Intramolecular Cross-linking of Beta Subunits and PEGylation of Bovine Stroma Free Hemoglobin For Use as a Hemoglobin Based Oxygen Carrier

Purified Bovine Stroma-Free Hemoglobin\u27s (BSFHb) two beta subunits where intramolecularly cross-linked (BXLHb) using bis(3,5-dibromosalicyl) fumarate (DBBF) and further modified with Polyethylene glycol (BPEGXLHb) for possible use as a Hemoglobin Based Oxygen Carrier (HBOC). Each stage of modification was characterized by size exclusion chromatography and fluorescence methods. We prepared several different molar ratios of DBBF and BSFHb to acquire the highest yield of BXLHb. Cross-linking of the beta subunits will stabilize the whole Hb tetramer from dissociation and prevent unwanted degradation of the HBOC. We prepared a sample modified with PEG (PEGylation) that had a molecular weight of 5kDa. PEGylation increases the total molecular weight of the HBOC which increases the oncotic pressure while the molecule is in the circulatory system. We also tested the stability of each stage of modification through peroxide challenge. Different concentration ratios of peroxide and each Hb were observed and degradation and dissociation was monitored using fluorescence absorbance and electron spin resonance (ESR). Oxygen affinity was tested using HemoxTM Analyzer and the partial pressure at 50 percent (p50) saturation was compared. The data showed that modification of cross-linking the beta subunits (BXLHb) and BPEGXLHb were very stable compared to BSFHb. Although it was observed that the BXLHb modification alone showed a bit more stability than that of the BPEGXLHb, PEGylation is still an essential modification that allows the HBOC to have the proper oncotic pressure and size to remain in the circulatory system where is can function and provide the necessary benefit of carrying oxygen to tissue