Participation of Hemoglobin a in the Aggregation of Deoxyhemoglobin

A diluted solution of a mixture of Hb S and Hb A in concentrated phosphate buffer was found to aggregate with a clear demonstration of delay time. The mechanism of the participation of Hb A in the aggregation of deoxy Hb S in concentrated phosphate buffer has been investigated. Both the delay time and the aggregation time in the polymerization reaction of mixtures of Hb A and Hb S were prolonged as the fraction of Hb A was increased. The logarithmic plot of reciprocal delay time versus hemoglobin concentration of the mixtures of deoxy Hb S and deoxy Hb A showed linear lines with slopes (n values) of 1.4, 1.6, and 1.9 in 3:1, 1:1, and 1:3 mixtures of deoxy Hb S and deoxy Hb A in 2.0 M potassium phosphate buffer. When the same data were plotted against the concentration of deoxy Hb S instead of the total hemoglobin concentration, the last two mixtures showed almost the same line, indicating that deoxy Hb S behaves similarly in mixtures in which t e fraction of Hb S is less than 50%. The intensity of turbidity (A700) increases linearly with an increase in total hemoglobin concentration. The solubilization curve of aggregated hemoglobin measured by repetitive additions of aliquots of water was shifted to the left as the fraction of Hb A was increased until it reached 50%. Aggregates containing more than 50% deoxy Hb A were solubilized rapidly and were not greatly affected by decreasing the fraction of deoxy Hb S. No difference was recognized in the kinetics of the aggregation and solubilization reactions when Hb A and Hb S were mixed in either the oxy or deoxy form, suggesting that the aggregation in concentrated phosphate buffer has no relationship to the formation of an AS hybrid ((~~j3*/3~). There was a linear relationship between the log of reciprocal delay time and log of total hemoglobin concentration with slopes (n values) of 1.6, 3.0, 7.0, and 16 in 2.0, 1.8, 1.5, and 1.0 M potassium phosphate buffers, respectively. The plotting of n values versus phosphate concentration gave a straight line identical with that obtained for pure deoxy Hb S. This suggests that the size of the nuclei produced in the mixture of deoxy Hb S and deoxy Hb A is unaffected by the presence of deoxy Hb A. Electrophoretic analysis showed that Hb A was clearly incorporated in the aggregates and that the percentage of Hb S in aggregates increased with the increase in the Hb S/Hb A ratio of the starting mixture. The fraction of Hb S in the the aggregates