Aggregation and crystallization of hemoglobins A, S, and C. Probable formation of different nuclei for gelation and crystallization.

The oxy and carbonmonoxy forms of Hb A and Hb S formed aggregates or gels when dissolved in phosphate buffers at concentrations above their solubility and warmed rapidly to 30 degrees C from 0 degrees C. Kinetic studies showed that although deoxy-Hb A and deoxy-Hb S aggregated with a clear exhibition of a delay time, the oxy and carbonmonoxy forms of Hb A and Hb S did not show a delay time. These results suggest that the deoxy forms of Hb A and Hb S aggregate according to the nucleation-controlled mechanism, while oxy- and carbonmonoxyhemoglobins aggregate by the simple linear aggregation mechanism. It was also found that the gels or aggregates of deoxy-Hb A and carbonmonoxy-Hb S could be converted to crystals by further incubation. The rate of crystallization depended upon the concentration of hemoglobin in the supernatant, with faster crystallization at higher concentrations. Similar experiments with deoxy-Hb C (beta 6 Glu leads to Lys) showed that this hemoglobin also crystallized after aggregation, with both reactions accompanied by a delay time. The activation energy for the crystallization reaction of deoxy-Hb C (100 approximately 150 kcal/mol) was much higher than that for the aggregation reaction (20 kcal/mol). These results suggest that deoxy-Hb A, deoxy-Hb S, and deoxy-Hb C form two types of nuclei that are specific to the formation of gels (or aggregates) or crystals. The concentration of hemoglobin measured after completion of crystallization was much lower than that measured after gelation (or aggregation) and was independent of the initial hemoglobin concentration. This concentration is assumed to be the real solubility of hemoglobin.