Role of the β4Thr-β73Asp hydrogen bond in HbS polymer and domain formation from multinucleate-containing clusters

Fiber formation and domain formation from deoxy-HbS as well as from beta 4 and beta 73 HbS variants were investigated after temperature jump using DIC microscopy to gain a basic understanding of the determinants involved. Oversaturated deoxy-HbS generated numerous 14-stranded fibers and formed ovoid-shaped, multispherulitic domains. Domain number increased linearly as a function of time. Oversaturated deoxy-alpha(2)beta(E6V,T4S)(2) also generated time-dependent, ovoid-shaped spherulitic domains like HbS and alpha(2)beta(E6V,D73H)(2) in the deoxy form. In contrast, alpha(2)beta(E6V,T4Y)(2) and HbC-Harlem (alpha(2)beta(E6V,D73N)(2)) in the deoxy form generated time-dependent, ball-shaped domains containing many straight, crystalline-like fibers without evidence of branching. Some of these domains formed large needlelike crystals after overnight incubation. The inhibitory effect on polymer formation by beta 4Tyr in HbS was stronger than that by beta 4Ser but weaker than that by beta 73ASn or beta 73Leu. In contrast, both deoxy- and oxy-alpha(2)beta(E6V,T4V)(2) promoted formation of tiny, disordered amorphous aggregates without a delay time like oxy-HbS, which is in contrast to formation after a delay time of needlelike fibers for alpha(2)beta(E6V,D73L)(2). Solubilities for both deoxy- and oxy-alpha(2) beta(E6V,T4V)(2) were similar to that of deoxy-alpha(2)beta(E6V,D73H)(2) but similar to 10-fold lower than that of deoxy-HbS. These results suggest that the strength of the hydrogen bond between beta 4Thr and beta 73Asp and the balance between the hydrogen bond and beta 6Val hydrophobic interactions in deoxy-HbS polymers control formation of different types of fibers in a single domain or lead to formation of disordered, non-nucleated amorphous aggregates. These results also lead to a model in which multinucleation rather than a single-nucleation event occurs in a single cluster to generate numerous fibers growing from a single domain.