of the loss of the negative charge at 43(beta), a residue proximal to the alpha(1)beta(2) interface. The copolymerization studies of amidated HbS with HbA, and HbS with amidated HbA demonstrate that cis Glu-43(beta) 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).


INTRODUCTION

The mutation of Glu to Val that is located at the sixth position of beta-chain in sickle cell hemoglobin molecule is responsible for its polymerization in the deoxy conformation. The assembly of deoxyhemoglobin S (HbS) (^1)tetramers into fiber involves the cooperative participation of number of residues of alpha- as well as beta-chains of the molecule, in addition to Val-6(beta). 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 beta-chain segment containing Val-6(beta), 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 beta-chain of another tetramer located in the second strand of the double strand. However, only one of the two Val-6(beta) 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(beta). When the inhibitory mutation/modification is located on the alpha- and beta-chains of alphabeta dimer that provides the Val-6(beta) for the polymerization reaction, it is referred to as a ``cis'' residue. When the mutation/modification is located on the other alphabeta 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(beta), CD2 residue of beta-chain, is located near the alpha(1)beta(2) 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(beta) interacts with Ala-53(alpha) and Gln-54(alpha) (Wishner et al., 1975; Padlan and Love, 1985) and with His-45(alpha) (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(beta).

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(beta) 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(beta). This observation is consistent with the implication of Glu-43(beta) 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(beta) 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(beta). Increasing the average modification of Glu-43(beta) 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(beta) 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(beta) 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(beta) 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(beta) of HbS that gets converted into amide or substituted amide.




MATERIALS AND METHODS

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).

Isoelectric Focusing of HbS and Its Disubstituted Derivatives

Agarose IEF gels were used to establish the purity of disubstituted derivatives. HbS and diMA-HbS, diGEE-HbS, and diGA-HbS were applied onto agarose IEF gels with a blend of pH 6-8 Resolve ampholytes. Gels were electrofocused for 60 min to resolve the components in the sample completely.

Kinetics of Polymerization in High Phosphate Buffer

Kinetic studies of polymerization of HbS and diGEE-HbS was carried out in 1.8 M phosphate buffer, pH 7.2, at 30 °C. The concentration of the deoxy Hb was estimated using the millimolar extinction coefficient 12.5 at 555 nm (Adachi and Asakura, 1979). The development of turbidity as a consequence of polymerization was recorded using a Shimadzu UV 265 spectrophotometer after a temperature jump from 0 to 30 °C. The solubility is estimated by determining the concentration of Hb in solution after the completion of the polymerization.

First Derivative UV Spectra of HbS and Its Disubstituted Derivatives

The first derivative spectra of proteins in the UV region (260 to 340 nm) in the oxy and deoxy conformation were recorded on Shimadzu UV-265 spectrophotometer. Proteins concentration was maintained at 55 µM on heme basis, in 50 mM BisTris, pH 7.4, at 25 °C. Spectra of proteins were recorded in the first derivative mode in the spectrophotometer. Deoxygenation is carried out by repeated evacuation and flushing with pure nitrogen.


RESULTS

Purification of DiMA-HbS, DiGEE-HbS, and DiGA-HbS

Fig. 2illustrates the typical purification chromatogram that one gets for the purification of the disubstituted derivatives of HbS generated on amidation. The figure shown here is obtained for the amidated product obtained with galactosamine as the nucleophile. Three distinct components were present in the amidated sample and accounted for nearly 65, 25, and 10%, of the protein eluted from the column. These were designated as A, B, and C, in the order of their elution. Component A, the major component of the chromatogram, elutes at a position corresponding to that of unmodified HbS and established as HbS by isoelectric focusing and tryptic peptide mapping. Component B is the disubstituted derivative of HbS. The smaller component, C, eluting after the disubstituted derivative has been tentatively identified as a tetrasubstituted derivative. The disubstituted derivative was subjected to a second purification on CM-cellulose column using similar chromatographic conditions. The derivative thus purified is referred to as diGA-HbS. The disubstituted derivatives from the reactions with methylamine and glycine ethyl ester were also isolated similarly and referred as diMA-HbS and diGEE-HbS, respectively. The purity of the disubstituted derivatives of HbS thus isolated are homogeneous as seen by their isoelectric focusing patterns (Fig. 2, inset).


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.



Chemical Characterization of Disubstituted Derivatives of HbA and HbS

The globin chains from the disubstituted derivatives have been digested with trypsin and tryptic peptides were mapped using the procedures described earlier (Rao and Acharya, 1991). RP-HPLC map of the tryptic digest of diGA-HbS is shown in Fig. 3. The tryptic map of diGA-HbS contains one radioactive peptide representing more than 98% of total radioactivity and this elutes at 108 min. Another peptide representing a very small amount of radioactivity (less than 1%) elutes at 80 min. The major peptide eluting at 108 min was isolated and purified further by RP-HPLC at pH 6.0 using a 10 mM ammonium acetate-acetonitrile system (Seetharam et al., 1983; Acharya and Seetharam, 1985). Upon amino acid sequencing of this purified peptide, the radioactivity was recovered at the third cycle identifying the site of substitution as Glu-43(beta). The disubstituted derivatives from the reactions with methylamine and glycine ethyl ester have also been similarly digested with trypsin, mapped by RP-HPLC. The radioactive peptides have been isolated and sequenced similarly. The results have established the modification site in the amidated derivatives is Glu-43(beta).


