NUCLEATION, FIBER GROWTH AND MELTING, AND DOMAIN FORMATION AND STRUCTURE IN SICKLE-CELL HEMOGLOBIN GELS

Pathogenesis in sickle cell disease depends on polymerization and gela tion of deoxyhemoglobin S. Under the double nucleation model, polymeri zation is initiated by homogeneous nucleation, followed by heterogeneo us nucleation on pre-existing fibers. Fibers grow by non-cooperative a ddition of hemoglobin. The model derives from macroscopic results rath er than direct observation of individual events. We observe individual events and structures by differential interference contrast (DIG) mic roscopy to show consistency with the model, to define structure and de velopment of gel domains and their relation to kinetics, and to demons trate the mechanism of fiber melting. Kinetics were controlled by prod ucing deoxyhemoglobin by photolysis of CO hemoglobin under DIC observa tion. The first visible polymers appeared randomly and were usually li near aggregates less than 1 mu m long, consistent with homogeneous nuc leation and immediate post-nucleation aggregates. Aggregates then bran ched extensively, consistent with heterogeneous nucleation. This branc hing of new fibers was also induced at countable rates on isolated sin gle fibers. Branching and fiber growth rapidly produced dense domains. Changes in photolytic intensity altered domain growth rates and domai n structure. At low intensity and slow growth, fibers grew radially wi thout branching. Domains lacked cross-links and polymer density was lo w. High intensity produced faster growth, much heterogeneous nucleatio n and highly cross-linked, dense, domains. At still higher intensity, homogeneous nucleation was very rapid, producing many small domains. T hese results show a hierarchy of processes: as deoxyhemoglobin concent ration increases, growth occurs without observable nucleations, and th en heterogeneous and finally homogeneous nucleation become dominant. T his is consistent with the double nucleation model under which the con centration 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 pho tolysis reversed this, producing growth. Isolated fibers melted and gr ew at both ends. The results are consistent with a fiber melting mecha nism that is the reverse of growth.