A Role for the α113 (GH1) Amino Acid Residue in the Polymerization of Sickle Hemoglobin
2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês
10.1074/jbc.m101788200
ISSN1083-351X
AutoresMylavarapu V.S. Sivaram, Rajamani Sudha, Rajendra P. Roy,
Tópico(s)Neonatal Health and Biochemistry
ResumoA cluster of amino acid residues located in the AB-GH region of the α-chain are shown in intra-double strand axial interactions of the hemoglobin S (HbS) polymer. However, αLeu-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 (βGlu-6→Val, αLeu-113→His) to ascertain the hitherto unknown role of the α113 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 α113 site in the polymerization process as well as to evaluate its relative inhibitory strength, we constructed HbS tetramers in which the α113 mutation was coupled individually with two established fiber contact sites (α16 and α23) located in the AB region of the α-chain: HbS(αLys-16→Gln, αLeu-113→His), HbS(αGlu-23→Gln, αLeu-113→His). The single mutants at α16/α23 sites were also engineered as controls. The Csat values of the HbS point mutants involving sites α16 or α23 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 α113 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. A cluster of amino acid residues located in the AB-GH region of the α-chain are shown in intra-double strand axial interactions of the hemoglobin S (HbS) polymer. However, αLeu-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 (βGlu-6→Val, αLeu-113→His) to ascertain the hitherto unknown role of the α113 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 α113 site in the polymerization process as well as to evaluate its relative inhibitory strength, we constructed HbS tetramers in which the α113 mutation was coupled individually with two established fiber contact sites (α16 and α23) located in the AB region of the α-chain: HbS(αLys-16→Gln, αLeu-113→His), HbS(αGlu-23→Gln, αLeu-113→His). The single mutants at α16/α23 sites were also engineered as controls. The Csat values of the HbS point mutants involving sites α16 or α23 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 α113 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. sickle hemoglobin high pressure liquid chromatography reverse phase HPLC N-(9-fluorenyl)methoxycarbonyl fast protein liquid chromatography Sickle cell anemia is a consequence of a point mutation (Glu-6→Val) at the sixth position in the β-chain of the hemoglobin molecule (1Ingram V.M. Nature. 1956; 178: 792-794Crossref PubMed Scopus (471) Google Scholar). 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 (2Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8272-8279Abstract Full Text PDF PubMed Google Scholar, 3Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar, 4Dykes G.W. Crepeau R.H. Edelstein S.J. Nature. 1978; 272: 506-510Crossref PubMed Scopus (113) Google Scholar, 5Dykes G.W. Crepeau R.H. Edelstein S.J. J. Mol. Biol. 1979; 130: 451-472Crossref PubMed Scopus (149) Google Scholar). The model of the fiber structure derived from these analyses consists of seven Wishner-Love double strands (6Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar, 7Cretegny I. Edelstein S.J. J. Mol. Biol. 1993; 230: 733-738Crossref PubMed Scopus (51) Google Scholar, 8Watowich S.J. Gross L.J. Josephs R. J. Struct. Biol. 1997; 111: 161-179Crossref Scopus (45) Google Scholar, 9Eaton W.A. Hofrichter J. Adv. Protein Chem. 1990; 40: 63-279Crossref PubMed Scopus (518) Google Scholar). The polymerization process is triggered by lateral interactions of the donor Val-6β of a tetramer of one strand of the double strand with the acceptor pocket at the EF corner (elicited mainly by βPhe-85 and βLeu-88) of the β-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 α- and β-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 (10Nagel R.L. Johnson J. Bookchin R.M. Garel M.C. Rosa J. Schiliro G. Wajcman H. Labie D. Moo-Penn W. Castro O. Nature. 1980; 283: 832-834Crossref PubMed Scopus (70) Google Scholar, 11Benesch R.E. Kwong S. Benesch R. Edalji R. Nature. 1977; 269: 772-775Crossref PubMed Scopus (44) Google Scholar, 12Benesch R.E. Yung S. Benesch R. Mack J. Schneider R. Nature. 