Nucleation, fiber growth and melting, and domain formation and structure in sickle cell hemoglobin gels

Abstract

Pathogenesis in sickle cell disease depends on polymerization and gelation of deoxyhemoglobin S. Under the double nucleation model, polymerization is initiated by homogeneous nucleation, followed by heterogeneous nucleation on pre-existing fibers. Fibers grow by non-cooperative addition of hemoglobin. The model derives from macroscopic results rather than direct observation of individual events. We observe individual events and structures by differential interference contrast (DIC) microscopy to show consistency with the model, to define structure and development of gel domains and their relation to kinetics, and to demonstrate the mechanism of fiber melting. Kinetics were controlled by producing deoxyhemoglobin by photolysis of CO hemoglobin under DIC observation. The first visible polymers appeared randomly and were usually linear aggregates less than 1 micron long, consistent with homogeneous nucleation and immediate post-nucleation aggregates. Aggregates then branched extensively, consistent with heterogeneous nucleation. This branching of new fibers was also induced at countable rates on isolated single fibers. Branching and fiber growth rapidly produced dense domains. Changes in photolytic intensity altered domain growth rates and domain structure. At low intensity and slow growth, fibers grew radially without branching. Domains lacked cross-links and polymer density was low. High intensity produced faster growth, much heterogeneous nucleation and highly cross-linked, dense, domains. At still higher intensity, homogeneous nucleation was very rapid, producing many small domains. These results show a hierarchy of processes: as deoxyhemoglobin concentration increases, growth occurs without observable nucleations, and then heterogeneous and finally homogeneous nucleation become dominant. This is consistent with the double nucleation model under which the concentration dependence of growth is low, and that of heterogeneous and homogeneous nucleation successively higher. Under decreased photolysis, fiber ends melted continuously without fiber breakage; increased photolysis reversed this, producing growth. Isolated fibers melted and grew at both ends. The results are consistent with a fiber melting mechanism that is the reverse of growth.