Resolution of Gγ and Aγ foetal haemoglobin tetramers in immobilized pH gradients

Intact tetramers of foetal haemoglobin (Gγ, Aγ and the mutant AγT) can be separated by isoelectric focusing in immobilized pH gradients over a very shallow pH interval (pH 7.35–7.55). The Gγ tetramer exhibits a lower pI (7.450) than the Aγ tetramer (pI 7.453); the ΔpI between the two species is barely 0.003 of a pH unit, close to the theoretical resolution limit of the technique, ΔpI = 0.001. Haem-free, denatured γ chains exhibit a reversal in pI order, the Aγ chain being more acidic than the Gγ chains; this is attributed to preferential binding of detergent micelles to the more hydrophobic Aγ polypeptide. The advantage of the present technique is the simultaneous analysis of several samples (30–40 per gel slab) and the recovery of intact, haemoglobin tetramers for subsequent studies.