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

Artif Organs. 2017 Apr;41(4):351-358. doi: 10.1111/aor.12928. Epub 2017 Mar 20.

Abstract

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.

Keywords: Cross-linked hemoglobin; Hemoglobin-based oxygen carrier; Multiarm PEG: inside-out PEGylation; Thiol-maleimide click chemistry.

MeSH terms

  • Animals
  • Blood Substitutes / chemical synthesis
  • Blood Substitutes / chemistry*
  • Cattle
  • Chromatography, Gel
  • Cross-Linking Reagents / chemistry*
  • Drug Compounding / methods*
  • Hemoglobins / chemical synthesis
  • Hemoglobins / chemistry*
  • Maleimides / chemistry*
  • Molecular Weight
  • Oxygen / metabolism
  • Polyethylene Glycols / chemical synthesis
  • Polyethylene Glycols / chemistry*
  • Protein Denaturation
  • Ultracentrifugation

Substances

  • Blood Substitutes
  • Cross-Linking Reagents
  • Hemoglobins
  • Maleimides
  • PEG-hemoglobin
  • maleimide
  • Polyethylene Glycols
  • Oxygen