An urgency to create an effective blood substitute arises from the lack of blood supply from human donors and the risk of blood pathogens. A hemoglobin-based oxygen carrier (HBOC) from a non-human source would carry oxygen to hypoxic tissue treating hemorrhagic trauma and other distresses that provoke oxygen deprivation of vital organs and tissue. However, the injection of unmodified hemoglobin (Hb) into a living system can cause severe complications such as nephritic toxicity and vascular constriction. Crosslinking the bovine hemoglobin with 3,5-dibromosalicylic fumarate (DBSF) at the Lys β82 residues under oxy-conditions is the essential reaction to prevent such toxicity. Crosslinking the tetramer via the β- chains (as opposed to the α-chains) produced a high oxygen affinity product, as expected. In addition to nephritic toxicity, vascular constriction is a concern when hemoglobin tetramers are used to replace blood. This can be prevented by producing a high molecular weight complex thru “Inside-out PEGylation” in which the crosslinked Hb (ββXL-Hb) was attached to the PEG at the Cys β93 residues via sulfhydryl-maleimide click chemistry. The “Inside-out” PEGylation is unique in that it covers an 8-arm PEG backbone with multiple Hb tetramers as compared to previous PEG HBOC’s where the Hb tetramer is concealed within multiple PEG chains. Size exclusion chromatography, SDS-PAGE, and analytical ultracentrifugation (AUC) suggest that the final PEGylated species has 3 or 4 ββXL-Hb attached to each 8-arm PEG. Circular dichroism (CD) and oxygen binding studies validate that PEGylation does not drastically alter the secondary structure or functional properties of the protein. With the successful characterization results, we believe this is the first step to creating a viable HBOC.

“Inside-out” site directed PEGylation of cross-linked hemoglobin

251st National Meeting (2016)

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