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A Recombinant Sickle Hemoglobin Triple Mutant with Independent Inhibitory Effects on Polymerization

1996; Elsevier BV; Volume: 271; Issue: 41 Linguagem: Inglês

10.1074/jbc.271.41.25152

ISSN

1083-351X

Autores

Juha‐Pekka Himanen, Urooj A. Mirza, Brian T. Chait, Robert M. Bookchin, James M. Manning,

Tópico(s)

Iron Metabolism and Disorders

Resumo

As part of a comprehensive effort to map the most important regions of sickle hemoglobin that are involved in polymerization, we have determined whether two sites previously shown to be involved, Leu-88(β) and Lys-95(β), had additive effects when substituted. The former site is part of the hydrophobic pocket that binds Val-6(β), the natural mutation of HbS, and the latter site is a prominent part of the hemoglobin exterior. A sickle hemoglobin triple mutant with three amino acid substitutions on the β-chain, E6V/L88A/K95I, has been expressed in yeast and characterized extensively. Its oxygen binding curve, cooperativity, response to allosteric effectors, and the alkaline Bohr effect showed that it was completely functional. The polymer solubility of the deoxy triple mutant, measured by a new micromethod requiring reduced amounts of hemoglobin, was identical to that of the E6V(β)/K95I(β) mutant, i.e. when the K95I(β) substitution was present on the same tetramer together with the naturally occurring E6V(β) substitution, the L88A(β) replacement had no additive effect on polymer inhibition. The results suggest that Lys-95(β) on the surface of the tetramer and its complementary binding region on the adjoining tetramer are potential targets for the design of an effective antisickling agent. As part of a comprehensive effort to map the most important regions of sickle hemoglobin that are involved in polymerization, we have determined whether two sites previously shown to be involved, Leu-88(β) and Lys-95(β), had additive effects when substituted. The former site is part of the hydrophobic pocket that binds Val-6(β), the natural mutation of HbS, and the latter site is a prominent part of the hemoglobin exterior. A sickle hemoglobin triple mutant with three amino acid substitutions on the β-chain, E6V/L88A/K95I, has been expressed in yeast and characterized extensively. Its oxygen binding curve, cooperativity, response to allosteric effectors, and the alkaline Bohr effect showed that it was completely functional. The polymer solubility of the deoxy triple mutant, measured by a new micromethod requiring reduced amounts of hemoglobin, was identical to that of the E6V(β)/K95I(β) mutant, i.e. when the K95I(β) substitution was present on the same tetramer together with the naturally occurring E6V(β) substitution, the L88A(β) replacement had no additive effect on polymer inhibition. The results suggest that Lys-95(β) on the surface of the tetramer and its complementary binding region on the adjoining tetramer are potential targets for the design of an effective antisickling agent. INTRODUCTIONSickle cell anemia results from a single point mutation in the gene encoding β-globin, whereby the Glu-6(β) residue in hemoglobin A (HbA) is substituted by Val in sickle hemoglobin (HbS) (1Pauling L. Itano H. Singer S.J. Wells J.C. Science. 1949; 110: 543-548Crossref PubMed Scopus (1175) Google Scholar, 2Ingram V.M. Nature. 1956; 178: 792-794Crossref PubMed Scopus (468) Google Scholar). This hydrophobic side chain initiates a process by which the densely packed deoxyhemoglobin tetramers inside the red blood cells interact through other sites to form long polymer fibers that distort the cells into a characteristic sickle shape. Although the identity of many of these amino acid sites involved in polymer formation and the extent to which they participate is known (3Nagel R.L. Bookchin R.M. Caughey W.S. Biochemical and Clinical Aspects of Hemoglobin Abnormalities. Academic Press, New York1978: 195Crossref Google Scholar, 4Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar, 5Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar, 6Martin de Llano J.J. Manning J.M. Protein Sci. 1994; 3: 1206-1212Crossref PubMed Scopus (33) Google Scholar, 7Dickerson R.E. Geis I. Hemoglobin: Structure, Function, Evolution and