“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.