Hemoglobin (Hb) E (␤-Glu26Lys) remains an enigma in terms of its contributions to red blood cell (RBC) pathophysiological mechanisms; for example, EE individuals exhibit a mild chronic anemia, and HbE/␤-thalassemia individuals show a range of clinical manifestations, including high morbidity and death, often resulting from cardiac dysfunction.The purpose of this study was to determine and evaluate structural and functional consequences of the HbE mutation that might account for the pathophysiology. Functional studies indicate minimal allosteric consequence to both oxygen and carbon monoxide binding properties of the ferrous derivatives of HbE. In contrast, redoxsensitive reactions are clearly impacted as seen in the following: 1) the ϳ2.5 times decrease in the rate at which HbE catalyzes nitrite reduction to nitric oxide (NO) relative to HbA, and 2) the accelerated rate of reduction of aquometHbE by L-cysteine (L-Cys). Sol-gel encapsulation studies imply a shift toward a higher redox potential for both the T and R HbE structures that can explain the origin of the reduced nitrite reductase activity of deoxyHbE and the accelerated rate of reduction of aquometHbE by cysteine. Deoxy-and CO HbE crystal structures (derived from crystals grown at or near physiological pH) show loss of hydrogen bonds in the microenvironment of ␤Lys-26 and no significant tertiary conformational perturbations at the allosteric transition sites in the R and T states. Together, these data suggest a model in which the HbE mutation, as a consequence of a relative change in redox properties, decreases the overall rate of Hb-mediated production of bioactive NO.

Earlier observations indicated that volume exclusion by admixed non-hemoglobin macromolecules lowered the polymer solubility ("C sat ") of deoxyhemoglobin (Hb) S, presumably by increasing its activity. In view of the potential usefulness of these observations for in vitro studies of sickling-related polymerization, we examined the ultrastructure, solubility behavior, and phase distributions of deoxygenated mixtures of Hb S with 70-kDa dextran, a relatively inert, low ionic strength space-filling macromolecule. Increasing admixture of dextran progressively lowered the C sat of deoxyHb S. With 12 g/dl dextran, a 5-fold decrease in apparent C sat ("dextran-C sat ") was obtained together with acceptable sensitivity and proportionality with the standard C sat when assessing the effects of non-S Hb admixtures (A, C, and F) or polymerization inhibitors (alkylureas or phenylalanine). The volume fraction of dextran excluding Hb was 70 -75% of total deoxyHb-dextran (12 g/dl) volumes. Electron microscopy showed polymer fibers and fiber-tocrystal transitions indistinguishable from those formed without dextran. Thus when Hb quantities are limited, as with genetically engineered recombinant Hbs or transgenic sickle mice, the dextran-C sat provides convenient and reliable screening of effects of Hb S modifications on polymerization under near-physiological conditions, avoiding problems of high ionic strength.The solubility of deoxyHb 1 S polymers at equilibrium with the tetrameric "monomers" is a basic thermodynamic property of the polymerization process that underlies red cell sickling. Measurement of the polymer solubility, "C sat "(1) (also termed C s (2) or C sol (3)), of Hb S in conditions similar to those within the cells has served as a critical estimate of the relative polymerization tendency when comparing the native or modified Hb S, alone or in mixtures with non-S Hbs or inhibitors of polymerization. A concentrated solution of Hb S is fully deoxygenated to form a gel, and after high speed centrifugation to sediment the polymerized solid phase, the concentration of soluble Hb in the supernatant is the measured C sat .It is well documented that volume exclusion by macromolecules admixed with proteins results in their increased activity and corresponding interactions (4). Thus, when protein macromolecules capable of interacting, such as deoxyHb S, occupy a substantial fraction of the total volume of their medium, the addition of a second species of noninteracting macromolecules crowds the protein molecules closer together, increasing their interactions. More specifically, addition of macromolecules that do not copolymerize with deoxyHb S reduces its polymer solubility by this mechanism (5, 6). However, for the use of such observations in further investigations on the properties of deoxyHb S polymers, it is first necessary to characterize the morphology and properties of the polymer fibers generated in volume-excluded domains by comparison with those previously characterized for standard conditions.In the course of our ...

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