Gelation of deoxyhemoglobin A in concentrated phosphate buffer. Exhibition of delay time prior to aggregation and crystallization of deoxyhemoglobin A.

We found that deoxy Hb A aggregated or formed gels with a clear exhibition of a delay time when a solution of deoxy Hb A with a concentration of 120% of its solubility was incubated at 30°C. Since the solubility of deoxy Hb A is much higher than that of deoxy Hb S, this condition was created by increasing the phosphate concentration. The length of the delay and aggregation times and the amounts of aggregates produced depended on the initial hemoglobin concentration. Deoxy Hb A formed gels in 1.5 M phosphate buffer, but formed a suspension of amorphously aggregated hemoglobins in 1.8 M phosphate or above. The gels or aggregates could be totally reliquefied by cooling. These results indicate that the hydrophobic amino acid substitution (Val) at the p-6 position is not essential for the gelation of hemoglobin. The slope of the curve (n value) obtained by the logarithmic plotting of the delay time uewus hemoglobin concentration in 1.8 M phosphate buffer, pH 7.4, was 5.6 instead of the 2.8 of deoxy Hb S. The nuclei formed prior to the aggregation of deoxy Hh A in 1.8 M phosphate buffer may be larger than those of deoxy Hb S. The gel or aggregates of deoxy Hb A thus produced were transformed to three-dimensional crystals by further incubation over a 12-h period. The rate of crystallization was much faster with deoxy Hb A than with deoxy Hb S. No crystallization occurred if deoxy Hb A or deoxy Hb S was merely salted out in 2.5 M phosphate buffer.