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.



Oxygen Equilibrium Properties of the Disubstituted Derivatives of HbS

The oxygen affinity of the amidated derivatives of HbS has been determined at pH 7.2 and 37 °C using a Hem-O-Scan (Aminco). The amidation of -carboxyl group of Glu-43(beta) of HbS increased the oxygen affinity of the protein. P values decreased from a control value of 7.3 to 5.5. P values of all three disubstituted derivatives were nearly the same (Table 1). The Hill coefficient of all three derivatives of HbS are also normal demonstrating the limited influence of the modification on the quaternary structure of the protein.



Equilibrium Solubility (C) of Disubstituted Derivatives of HbS

The equilibrium solubility of the amidated derivatives is higher than that of the control HbS (Table 1). The C of each of the amidated derivative is distinct and exhibited some degree of sensitivity to the alkyl chains (Fig. 1) present on the amide nitrogen of Gln-43(beta). The derivative with a methyl group on the Gln-43(beta), namely diMA-HbS, exhibited the lowest increase in the solubility. The increase in the solubility is about 36% as compared to the control HbS. On the other hand, the galactosamine derivative is the most soluble of the three amidated derivatives. Its solubility is nearly 47% higher than that of the control protein. The solubility of the diGEE-HbS is intermediate. Thus the increase in the chain length of the alkyl chain on Gln-43(beta) from one carbon (methyl) to a six-carbon chain (hexitolyl) contributes only about 10% to the relative increase in the solubility of diMA-HbS.

Preparation of HbS Hoshida

All the three amidated derivatives discussed above can be considered as different substitutions on the amide nitrogen of Gln-43(beta) of HbS (Fig. 1). A mutant HbS in which the Glu-43(beta) is modified (mutated) to Gln should be called HbS Hoshida, in analogy with Hb-Hoshida (Iuchi et al., 1978). Thus, the amidated derivatives discussed above can be considered as amide N-substituted HbS Hoshida. The solubility data presented above can be interpreted as that most of the solubility increase on amidation of HbS is occurring primarily as a result of the conversion of the -carboxyl group of Glu-43(beta) into an amide. However, the solubilizing influence seen with diMA-HbS could be an additive or synergistic aspects of the loss of the negative charge of Glu-43(beta) as well as the presence of methyl group on the amide nitrogen of diMA-HbS (Fig. 1). In order to confirm whether the conversion of Glu-43(beta) of HbS into Gln by itself is sufficient to increase the solubility of HbS to the level of diMA-HbS, we have also prepared HbS Hoshida by the amidation procedure using ammonium chloride as the nucleophile. Ammonium chloride being a very poor nucleophile, the yield of the amidated product, HbS Hoshida, is considerably lower (10-12%) than that obtained with methylamine as the nucleophile (25%). Accordingly, for the solubility measurements, the oxygen affinity method developed by Benesch et al.(1978), rather than the ultracentrifugation method is used. This approach is particularly suited when one is dealing with smaller amounts of modified or mutated Hb (Benesch et al., 1978; Seetharam et al., 1983). The oxygen affinity of HbS Hoshida is comparable to that of diMA-HbS, thereby establishing that the oxygen affinity changes seen on amidation is certainly a consequence of the loss of the negative charge of Glu-43(beta).

Furthermore, as shown in Fig. 4, the modification of Glu-43(beta) 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(beta) 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(beta) 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.



First Derivative UV Spectra of HbS and Its Disubstituted Derivatives

The first derivative UV spectra (260-340 nm) of oxy and deoxy HbS, and the three disubstituted derivatives of HbS, are shown in Fig. 5. The fine structure of all the amidated derivatives of HbS as reflected in the derivative UV spectra is indistinguishable from that of the native protein in the oxy conformation. On the other hand, the derivative spectra of the amidated products are distinct from one another in the deoxy conformation as well as from that of native protein except for that of HbS Hoshida, which is almost identical to that of diMA-HbS. The results demonstrate the presence of localized conformational differences at their alpha(1)beta(2) interface in their deoxy conformation. As the size of the substituent on the Gln-43(beta) of HbS Hoshida increases, the perturbation at the alpha(1)beta(2) interface also increases (as reflected by the UV first derivative spectra. This deoxy conformation-dependent difference at the alpha(1)beta(2) interface is minimal in the sample amidated with methylamine as compared to that of control HbS, whereas it is maximal in the sample amidated with galactosamine. Deoxy conformational difference is intermediate in HbS amidated with glycine ethyl ester as compared with that of other three amidated derivatives.


