"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.
Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL.
Kyle D
KD
Webster
Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL.
Dana
D
Dahhan
Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL.
Abigail M
AM
Otto
Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL.
Cheyanne L
CL
Frosti
James Graham Brown Cancer Center.
William L
WL
Dean
James Graham Brown Cancer Center.|Department of Medicine, University of Louisville, Louisville, KY, USA.
Jonathan B
JB
Chaires
Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL.
Kenneth W
KW
Olsen
D000818:Animals; D001802:Blood Substitutes / Q000138:chemical synthesis / Q000737:chemistry*; D002417:Cattle; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004339:Drug Compounding / Q000379:methods*; D006454:Hemoglobins / Q000138:chemical synthesis / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry*; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000737:chemistry*; D011489:Protein Denaturation; D014461:Ultracentrifugation
D001802:Blood Substitutes; D003432:Cross-Linking Reagents; D006454:Hemoglobins; D008301:Maleimides; C109960:PEG-hemoglobin; C043592:maleimide; D011092:Polyethylene Glycols; D010100:Oxygen
Cross-linked hemoglobin; Hemoglobin-based oxygen carrier; Multiarm PEG: inside-out PEGylation; Thiol-maleimide click chemistry