title: CO binding improves the structural, functional, physical and anti-oxidation properties of the PEGylated hemoglobin.
abstract: BACKGROUND
  PEGylated hemoglobin (Hb) is a promising oxygen therapeutic agent for clinical application. However, it suffered from structural perturbation, functional instability and methemoglobin (metHb) formation.
  
  
  OBJECTIVE
  To improve the structural, functional, physical and anti-oxidation properties of the PEGylated Hb.
  
  
  METHODS
  PEGylation of Hb with CO binding (HbCO) was conducted using maleimide and acylation chemistry, respectively. Physical and chemical parameters were measured for Hb samples. The circular dichroism spectra, dynamic light scattering and analytical ultracentrifugation were used to investigate the structure and conformation of PEGylated HbCO.
  
  
  RESULTS
  CO binding can inhibit the autoxidation of the PEGylated Hb, structurally stabilize its tetramer and improve its thermal and pH stability. Importantly, the circular dichroism spectra showed that CO binding can decrease the structural perturbation of Hb induced by PEGylation. The PEGylated HbCO with CO release showed slightly higher oxygen-delivery capacity than the PEGylated Hb. The PEGylated HbCO did not show metHb formation after 30-day storage at 4°C.
  
  
  CONCLUSIONS
  CO binding structurally stabilized the PEGylated Hb, abolished its metHb formation, and significantly increased its physical stability. In particular, it also avoided the perturbation of PEG chains on the heme microenvironment. The functional property of the PEGylated HbCO can be maintained during its long-term storage, which is of great significance for field transfusion.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , Beijing , P. R. China.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Wang
authors.forename: Tao
authors.initials: T
authors.lastname: Hu
authors.forename: Lijing
authors.initials: L
authors.lastname: Sun
authors.forename: Shaoyang
authors.initials: S
authors.lastname: Ji
authors.forename: Dawei
authors.initials: D
authors.lastname: Zhao
authors.forename: Jiaxin
authors.initials: J
authors.lastname: Liu
authors.forename: Guanghui
authors.initials: G
authors.lastname: Ma
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
mesh_terms: D001802:Blood Substitutes / Q000737:chemistry*; D002248:Carbon Monoxide / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008706:Methemoglobin / Q000037:antagonists & inhibitors; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D055550:Protein Stability
chemical_list: D001802:Blood Substitutes; D006454:Hemoglobins; D011092:Polyethylene Glycols; D002248:Carbon Monoxide; D008706:Methemoglobin; D010100:Oxygen
keywords: PEGylation; autoxidation; heme environment; oxygen carrier; thermal stability