"Inside-Out" PEGylation of Bovine β-Cross-Linked Hemoglobin

The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside-out PEGylation, has been used here to conjugate a multiarm maleimide-PEG (Mal-PEG) to β-cross-linked (βXL-Hb) hemoglobin (Hb) tetramers through the Cys β93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three βXL-Hb to one Mal-PEG. Thermal denaturation studies showed that the cross-linked and PEGylated species were more stable than native Hb. Cross-linking under oxy-conditions produced a high oxygen affinity Hb species (P₅₀  = 9.18 Torr), but the oxygen affinity was not significantly altered by PEGylation (P₅₀  = 9.67 Torr). Inside-out PEGylation can be used to produce a hemoglobin-based oxygen carrier and potentially for other multiprotein complexes.