Participation of hemoglobins A and F in polymerization of sickle hemoglobin.

We have measured the solubility of sickle hemoglobin by ultracentrifugation of concentrated solutions of deoxyhemoglobins S + A and S + F in 0.06 I phosphate, pH 7.0. Cyanmethemoglobin derivatives were deoxygenated, mixed, and reduced with dithionite in such a way as to produce gelled deoxyhemoglobin solutions containing 50% asymmetrical hybrid tetramers (a~3~/3* or CU#+~) and solutions containing no hybrid. Centrifugation at 140,000 x g for 240 min at 25” permitted the clear separation of sol from gel. The concentration of hemoglobin in the sol phase provided a very precise measure of solubility. The distribution of S and nonS hemoglobins was measured by gel electrofocusing and densitometric scanning. In mixtures containing equal amounts of Hbs S and A, the solubility was 23 g/100 ml, unaffected by the presence or absence of hybrid. In both hybrid and nonhybrid mixtures, the amount of Hb S contained in the gel was directly proportional to the initial percentage of Hb S in the mixture. In contrast, mixtures of Hbs S + F containing 50% hybrid tetramers had a higher solubility (29 g/100 ml) than mixtures containing no hybrid (26 g/100 ml). Hb S was enriched in the gel phase of hybrid and nonhybrid mixtures of both Hbs S + A and Hbs S + F. Nevertheless, our results, corrected for the co-existence of soluble hemoglobin and fiber in the gel phase, indicate that hybrid tetramers (a@/?’ and (~@y) as well as Hb A and even Hb F can co-polymerize with Hb S. The inhibition of sickling by Hb F is due to the presence of y chain in an orientation trans to the subunit having the /36 contact site.