Purification of human hemoglobin valence intermediates by preparative immobilized pH gradients

We compare three separation techniques for preparative purposes, i.e. ion-exchange chromatography on CM-cellulose, conventional isoelectric focusing in polyacrylamide gel slabs and immobilized pH gradients. The biological system used to test the three methods is a solution containing four hemoglobin (Hb) valence intermediates, i.e. metHb, oxyHb, (alpha + beta O2)2 and (alpha O2 beta +)2. The delta pI between the two valence intermediates is 0.04 pH units. Immobilized pH gradients give the best performance in terms of resolving power, total amount of protein which can be loaded and retention of biological activity by the protein (the latter assessed by determination of CO dissociation rates).