Pathology. Benjamin Cummings, Reading, MA1983: 133Google Scholar, 8Nagel R.L. Bookchin R.M. Levere R.D. Sickle Cell Anemia and Other Hemoglobinopathies. Academic Press, New York1974: 51Google Scholar), the quantitative contributions to polymerization of many other sites are unknown. A goal of this study was to provide such information for selected polymerization contact sites for which natural mutants either do not exist or have not been reported. Recombinant sickle double and triple mutants are used for this purpose.Studies describing the hydrophobicity and stereochemistry of deoxy HbS have shown that Val-6(β) binds tightly between Phe-85 and Leu-88 in the acceptor pocket on an adjacent β-chain. According to computer-generated models, the three-dimensional fit of the side chain of Val into the acceptor pocket is much better than that of Ala (7Dickerson R.E. Geis I. Hemoglobin: Structure, Function, Evolution and Pathology. Benjamin Cummings, Reading, MA1983: 133Google Scholar), explaining the inability of Hb Makassar with Ala-6(β) to polymerize (8Nagel R.L. Bookchin R.M. Levere R.D. Sickle Cell Anemia and Other Hemoglobinopathies. Academic Press, New York1974: 51Google Scholar), even though the hydrophobicity of Ala and Val do not differ drastically. Other studies have suggested that substitutions by larger hydrophobic residues at the position 6, readily promote polymerization (9Baudin-Chich V. Pagnier J. Marden M. Cohn B. Loraze N. Kister J. Schaad O. Edelstein S.J. Poyart C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1845-1849Crossref PubMed Scopus (21) Google Scholar). These findings point out the complexity of the polymerization process, which cannot be explained simply by the hydrophobicity and stereochemistry of the β-6 site and its corresponding acceptor pocket. Indeed, it has been established that other contact sites in the gelation process reinforce the initial contact (3Nagel R.L. Bookchin R.M. Caughey W.S. Biochemical and Clinical Aspects of Hemoglobin Abnormalities. Academic Press, New York1978: 195Crossref Google Scholar, 4Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar, 5Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar, 9Baudin-Chich V. Pagnier J. Marden M. Cohn B. Loraze N. Kister J. Schaad O. Edelstein S.J. Poyart C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1845-1849Crossref PubMed Scopus (21) Google Scholar, 10Adachi K. Konitzer P. Kim J. Welch N. Surrey S. J. Biol. Chem. 1993; 268: 21650-21656Abstract Full Text PDF PubMed Google Scholar). In addition, studies with noncovalent chemical inhibitors have shown that these compounds do not act as predicted by their hydrophobic nature (11Ross P.D. Subramanian S. Caughey W.W. Biochemical and Clinical Aspects of Hemoglobin Abnormalities. Academic Press, New York1978: 629Crossref Google Scholar), implying a significant contribution of other interactions.In our efforts to understand the mechanism of sickle hemoglobin gelation and to identify the critical sites in the gelation process, we use a yeast expression system (6Martin de Llano J.J. Manning J.M. Protein Sci. 1994; 3: 1206-1212Crossref PubMed Scopus (33) Google Scholar, 12Himanen J.-P. Schneider K. Chait B. Manning J.M. J. Biol. Chem. 1995; 270: 13885-13891Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 13Trudel M. Saadane N. Garel M.-C. Bardakdjian-Michau J. Blouquit Y. Guerquin-Kern J.-L. Rouyer-Fessard P. Vidaud D. Pachnis A. Romeo P.-H. Beuzard Y. Costantini F. EMBO J. 1991; 10: 3157-3165Crossref PubMed Scopus (110) Google Scholar, 14Wagenbach M. O'Rourke K. Vitez L. Wieczorek A. Hoffman S. Durfee S. Tedesco J. Stetler G. Bio/Technology. 1991; 9: 57-61Crossref PubMed Scopus (111) Google Scholar, 15Martin de Llano J.J. Jones W. Schneider K. Chait B.T. Manning J.M. Rodgers G. Benjamin L.J. Weksler B. J. Biol. Chem. 1993; 268: 27004-27011Abstract Full Text PDF PubMed Google Scholar) to produce HbS double and triple mutants as an adjunct to chemical modification studies (16Cerami A. Manning J.M. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 1180-1183Crossref PubMed Scopus (220) Google Scholar, 17Njikam N. Jones W.M. Nigen A.M. Gillette P.N. Williams Jr., R.C. Manning J.M. J. Biol. Chem. 1973; 248: 8052-8056Abstract Full Text PDF PubMed Google Scholar, 18Manning J.M. Adv. Enzymol. Mol. Biol. 1991; 64: 55PubMed Google Scholar). Unlike the Escherichia coli expression system, the yeast system produces a native hemoglobin molecule, as judged by many biochemical criteria (15Martin de Llano J.J. Jones W. Schneider K. Chait B.T. Manning J.M. Rodgers G. Benjamin L.J. Weksler B. J. Biol. Chem. 1993; 268: 27004-27011Abstract Full Text PDF PubMed Google Scholar). In addition, since yeast incorporates its own heme group into globin, there are no time-consuming manipulations, such as reconstituting hemoglobin with exogenous heme. Thus, it is feasible to study the involvement of any site on the hemoglobin molecule in the gelation process and to judge the significance of any differences between the crystal structure (5Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar) and the electron microscope structure (4Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar, 19Edelstein S.J. Crepeau R.H. J. Mol. Biol. 1979; 134: 851-855Crossref PubMed Scopus (17) Google Scholar) of HbS. For example, we recently determined that the contact site Lys-95(β) on the outside of the tetramer distant from the hydrophobic pocket, which was implicated in one structure (4Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar) but not the other (5Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar), was significantly involved in the gelation process (12Himanen J.-P. Schneider K. Chait B. Manning J.M. J. Biol. Chem. 1995; 270: 13885-13891Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Indeed, its substitution by Ile inhibits gelation twice as much as a mutation at a site in the acceptor pocket, L88A(β) (6Martin de Llano J.J. Manning J.M. Protein Sci. 1994; 3: 1206-1212Crossref PubMed Scopus (33) Google Scholar). The diverse locations of these two sites prompted us to design a recombinant Hb having both K95I(β) and L88A(β) in addition to the Val-6(β) mutation in order to measure whether the influence of the two substitutions on gelation is additive. Such a study may reveal important details of the gelation process and could influence efforts for developing well targeted clinically effective inhibitors. For these studies, we employ a new method based on the drastic decrease in the solubility of hemoglobin S upon addition of dextran (20Bookchin R.M. Balazs T. Lew V.L. Blood. 1994; 86: 473aGoogle Scholar).DISCUSSIONIn this study the recombinant triple mutant E6V(β)/L88A(β)/K95I(β) produced in yeast was shown to have the predicted amino acid composition, molecular mass, isoelectric point, and trypsin cleavage sites. Its oxygen affinity, cooperativity, response to negatively charged effectors, alkaline Bohr effect, and the tetramer/dimer dissociation constant were the same as those for HbS. These results, together with extensive characterization of recombinant hemoglobins by a variety of biochemical criteria (15Martin de Llano J.J. Jones W. Schneider K. Chait B.T. Manning J.M. Rodgers G. Benjamin L.J. Weksler B. J. Biol. Chem. 1993; 268: 27004-27011Abstract Full Text PDF PubMed Google Scholar, 25Yanase H. Cahill S. Martin de Llano J.J. Manning L.R. Schneider K. Chait B.T. Vandegriff K.D. Winslow R.M. Manning J.M. Protein Sci. 1994; 3: 1213-1223Crossref PubMed Scopus (22) Google Scholar, 26Martin de Llano J.J. Schneewind O. Stetler G. Manning J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 918-922Crossref PubMed Scopus (41) Google Scholar, 29Manning L.R. Jenkins W.T. Hess J.R. Vandegriff K. Winslow R. Manning J.M. Protein Sci. 1996; 5: 775-781Crossref PubMed Scopus (76) Google Scholar), are consistent with the expression by the yeast system of a native hemoglobin molecule with the correct N-terminal processing. Thus, we have no evidence for any misfolding of the triple mutant as reported for other recombinant hemoglobins made using E. coli as a production host (30Hernan R.A. Sligar S.G. J. Biol. Chem. 1995; 270: 26257-26264Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Hence, the gelation of the native HbS and the recombinant double and triple mutants by the procedure described here can be taken as reliable measurements of the gelation concentrations.We reported previously that Lys-95(β), which is distant from the hydrophobic pocket in the region of Phe-85(β)-Leu-88(β) comprising the acceptor site for Val-6(β), inhibits gelation much more than the substitution of a residue in the pocket itself (6Martin de Llano J.J. Manning J.M. Protein Sci. 1994; 3: 1206-1212Crossref PubMed Scopus (33) Google Scholar, 12Himanen J.