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 (bullet bullet bullet), and deoxy diGA-HbS (- bullet -).



Kinetics of Polymerization and the Solubility of DiGEE-HbS in High Phosphate Buffer

The polymerization behavior of diGEE-HbS has also been studied in high phosphate buffer, 1.8 M, pH 7.2, and 30 °C. Amidation of Glu-43(beta) of HbS with GEE increased the delay time of HbS from 4.2 to 6.8 min (Fig. 6). A direct correlation is also seen between the initial concentrations of the proteins with the respective delay times (Fig. 6, inset). The slope of the line for the derivative is nearly the same as that of the control HbS. The solubility of the derivative was independent of the initial concentration of the protein. The solubility of the disubstituted derivative is about 26% higher than that of the control HbS. However, this increase in solubility (26%) observed for disubstituted derivative in the high phosphate buffer is lower as compared to the increase in the solubility of diGEE-HbS assayed in 100 mM phosphate buffer and at pH 6.8 (41%). This difference suggests that the influence of the modification of Glu-43(beta) on the solubility of HbS is modulated by the ionic strength and/or pH of the buffer used in the polymerization process.


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.



Active Configuration of Glu-43(beta)

The delineation of the cis/trans configuration of a contact residue in the polymer by copolymerization approach was developed by Bookchin et al. (1967, 1970), and has been now applied to a number of sites in the polymer (Monplaisir et al., 1986; Caburi-Martin et al., 1986). When an equimolar mixture (1:1) of HbA and HbS is deoxygenated, distribution of the tetramers in the sample will be 25% HbA, 25% HbS, and 50% asymmetrical hybrid tetramer alpha(2)beta^Abeta^S (Bookchin et al., 1975, 1977). The decrease in the rate of dissociation of the tetramers in the deoxy state is primarily responsible for this distribution. Accordingly, an equimolar mixture of diGEE-HbS and HbA in the deoxy conformation will contain only 25% diGEE-HbS and 25% HbA, and 50% of the population will be asymmetric hybrids containing one modified beta^S chain and one unmodified beta^A chain. In such a mixture, 75% of the tetramers will carry the Val-6(beta) mutation. In all of these, the Glu-43(beta) of the beta^S chain is amidated, i.e. the modification is selective for the cis Glu-43(beta). Accordingly, in such mixtures, trans Glu-43(beta) of the asymmetric hybrids remains underivatized. Similarly, in an equimolar mixture of HbS and diGEE-HbA, the asymmetric hybrids will have only their trans Glu-43(beta) derivatized. Since all beta^S chain-containing tetramers have their Glu-43(beta) unmodified, and only the Glu-43 of beta^A chains are derivatized, nearly 75% of the population (beta^S containing tetramers) will have unmodified Glu-43(beta) in their cis position. Thus a comparison of the equilibrium solubility of these two mixtures with that of an equimolar mixture of HbS and HbA will provide an insight into the relative roles of cis and the trans Glu-43(beta) in facilitating the polymerization reaction.

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(beta) 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(beta) is small when it is on the beta^A 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).




DISCUSSION

HbS Hoshida and the three substituted forms of HbS Hoshida (the substituents being on the amide nitrogen of Gln-43(beta)) 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(beta). This conclusion is consistent with the observation that the oxygen affinities of naturally occurring mutants of Glu-43(beta), Hb-Galvaston (beta43 Glu Ala) (Bowman et al., 1964), and Hb Hoshida (beta43 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(beta), most of the solubility increase of HbS is achieved by converting the -carboxyl group of Glu-43(beta) to an amide.

The UV derivative spectroscopic investigations of the amidated derivatives have demonstrated the presence of some amount of conformational perturbations of the alpha(1)beta(2) interface region in the deoxy structure of the protein resulting as a consequence of amidation of Glu-43(beta). 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(beta) 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(beta) 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(beta). 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(beta) 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(beta) position of HbA. Although the original molecular modeling studies implicated a perfect fit between the side chain of Val-6(beta) 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(beta) 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(beta) 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 (beta43 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(beta) to Ala thus appears to have very little influence on the copolymerization. Glu-43(beta) is located in the CD region of the beta 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(beta)) and the D helix (58(beta) and 59(beta)) of trans beta-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(beta) 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(beta) in the polymerization of HbS (Edelstein, 1981; Watowich et al., 1989). The Glu-43(beta) is the only known amino acid residue of CD region of the beta-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.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL 38655 and a grant-in-aid from the American Heart Association (National). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Hematology, U911-C, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2133; Fax: 718-824-3153.

(^1)
The abbreviations used are: HbS, sickle cell hemoglobin; HbA, normal human hemoglobin; GEE, glycine ethyl ester; GA, galactosamine; MA, methylamine; MES, 4-morpholineethane sulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; IEF, isoelectric focusing; RP-HPLC, reverse phase high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. Ronald L. Nagel for the facilities extended to us and for many stimulating discussions on the manuscript. We also thank Dr. Robert Josephs, University of Chicago, Chicago, for valuable comments on this paper.


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