Polymerization of recombinant hemoglobin F γE6V and hemoglobin F γE6V, γQ87T alone, and in mixtures with hemoglobin S

Abstract

To further understand determinants for Hemoglobin (Hb) S polymerization, as wall as the inhibitory mechanism of Hb F on Hb S polymerization, Hb F variants containing Val-γ6 (Hb F γE6V) or Val-γ6, Thr-γ87 (Hb F γE6V, γQ87T) were expressed in yeast. The oxy form of Hb F γE6V was about 10- fold less stable to mechanical agitation than native oxy Hb F, which is similar to stability differences comparing oxy Hb S and oxy Hb A. Deoxy Hb F γE6V showed ~20-fold decreased solubility compared with native deoxy Hb F in high phosphate buffer and formed gels like deoxy Hb S in low phosphate buffer, indicating that the Val-γ6 substitution decreases solubility of Hb F like Val-β6 in deoxy Hb S. Oversaturated deoxy Hb F γE6V polymerized without a delay time in low and high phosphate buffers, in contrast to deoxy Hb S, which is accompanied by a distinct delay time before polymerization. Deoxy Hb F γE6V, γQ87T also polymerized without a delay time like deoxy Hb F γE6V. These results suggest that deoxy Hb F γE6VγQ87T polymers are different from those of deoxy Hb S, and that contact sites differ from those of deoxy Hb S, even though both have the same primary donor (A3) and acceptor sites in the EF helix. These results also suggest that other amino acids in addition toβ6 Val and amino acids in the F helix are critical for nucleation-controlled polymerization of deoxy Hb S. 1:1 mixtures of deoxy Hb S and either Hb F variant polymerized with a delay time when the concentrations for the Hb S/Hb F γE6V and Hb S/Hb F γE6V, γQ87T mixtures were about 2- and 1,5-fold, respectively, higher than that for Hb S. Logarithmic plots of delay time versus concentration for Hb S/Hb F γE6V mixtures showed the same straight line as the line for Hb S/Hb S βT87Q mixtures, but values for Hb S/Hb F γE6V, γQ87T mixtures were intermediate between those for Hb S and Hb S/Hb F γE6V mixtures. A 1:1 mixture of deoxy Hb A and Hb F γE6V, γQ87T also polymerized, but exhibited biphasic kinetics, when the concentration was increased to more than 3.5-fold higher than that required for Hb S polymer formation. These results suggest that Gln-γ87 is a critical amino acid for exclusion of FS hybrids (α2β(s)γ) from nuclei formation with Hb S. Our findings also show that Val-γ6 in hybrids that form in mixtures of the Hb F variants with either Hb S or Hb A interacts with the hydrophobic acceptor pocket on the EF helix of an adjacent tetramer containing Thr-β87.