-P. Schneider K. Chait B. Manning J.M. J. Biol. Chem. 1995; 270: 13885-13891Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Our results agreed with some previous reports implicating Lys-95(β) in the gelation process (33Bookchin R.M. Nagel R.L. Balazs T. Harris J.W. Clin. Res. 1974; 22: 384AGoogle Scholar) and as an intermolecular contact site in the polymer (3Nagel R.L. Bookchin R.M. Caughey W.S. Biochemical and Clinical Aspects of Hemoglobin Abnormalities. Academic Press, New York1978: 195Crossref Google Scholar, 4Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar), although this site was not involved in the Wishner-Love double strand crystal of deoxy-HbS (5Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar). The strong influence of the β-95 site, which is located on the exterior of the tetramer at the lateral contact site of the HbS tetramer, on gelation strongly suggests that the K95I(β) mutant of HbS has different protein self-assembly properties than HbS itself (32Eaton W.A. Hofrichter J. Embury S.H. Hebbel R.P. Mohandas N. Steinberg M.H. Sickle Cell Disease: Basic Principles and Clinical Practice. Raven Press, New York1994: 53Google Scholar). The role of the Val-6(β) and its hydrophobic acceptor pocket may be to provide a molecular switch to turn the gelation either on or off. If this position is mutated to Ala (Hb Makassar), no gelation occurs because Ala prevents sufficient stabilization of the primary nuclei. Our results on the gelation of E6V(β)/L88A(β) mutant (6Martin de Llano J.J. Manning J.M. Protein Sci. 1994; 3: 1206-1212Crossref PubMed Scopus (33) Google Scholar, 34Liao D. Martin de Llano J.J. Himanen J.P. Manning J.M. Ferrone F.A. Biophys. J. 1996; 70: 2442-2447Abstract Full Text PDF PubMed Scopus (12) Google Scholar) also suggest that the Leu to Ala substitution in the acceptor pocket mainly affects the initial nucleation process, but once nucleation has taken place other residues stabilize the polymer. These findings also further emphasize the importance of certain ionizable surface amino acids. Their potential importance as well as that of their complementary sites on adjacent tetramers lies in the possible development of clinical intervention against sickle cell disease. The results presented here demonstrate that two sites on the HbS tetramer exert significantly different and independent effects on the inhibition of polymerization.Since the polymer solubility of the triple mutant was the same as that of the double mutant without the L88A(β) substitution, i.e. E6V(β)/K95I(β), the present results demonstrate that the inhibitory effects of the two β-chain substitutions (L88A and K95I) on HbS, are not additive. Although the L88A(β) mutant, in which the substitution is in the hydrophobic acceptor pocket, has a gelation concentration about midway between the K95I(β) mutant and HbS itself, it does not appear to influence the overall behavior of the triple mutant.The results of recent studies on recombinant mutants are consistent with the notion that once the initial contact site is established by the Glu-6 → Val substitution in the sickle Hb tetramer, then additional substitutions may strengthen or weaken the polymerization tendency. The only previous study involving two β-chain mutations of HbS was by Trudel et al. (13Trudel M. Saadane N. Garel M.-C. Bardakdjian-Michau J. Blouquit Y. Guerquin-Kern J.-L. Rouyer-Fessard P. Vidaud D. Pachnis A. Romeo P.