Determination of the Molecular Weight Distribution of the PEGylated Bovine Hemoglobin (PEG-bHb)

Abstract

The method to determine the average molecular weight and distribution of molecular weight of the PEGylated hemoglobin (PEG-bHb) and their molecular weight of polypeptides in PEG-bHb with UV absorbance (UV), light scattering (LS) and refractive index (RI) detectors is described in this paper. The results indicate that when molar ratio of PEG to hemoglobin is 7:1, the average molecular weight of PEG-bHb (M(CP) is 95 kDa, the average molecular weigh of protein moiety in PEG-bHb (M(P)) is 68 kDa, and their distribution is from 48-157 kDa and 44-121 kDa, respectively; when the molar ratio is 10:1, the M(CP) is 106 kDa, the M(P) is 70kDa, and their distribution is from 56-216kDa, 49-135kDa, respectively; when the molar ratio is 13:1, the M(CP) is 73kDa, and their distribution is from 81-272 kDa and 41-144 kDa, respectively. It has been proved that this method is a simple and reliable way for the determination of average molecular weight and distribution of molecular weight of PEGlyted hemoglobin. This experiment also suggests that there exists not only aggregation in the frontal of elution peak of PEG-bHb, but dissociation of tetramer of hemoglobin into dimmer in the tail of elution peak, and the degree of aggregation is depended on the molar ratio of PEG to hemoglobin due to the present of diol PEG and the dissociation is dependant on the concentration of hemoglobin.