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Webster, Kyle D; Dahhan, Dana; Otto, Abigail M; Frosti, Cheyanne L; Dean, William L; Chaires, Jonathan B; Olsen, Kenneth W
Artificial organs, 04/2017, Letnik: 41, Številka: 4Journal Article
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 beta-cross-linked (betaXL-Hb) hemoglobin (Hb) tetramers through the Cys beta93 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 betaXL-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.
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