-H. Beuzard Y. Costantini F. EMBO J. 1991; 10: 3157-3165Crossref PubMed Scopus (110) Google Scholar) using a transgenic mouse system, with the purpose of promoting polymerization to obtain a better transgenic mouse model of sickle cell anemia. In that study, there was no quantitation of the individual effects of the substitutions on polymer solubility. The present study was aimed at furthering our understanding of the mechanism of gel formation by inhibiting polymerization and to identify the most important sites that influence the polymerization process significantly. The results indicate that amino acid replacements at Leu-88(β) and Lys-95(β) act independently in inhibiting polymerization, i.e. certain sites can influence the overall prevention of polymerization to a greater extent than others. Such sites might be potentially accessible to anti-sickling agents that could be designed to fit their particular environment as well as that of their complementary binding site on adjacent tetramers. The Lys-95(β) site and the site to which it binds appear to fulfill such criteria. INTRODUCTIONSickle cell anemia results from a single point mutation in the gene encoding β-globin, whereby the Glu-6(β) residue in hemoglobin A (HbA) is substituted by Val in sickle hemoglobin (HbS) (1Pauling L. Itano H. Singer S.J. Wells J.C. Science. 1949; 110: 543-548Crossref PubMed Scopus (1175) Google Scholar, 2Ingram V.M. Nature. 1956; 178: 792-794Crossref PubMed Scopus (468) Google Scholar). This hydrophobic side chain initiates a process by which the densely packed deoxyhemoglobin tetramers inside the red blood cells interact through other sites to form long polymer fibers that distort the cells into a characteristic sickle shape. Although the identity of many of these amino acid sites involved in polymer formation and the extent to which they participate is known (3Nagel R.L. Bookchin R.M. Caughey W.S. Biochemical and Clinical Aspects of Hemoglobin Abnormalities. Academic Press, New York1978: 195Crossref Google Scholar, 4Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar, 5Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar, 6Martin de Llano J.J. Manning J.M. Protein Sci. 1994; 3: 1206-1212Crossref PubMed Scopus (33) Google Scholar, 7Dickerson R.E. Geis I. Hemoglobin: Structure, Function, Evolution and Pathology. Benjamin Cummings, Reading, MA1983: 133Google Scholar, 8Nagel R.L. Bookchin R.M. Levere R.D. Sickle Cell Anemia and Other Hemoglobinopathies. Academic Press, New York1974: 51Google Scholar), the quantitative contributions to polymerization of many other sites are unknown. A goal of this study was to provide such information for selected polymerization contact sites for which natural mutants either do not exist or have not been reported. Recombinant sickle double and triple mutants are used for this purpose.Studies describing the hydrophobicity and stereochemistry of deoxy HbS have shown that Val-6(β) binds tightly between Phe-85 and Leu-88 in the acceptor pocket on an adjacent β-chain. According to computer-generated models, the three-dimensional fit of the side chain of Val into the acceptor pocket is much better than that of Ala (7Dickerson R.E. Geis I. Hemoglobin: Structure, Function, Evolution and Pathology. Benjamin Cummings, Reading, MA1983: 133Google Scholar), explaining the inability of Hb Makassar with Ala-6(β) to polymerize (8Nagel R.L. Bookchin R.M. Levere R.D. Sickle Cell Anemia and Other Hemoglobinopathies. Academic Press, New York1974: 51Google Scholar), even though the hydrophobicity of Ala and Val do not differ drastically. Other studies have suggested that substitutions by larger hydrophobic residues at the position 6, readily promote polymerization (9Baudin-Chich V. Pagnier J. Marden M. Cohn B. Loraze N. Kister J. Schaad O. Edelstein S.J. Poyart C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1845-1849Crossref PubMed Scopus (21) Google Scholar). These findings point out the complexity of the polymerization process, which cannot be explained simply by the hydrophobicity and stereochemistry of the β-6 site and its corresponding acceptor pocket. Indeed, it has been established that other contact sites in the gelation process reinforce the initial contact (3Nagel R.L. Bookchin R.M. Caughey W.S. Biochemical and Clinical Aspects of Hemoglobin Abnormalities. Academic Press, New York1978: 195Crossref Google Scholar, 4Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar, 5Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar, 9Baudin-Chich V. Pagnier J. Marden M. Cohn B. Loraze N. Kister J. Schaad O. Edelstein S.J. Poyart C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1845-1849Crossref PubMed Scopus (21) Google Scholar, 10Adachi K. Konitzer P. Kim J. Welch N. Surrey S. J. Biol. Chem. 1993; 268: 21650-21656Abstract Full