Resolution of G gamma and A gamma foetal haemoglobin tetramers in immobilized pH gradients

Abstract

Intact tetramers of foetal haemoglobin (G gamma, A gamma and the mutant A gamma T) can be separated by isoelectric focusing in immobilized pH gradients over a very shallow pH interval (pH 7.35-7.55). The G gamma tetramer exhibits a lower pI (7.450) than the A gamma tetramer (pI 7.453); the delta pI between the two species is barely 0.003 of a pH unit, close to the theoretical resolution limit of the technique, delta pI = 0.001. Haem-free, denatured gamma chains exhibit a reversal in pI order, the A gamma chain being more acidic than the G gamma chains: this is attributed to preferential binding of detergent micelles to the more hydrophobic A gamma 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.