1976; 260: 219-222Crossref PubMed Scopus (37) Google Scholar). The list of contact sites has been expanded by subsequent studies involving chemical modifications of HbS (13Manning J.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 55-91PubMed Google Scholar, 14Rao M.J. Iyer K.S. Acharya A.S. J. Biol. Chem. 1995; 33: 19250-19255Abstract Full Text Full Text PDF Scopus (6) Google Scholar) and site-directed mutagenesis (15Adachi K. Reddy L.R. Reddy K.S. Surrey S. Protein Sci. 1995; 4: 1272-1278Crossref PubMed Scopus (10) Google Scholar, 16Ho C. Willis B.F. Shen T.J. Ho N.T. Sun D.P. Tam M.F. Suzuka S.M. Fabry M.E. Nagel R.L. J. Mol. Biol. 1996; 263: 475-485Crossref PubMed Scopus (21) Google Scholar, 17Himanen J.-P. Schneider K. Chait B.T. Manning J.M. J. Biol. Chem. 1995; 23: 13885-13891Abstract Full Text Full Text PDF Scopus (15) Google Scholar, 18Himanen J.-P. Popowicz A.M. Manning J.M. Blood. 1997; 89: 4196-4203Crossref PubMed Google Scholar, 19Li X. Mirza U.A. Chait B.T. Manning J.M. Blood. 1997; 90: 4620-4627Crossref PubMed Google Scholar). 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 (6Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar, 7Cretegny I. Edelstein S.J. J. Mol. Biol. 1993; 230: 733-738Crossref PubMed Scopus (51) Google Scholar, 8Watowich S.J. Gross L.J. Josephs R. J. Struct. Biol. 1997; 111: 161-179Crossref Scopus (45) Google Scholar, 9Eaton W.A. Hofrichter J. Adv. Protein Chem. 1990; 40: 63-279Crossref PubMed Scopus (518) Google Scholar). 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 (20Manning J.M. Dumoulin A. Li X. Manning L.R. J. Biol. Chem. 1998; 273: 19359-19362Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We have chosen three sites, namely α16, α23, and α113, for further delineation of their contributions to the polymerization of HbS. Whereas sites αLys-16 and αGlu-23, located in the AB region, are established contact points in the HbS polymer, the participation of αLeu-113 (GH1) in the polymerization process is unknown. The α113 site is of interest because of its unique structural location. First, α113 is in sequence contiguity with a cluster of GH corner residues, α114, α115, and α116, which are established or implicated intra-double strand contact sites of the HbS fiber (7Cretegny I. Edelstein S.J. J. Mol. Biol. 1993; 230: 733-738Crossref PubMed Scopus (51) Google Scholar, 11Benesch R.E. Kwong S. Benesch R. Edalji R. Nature. 1977; 269: 772-775Crossref PubMed Scopus (44) Google Scholar, 16Ho C. Willis B.F. Shen T.J. Ho N.T. Sun D.P. Tam M.F. Suzuka S.M. Fabry M.E. Nagel R.L. J. Mol. Biol. 1996; 263: 475-485Crossref PubMed Scopus (21) Google Scholar). Second, the three-dimensional structure of the hemoglobin brings the AB region of the α-chain in close proximity to the GH corner. The involvement of several residues of the AB corner (α16, α20, α23) in HbS polymerization has been deduced from crystal structures and also been validated in solution experiments (21Benesch R.E. Kwong S. Benesch R. Nature. 1982; 299: 231-234Crossref PubMed Scopus (30) Google Scholar, 22Rhoda M.D. Blouquit Y. Caburi-Martin J. Monplasir N. Galacteros F. Garel M.C. Rosa J. Biochim. Biophys. Acta. 1984; 786: 62-64Crossref PubMed Scopus (14) Google Scholar, 23Kraus L.M. Miyaji T. Iuchi I. Kraus A.P. Biochemistry. 1966; 5: 3701-3708Crossref PubMed Scopus (43) Google Scholar, 24Nagel R.L. Bookchin R.M. Caughey W.S. Biological and Clinical Aspects of Hemoglobin Abnormalities. Academic Press, New York1978: 195-201Google Scholar). However, αLeu-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 (αLeu-113→His), is reported (25Guis M. Mentzer W.C. Jue D.L. Johnson M.H. McGuffey J.E. Moo-Penn W.F. Hemoglobin. 1985; 9: 175-177Crossref PubMed Scopus (12) Google Scholar), 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 (αLeu-113→His, βGlu-6→Val) to establish the role of the α113 site in HbS fiber generation. Furthermore, we have combined the α113 mutation at the GH corner with mutations of contact sites involving residues α16 and α23 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-6β-dependent polymerization of HbS. We have adopted a chemo-enzymatic strategy for the construction of α-globin mutants. The propensity of V8 protease to catalyze the ligation of complementary fragments, α1–30 and α31–141, to generate a full-length α-globin (α1–141) has been utilized for this purpose (26Roy R.P. Acharya A.S. Methods Enzymol. 