Text PDF PubMed Google Scholar). In addition, studies with noncovalent chemical inhibitors have shown that these compounds do not act as predicted by their hydrophobic nature (11Ross P.D. Subramanian S. Caughey W.W. Biochemical and Clinical Aspects of Hemoglobin Abnormalities. Academic Press, New York1978: 629Crossref Google Scholar), implying a significant contribution of other interactions.In our efforts to understand the mechanism of sickle hemoglobin gelation and to identify the critical sites in the gelation process, we use a yeast expression system (6Martin de Llano J.J. Manning J.M. Protein Sci. 1994; 3: 1206-1212Crossref PubMed Scopus (33) Google Scholar, 12Himanen J.-P. Schneider K. Chait B. Manning J.M. J. Biol. Chem. 1995; 270: 13885-13891Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 13Trudel M. Saadane N. Garel M.-C. Bardakdjian-Michau J. Blouquit Y. Guerquin-Kern J.-L. Rouyer-Fessard P. Vidaud D. Pachnis A. Romeo P.-H. Beuzard Y. Costantini F. EMBO J. 1991; 10: 3157-3165Crossref PubMed Scopus (110) Google Scholar, 14Wagenbach M. O'Rourke K. Vitez L. Wieczorek A. Hoffman S. Durfee S. Tedesco J. Stetler G. Bio/Technology. 1991; 9: 57-61Crossref PubMed Scopus (111) Google Scholar, 15Martin de Llano J.J. Jones W. Schneider K. Chait B.T. Manning J.M. Rodgers G. Benjamin L.J. Weksler B. J. Biol. Chem. 1993; 268: 27004-27011Abstract Full Text PDF PubMed Google Scholar) to produce HbS double and triple mutants as an adjunct to chemical modification studies (16Cerami A. Manning J.M. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 1180-1183Crossref PubMed Scopus (220) Google Scholar, 17Njikam N. Jones W.M. Nigen A.M. Gillette P.N. Williams Jr., R.C. Manning J.M. J. Biol. Chem. 1973; 248: 8052-8056Abstract Full Text PDF PubMed Google Scholar, 18Manning J.M. Adv. Enzymol. Mol. Biol. 1991; 64: 55PubMed Google Scholar). Unlike the Escherichia coli expression system, the yeast system produces a native hemoglobin molecule, as judged by many biochemical criteria (15Martin de Llano J.J. Jones W. Schneider K. Chait B.T. Manning J.M. Rodgers G. Benjamin L.J. Weksler B. J. Biol. Chem. 1993; 268: 27004-27011Abstract Full Text PDF PubMed Google Scholar). In addition, since yeast incorporates its own heme group into globin, there are no time-consuming manipulations, such as reconstituting hemoglobin with exogenous heme. Thus, it is feasible to study the involvement of any site on the hemoglobin molecule in the gelation process and to judge the significance of any differences between the crystal structure (5Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar) and the electron microscope structure (4Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar, 19Edelstein S.J. Crepeau R.H. J. Mol. Biol. 1979; 134: 851-855Crossref PubMed Scopus (17) Google Scholar) of HbS. For example, we recently determined that the contact site Lys-95(β) on the outside of the tetramer distant from the hydrophobic pocket, which was implicated in one structure (4Watowich S.J. Gross L.J. Josephs R. J. Mol. Biol. 1989; 209: 821-828Crossref PubMed Scopus (41) Google Scholar) but not the other (5Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar), was significantly involved in the gelation process (12Himanen J.-P. Schneider K. Chait B. Manning J.M. J. Biol. Chem. 1995; 270: 13885-13891Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Indeed, its substitution by Ile inhibits gelation twice as much as a mutation at a site in the acceptor pocket, L88A(β) (6Martin de Llano J.J. Manning J.M. Protein Sci. 1994; 3: 1206-1212Crossref PubMed Scopus (33) Google Scholar). The diverse locations of these two sites prompted us to design a recombinant Hb having both K95I(β) and L88A(β) in addition to the Val-6(β) mutation in order to measure whether the influence of the two substitutions on gelation is additive. Such a study may reveal important details of the gelation process and could influence efforts for developing well targeted clinically effective inhibitors. For these studies, we employ a new method based on the drastic decrease in the solubility of hemoglobin S upon addition of dextran (20Bookchin R.M. Balazs T. Lew V.L. Blood. 1994; 86: 473aGoogle Scholar).

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