Polymerization of deoxyhemoglobin C Harlem (β6 Glu → Val, β73 Asp → Asn) : The effect of β73 asparagine on the gelation and crystallization of hemoglobin

Abstract

The kinetics of aggregation and the solubility of deoxy Hb† C Harlem ( α 2 β 2 6 Val, 73 Asn) in concentrated phosphate buffers were studied in comparison with those of deoxy Hb S and deoxy Hb A. Deoxy Hb C Harlem aggregated with a clear exhibition of a delay time. The length of the delay and aggregation times and the degree of the aggregation depended upon the initial hemoglobin concentration. The initial hemoglobin concentration required for the aggregation of deoxy Hb C Harlem was approximately 200% of its solubility, a value much higher than that required for the aggregation of deoxy Hb S (120%). With the same hemoglobin concentration, the delay time for the aggregation of deoxy Hb C Harlem was approximately 100 times longer than that of deoxy Hb S. The logarithmic plotting of the delay time versus hemoglobin concentration in 1.8 m-phosphate buffer (pH 7.4) showed linear lines with a slope ( n) of 4.0 for deoxy Hb C Harlem. In contrast to the results for the aggregation of deoxy Hb S, n values for deoxy Hb C Harlem were unchanged with phosphate concentrations varying from 1.2 m to 2.0 m. The solubilities of deoxy Hb S and deoxy Hb C Harlem were increased exponentially by lowering the pH of the medium, with the increase being more conspicuous for Hb C Harlem. The gels (or aggregates) of Hb C Harlem were converted to crystals at a rate much faster than were those of Hb A and Hb S. The kinetics for gelation and crystallization of deoxy Hb C Harlem can be explained by the following scheme, where nuclei G and nuclei C are formed before gelation and crystallization, respectively. Monomenc deoxy Hb ▪ The hemoglobin concentration required for the crystallization of deoxy Hb C Harlem was about ten times lower than that required for deoxy Hb A. The solubility of deoxy Hb C Harlem after aggregation was about twice that of deoxy Hb S, suggesting that the substitution of Asn for Asp at the β73 residue inhibits the formation of nuclei G and accelerates the formation of nuclei C.