Ligand Binding to Polymerized Pyridoxylated Hemoglobin

Abstract

The physico-chemical properties of polymerized-pyridoxylated human hemoglobin (PPH) have been investigated with special attention to ligand binding equilibria and kinetics.

The preparations (8 different batches) were shown to be heterogeneous by alkaline and SDS electrophoresis, mainly due to the glutaraldehyde polymerization step. In agreement with previous results, the modified hemoglobin binds oxygen with low affinity and strongly reduced cooperativity with respect to HbA; the apparent H for oxygenation at pH 7.3 is very close to that of DPG saturated human hemoglobin A, while the Bohr effect is reduced to approximately half.

Carbon monoxide combination kinetics, studied by stopped flow, yields a biphasics time course; the relative amount of quickly and slowly reacting species (QRF and SRF) proved to be the most sensitive and reliable test for batch variability.