Polymer structure and solubility of deoxyhemoglobin S in the presence of high concentrations of volume-excluding 70-kDa dextran - Effects of non-S hemoglobins and inhibitors

Earlier observations indicated that volume exclusion by admired 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 deoxy Hb-dextran (12 g/dl) volumes. Electron microscopy showed polymer fibers and fiber-to-crystal 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.