1994; 231: 194-215Crossref PubMed Scopus (18) Google Scholar). Appropriate synthetic α1–30 segments were employed to incorporate desired mutations at sites α16 and α23. The Hb Twin Peaks mutation was introduced through the α31–141 segment of the marmoset (Callithrix argentata) α-chain, which contains a single amino acid substitution, αLeu-113→His, with respect to the human α31–141 segment (27Maita T. Hayashida M. Matsuda G. J. Biochem. ( Tokyo ). 1984; 95: 805-813Crossref PubMed Scopus (7) Google Scholar). HbS tetramers were assembled from βs-chain and respective single and double mutant α-chains. The structural/conformational, functional, and polymerization behavior of mutants was studied with a view to examining the hitherto unknown role of the α113 (GH1) site in the HbS gelation process as well as to quantifying its inhibitory strength relative to selected AB region α-chain contact residues. 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 α- and β-chains were prepared as described previously (28Bucci E. Methods Enzymol. 1981; 76: 97-105Crossref PubMed Scopus (49) Google Scholar). The chains were freed from heme by acid-acetone precipitation. The complementary segments of α-globin needed for the semisynthesis of mutant chains were prepared by V8 protease digestion (26Roy R.P. Acharya A.S. Methods Enzymol. 1994; 231: 194-215Crossref PubMed Scopus (18) Google Scholar). 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, α1–30 and α31–141, 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 α-globin digest (human or marmoset) showed only two peaks, α31–141 and α1–30, as expected from a single cleavage at the 30–31 peptide bond. The peak fractions from each digest were pooled separately and lyophilized. 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-α-Fmoc-Glu was used as the starting material. The stepwise coupling of Fmoc amino acids was performed with theN,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole activation procedure. The coupling of each step was monitored by a Kaiser test (29Kaiser E. Colescott R.L. Bossinger C.D. Cook P.I. Anal. Biochem. 1970; 84: 595-598Crossref Scopus (3501) Google Scholar), 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: α(Lys-16→Gln), observed mass of 3040.73 Da (theoretical mass, 3040.73); α(Glu-23→Gln), observed mass of 3040.50 (theoretical mass, 3039.39). V8 protease-mediated semisynthesis of α-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 α1–30 and human or marmoset α31–141 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 α-globin was isolated from the mixture by CM52-urea chromatography, extensively dialyzed against 0.1% trifluoroacetic acid, and lyophilized (26Roy R.P. Acharya A.S. Methods Enzymol. 1994; 231: 194-215Crossref PubMed Scopus (18) Google Scholar). The semisynthetic yield of the protein varied between 35 and 45%. The identity of the α-globin constructs were checked by mass spectrometry and tryptic peptide mapping. The semisynthetic α-globin was reconstituted with heme and the βs-chain into tetrameric hemoglobin through the “Alloplex pathway” as described previously (26Roy R.P. Acharya A.S. Methods Enzymol. 1994; 231: 194-215Crossref PubMed Scopus (18) Google Scholar). The reconstituted tetramers were purified by CM-52 chromatography. The heme stoichiometry in purified tetramers was ascertained by 280:540 nm absorbance ratios. TheA280: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 α- and β-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. 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. 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. 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 mmbased 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. The gelation concentration of HbS constructs was determined by the dextran-Csat method of Bookchin et al. (30Bookchin R.M. Balazs T. Wang Z. Josephs R. Lew V.L. J. Biol. Chem. 1999; 274: 6689-6697Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). 