NEW SUPPORT FOR THE AFFINITY-CHROMATOGRAPHY OF HEMOGLOBIN

A new support for affinity chromatography of hemoglobin was synthesise d from EAH Sepharose-4B containing a hexamethylamine spacer. Benzenete tracarboxylic (ETC) or benzenehexacarboxylic (BHC) acids were covalent ly bound to the spacer arm. At pH close to the pi of the protein, the biospecificity of the support due to the interactions of the allosteri c site of hemoglobin with immobilised polyanionic ligands was proved. When the allosteric site was blocked by covalently linked pyridoxalpho sphate, the protein showed no more affinity for the support. Further i nvestigations were done on the BHC support; the association constants between BHC support and the hemoglobin forms, oxyhemoglobin and deoxyh emoglobin, were determined. The deoxyhemoglobin affinity was ten times higher than that of oxyhemoglobin, both for fixed and for free ligand . The following values of binding constants K-PX and K-PL (1 mol(-1)) with fixed or free ligand respectively were found: for oxyhemoglobin, K-PX = 8.0.10(2) K-PL = 1.4.10(4); for deoxyhemogrobin, K-PX = 9.7.10( 4), K-PL = 2.3.10(5). The BHC support capacity was about 4.7.10(-5) mo l hemoglobin g(-1) of dry gel corresponding to 1.5.10(-6) mol hemoglob in g(-1) of hydrated gel or 0.1 g hemoglobin g(-1) of hydrated gel.