Differential pathways in oxy and deoxy HbC aggregation/crystallization

CC individuals, homozygous for the expression of beta (C)-globin, and SC in dividuals expressing both beta (S) and beta (C)-globins, are known to form intraerythrocytic oxy hemoglobin tetragonal crystals with pathophysiologies specific to the phenotype, To date, the question remains as to why HbC for ms in vivo crystals in the oxy state and not in the deoxy state. Our first approach is to study HbC crystallization in vitro, under non-physiological conditions. We present here a comparison of deoxy and oxy HbC crystal forma tion induced under conditions of concentrated phosphate buffer (2g% Db, 1.8 M, potassium phosphate buffer) and viewed by differential interference cont rast microscopy, Oxy HbC formed isotropic amorphous aggregates with subsequ ent tetragonal crystal formation. Also observed, but less numerous, were tw isted, macro-ribbons that appeared to evolve into crystals. Deoxy HbC also formed aggregates and twisted macro-ribbon forms similar to those seen in t he oxy liganded state. However, in contrast to oxy HbC, deoxy HbC favored t he formation of a greater morphologic variety of aggregates including polym eric unbranched fibers in radial arrays with dense centers, with infrequent crystal formation in close spatial relation to both the radial arrays and macroribbons, Unlike the oxy (R-state) tetragonal crystal, deoxy HbC formed hat, hexagonal crystals. These results suggest: (1) the Lys substitution a t beta6 evokes a crystallization process dependent upon ligand state confor mation [i.e., the R (oxy) or T (deoxy) allosteric conformation]; and (2) th e oxy ligand state is thermodynamically driven to a limited number of aggre gation pathways with a high propensity to form the tetragonal crystal struc ture. This is in contrast to the deoxy form of HbC that energetically equal ly favors multiple pathways of aggregation, not all of which might culminat e in crystal formation. Proteins 2001; 42:99-107. (C) 2000 Wiley-Liss, Inc.