Based upon existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the ␣ and ␤ chains), and conformational constraints differ in solution and in the crystalline state. Over the years, our laboratory and others have provided evidence of conformational changes in HbS and, more recently, in HbC.We now present data based upon allosteric perturbation monitored by front-face fluorescence, ultraviolet resonance Raman spectroscopy, circular dichroism, and oxygen equilibrium studies that confirm and significantly expand previous findings suggesting solution-active structural differences in liganded forms of HbS and HbC distal to the site of mutation and involving the 2,3-diphosphoglycerate binding pocket. The liganded forms of these hemoglobins are of significant interest because HbC crystallizes in the erythrocyte in the oxy form, and oxy HbS exhibits increased mechanical precipitability and a high propensity to oxidize. Specific findings are as follows: 1) differences in the intrinsic fluorescence indicate that the Trp microenvironments are more hydrophobic for HbS > HbC > HbA, 2) ultraviolet resonance Raman spectroscopy detects alterations in Tyr hydrogen bonding, in Trp hydrophobicity at the ␣ 1 ␤ 2 interface (␤37), and in the A-helix (␣14/␤15) of both chains, 3) displacement by inositol hexaphosphate of the Hb-bound 8-hydroxy-1,3,6-pyrenetrisulfonate (the fluorescent 2,3-diphosphoglycerate analog) follows the order HbA > HbS > HbC, and 4) oxygen equilibria measurements indicate a differential allosteric effect by inositol hexaphosphate for HbC ϳ HbS > HbA.Naturally occurring ␤6 hemoglobin mutants aggregate into defined structures in the erythrocyte. Sickle cell hemoglobin (HbS, ␤6 Glu 3 Val) forms polymers in the deoxy state, whereas HbC (␤6 Glu 3 Lys) forms crystals in the oxy liganded state. A complete understanding of the mechanisms giving rise to deoxy HbS polymers and oxy HbC crystals remains to be elucidated. In the last few years, our laboratory has pursued questions related to mechanisms involved in the ligand-specific, induced crystallization of HbC, starting with site-specific probing of the R-state tetrameric structure of HbC (1, 2). Here, we extend the studies to include a comparison of the R-state of HbS. The R-state of HbS is of particular relevance because oxy HbS exhibits unusual properties compared with HbA, such as mechanical precipitability (3-5), greater unfolding at an airwater interface (6, 7), and increased autooxidation (8 -10).According to existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, this ignores the following: 1) important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the ␣ and ␤ chains), and 2) crystals might constrain conformation compared with that in solution.Spectroscopic and biochemical findin...

Time-resolved fluorescence methods have been used to show that 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), a fluorescent analog of 2,3-diphosphoglycerate, binds to the central cavity of carboxyhemoglobin A (HbACO) at pH 6.35. A direct quantitative approach, based on the distinctive free and bound HPT fluorescent lifetimes of 5.6 ns and ϳ27 ps, respectively, was developed to measure the binding affinity of this probe. HPT binds to a single site and is displaced by inositol hexaphosphate at a 1:1 mol ratio, indicating that binding occurs at the 2,3-diphosphoglycerate site in the central cavity. Furthermore, the results imply that low pH HbACO exists as an altered R state and not an equilibrium mixture of R and T states. The probe was also used to monitor competitive effector binding and to compare the affinity of the binding site in several cross-bridged HbA derivatives.The solvent-accessible central cavity of hemoglobin A (HbA) 1 contains functionally important binding sites for several classes of allosteric effectors that facilitate the lowering of oxygen affinity (1, 2). The ␤-subunit end of the central cavity contains a cluster of eight positive charges that interact with the negative charges of 2,3-diphosphoglycerate (DPG) (3). This site also binds a variety of other negatively charged effectors such as inositol hexaphosphate (IHP), inorganic phosphate, chloride, and polyglutamic acid. The other end (␣-subunit) of the central cavity contains additional binding sites, particularly for chloride ions. Another class of potent effectors derived from clofibric acid and bezafibrate (e.g. L35) bind near the middle of the central cavity with their negative charges projecting toward the ␣-subunit end (4 -6). Binding of these effectors is also associated with a reduction in oxygen affinity. Study of these effectors is of practical interest since control of oxygen affinity is a necessary component for the design of acellular Hb-based oxygen carriers (7, 8).X-ray crystallographic studies are important both in pinpointing effector-binding sites and in characterizing the geometry of the effector-bound site (2). However, other methods must be used to determine the structural and functional interactions that are important in solution and to perform titration studies for obtaining binding constants as a function of solution conditions and/or structural state. Functionally relevant synergistic and antagonistic effects among effectors are also best elucidated through solution studies. Functional characterization of hemoglobin suggests that synergistic and competitive activity can occur when combinations of effectors are bound (4). Of the various allosteric effectors, the interactions of DPG (the natural allosteric effector found in the red blood cell) and its analogs in HbA have been investigated the most extensively. However, the binding of DPG can only be measured indirectly through its effects on ligand reactivity and on spectroscopically accessible chromophores such as the heme groups. In this report, we present an extension o...

Summary. The structural basis of the crystallizing tendencies of oxyHbC (b6Glu → Lys), that produces haemolytic anaemia in homozygotes, is unknown. Using a fluorescent organic phosphate analogue (8-hydroxy-1,3,6-pyrenetrisulphonate), and conventional oxygen equilibrium studies, data suggest that the binding of inositolhexaphosphate (IHP) to oxyHbC differs from HbA, indicating perturbations of the oxyHbC central cavity, which was predicted from our earlier spectroscopic findings. To define the relationship between this conformational change in oxyHbC and its tendency to crystallize, the effect of four central cavity ligands on the crystallization rate was studied: a peptide containing 11 residues from the N-terminal portion of band 3, the full cytoplasmic domain of band 3, 2,3-diphosphoglycerate and IHP. OxyHbC crystallization was accelerated by all these central cavity ligands and not by the appropriate controls. These central cavity changes become an excellent candidate for the dramatic increase in the crystallization rate of oxyHbC.

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We show here that αG-Phila.2βC2 has an increased rate of crystal nucleation compared to α2 βC2 (HbC). We conclude from this finding that position α68, the mutation site of αG-Phila.2 β2 (HbGPhiladelphia), is a contact site in the crystal of HbC. In addition, that HbS enhances HbC crystallization (additive to the effect of αG-Phila, as shown here) and that αG-Phila. inhibits polymerization of HbS are pathogenically relevant previously known facts. All of these findings help explain the phenotype of an individual simultaneously heterozygous for the βS, βC, and the αG-Phila. genes (SCα-G Philadelphia disease). This disease is characterized by a mild clinical course, abundant circulating intraerythrocytic crystals, and increased folded red cells. This phenotype seems to be the result of increased crystallization and decreased polymerization brought about by the opposite effects of the gene product of the αG-Phila. gene on the βC and βS gene products. Some of the intraerythrocytic crystals in this syndrome are unusually long and thin, resembling sugar canes, unlike those seen in SC disease. The mild clinical course associated with increased crystallization implies that, in SC disease, polymerization of HbS is pathogenically more important than the crystallization induced by βC chains. The SCα-G Philadelphia disease is an example of multiple hemoglobin chain interactions (epistatic effect among globin genes) creating a unique phenotype.