sepharose and pyromellitic-acid

A new support for affinity chromatography of hemoglobin was synthesised from EAH Sepharose-4B containing a hexamethylamine spacer. Benzenetetracarboxylic (BTC) or benzenehexacarboxylic (BHC) acids were covalently 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 allosteric site of hemoglobin with immobilised polyanionic ligands was proved. When the allosteric site was blocked by covalently linked pyridoxalphosphate, the protein showed no more affinity for the support. Further investigations were done on the BHC support; the association constants between BHC support and the hemoglobin forms, oxyhemoglobin and deoxyhemoglobin, 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)=8.0x10(2), K(PL)=1.4x10(4); for deoxyhemoglobin, K(PX)=9.7x10(4), K(PL)=2.3x10(5). The BHC support capacity was about 4.7x10(-5) mol hemoglobin g(-1) of dry gel corresponding to 1.5x10(-6) mol hemoglobin g(-1) of hydrated gel or 0.1 g hemoglobin g(-1) of hydrated gel.

Topics: Benzoates; Chromatography, Affinity; Hemoglobins; Humans; Ligands; Oxyhemoglobins; Sepharose; Spectrophotometry, Ultraviolet