A solid phase adsorption method for preparation of bovine serum albumin-bovine hemoglobin conjugate

A solid phase adsorption method was proposed to prepare well-defined bovine serum albumin–bovine hemoglobin (Hb) conjugate. After adsorption by the solid phase, Q Sepharose Fast Flow media, bovine serum albumin (BSA) molecules were allowed to react with glutaraldehyde. The spacing out of BSA molecules on the solid phase was assumed to limit polymerization of BSA molecules, except some molecules bound closely on the solid phase resulting in minor dimer formation. Following the elution procedure, the activated monomeric BSA was separated from the dimers by gel filtration chromatography on Superdex 200 and then reacted with bovine Hb at 4 °C and pH 9.5. The 1:1 (BSA:Hb) conjugate was obtained with the yield of 64%. The P₅₀ values of the conjugates, prepared under anaerobic and aerobic conditions, were 19.1 and 14.2 mmHg, respectively. The dependence of the P₅₀ on chloride ions for the conjugate was slightly diminished, presumably due to covalent attachment of BSA to bovine Hb.