Polyethylene Glycol Improves Conjugation of Bovine Hemoglobin and Human Serum Albumin in a Controlled Ratio

Abstract

Direct conjugation of bovine hemoglobin (Hb) and human serum albumin (HSA) with glutaraldehyde would result in a complex mixture of dimers Hb-Hb, Hb-HSA, HSA-HSA and other oligomers. To obtain a high yield of target Hb-HSA, modulation of the reaction environment was carried out. It was found that polyethylene glycol (PEG), a hydrophilic polymer, could improve the yield of Hb-HSA conjugate. The degree of improvement depended on the molecular weight and concentration of PEG. Under optimum condition of 9% (w/v) of PEG 4000, the reaction proceeded in a controlled mode with conversion yield of starting proteins to Hb-HSA increasing from 6% to 30%. The purity was about 88% of the total conjugates. Furthermore, the impurities were mainly tetrameric molecules of two Hb-HSA conjugates. The improvement could be attributed to the "micro-compartment" created by addition of polyethylene glycol, which brings HSA and Hb close together, thus increasing the chance of conjugation between the two molecules.