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 0.05m, 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 ofCsat by Drabkin's reagent. The delay time kinetics of deoxyhemoglobin were studied in 1.8 m phosphate buffer (pH 7.25) as described by Adachi and Asakura (31Adachi K. Asakura T. J. Biol. Chem. 1979; 254: 4079-4084Abstract Full Text PDF PubMed Google Scholar, 32Adachi K. Asakura T. J. Biol. Chem. 1979; 254: 7765-7771Abstract Full Text PDF PubMed Google Scholar) 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. The construction of the α-chain of Hb Twin Peaks (αLeu-113→His) through the α-globin semisynthetic strategy involves the stitching of human α1–30 with an α31–141 segment containing the Twin Peaks mutation. The two peaks isolated from G-50 chromatography of the V8 protease digest of the marmoset α-globin were subjected to electrospray mass spectrometry. The reported sequence of marmoset α-globin (27Maita T. Hayashida M. Matsuda G. J. Biochem. ( Tokyo ). 1984; 95: 805-813Crossref PubMed Scopus (7) Google Scholar) 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 α-globin (α1–30, 3028.32 Da; α31–141, 12127.99 Da). The semisynthetic Twin Peaks α-globin was obtained by ligation of human α1–30 and marmoset α31–141 fragments through the V8 protease-catalyzed reaction as described under “Materials and Methods.” The purified material was reconstituted with the βs-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 α113 site in HbS. The purified HbS Twin Peaks was analyzed by reverse-phase HPLC to establish the stoichiometry and chemical integrity of the globin chains. The α- and β-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 βs-chains from both samples and indicated correct stoichiometry of the α- and β-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 α-chain of HbS Twin Peaks eluted earlier than the βs-chain, suggesting that Leu to His substitution exerted considerable influence on the chromatographic behavior of the α-chain. To rule out the possibility that the above elution behavior was a consequence of chemical modifications during semisynthesis or tetramer assembly, α- and βs-chains of HbS Twin Peaks were isolated by reverse-phase HPLC and subjected to electrospray mass spectrometry. The molecular mass of the isolated α-chain (15150.03 Da) obtained by electrospray mass spectrometry agreed very well with the calculated value of 15150.36 Da for the α-chain of Hb Twin Peaks. Likewise, the experimental mass of the β-chain (15837.99 Da) was in accord with the calculated mass of the βs-chain (15837.25 Da). Taken together, the results unambiguously established the chemical integrity 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→His mutation does not have a deleterious effect on the folding of the heme pocket. 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 (33Briehl R.W. Hobbs J.F. J. Biol. Chem. 1970; 245: 544-554Abstract Full Text PDF PubMed Google Scholar, 34Imai K. Biochemistry. 1973; 12: 128-134Crossref PubMed Scopus (19) Google Scholar, 35Imai K. Tsuneshige A. Harano T. Harano K. J. Biol. Chem. 1989; 264: 11174-11180Abstract Full Text PDF PubMed Google Scholar). 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. 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 αLeu-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. The gelation concentration (Csat) of HbS Twin Peaks was measured in the presence of high concentrations of dextran developed by Bookchin et al. (30Bookchin R.M. Balazs T. Wang Z. Josephs R. Lew V.L. J. Biol. Chem. 1999; 274: 6689-6697Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) 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 aCsat of 29–31 mg/ml in the initial concentration range of 50–80 mg/ml HbS. In contrast, theCsat 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 α-chain and βA-chain (Glu-6β) 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 α113 site to inhibit βs-dependent polymerization of HbS. To further authenticate the participation of the α113 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. Th
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