A radical view of haemoglobin - protein engineering and characterisation

Marie Grey

Research output: ThesisDoctoral Thesis (compilation)


In recent years shortages of donated blood have become a growing problem for health care systems world-wide. Blood substitutes based on human haemoglobin (Hb) could potentially ameliorate this situation. However, clinical trials have indicated problems due to e.g. oxidative stress. This thesis describes ways to detoxify the Hb by modulating radical reactions or by reducing radical toxicity using a fusion strategy. In addition, production and downstream processing issues are addressed.
In order to modulate electron transfer, amino acid residues in the Hb involved in these pathways were mutated to either increase reduction of the cytotoxic ferryl Hb (HbFe4+=O2-) or to stabilise the radical formed. Moreover, an alternate approach explored to reduce radical toxicity was to fuse superoxide dismutase (SOD) to the Hb alpha chain. SOD–Hb was shown to retain the oxygen binding capacity and in addition decrease ferryl Hb formation when challenged with superoxide radicals. Furthermore, in order to facilitate production of Hb in eukaryotic systems, a diglobin was designed where the Hb alpha chain is genetically linked to the beta chain. The diglobin was shown to be functional and could be secreted in yeast – a feature particularly suitable for large scale production. In order to improve purification efficiency, a new histidine tag comprising both histidine and glutamine residues was attached to the Hb and the diglobin. Interestingly, the His-Gln tag increased Hb expression levels 2-3 fold. Finally, possible structural changes induced by addition of the tag or fusing of the chains were investigated using hydrophobic interaction chromatography and aqueous two-phase partitioning.
Original languageEnglish
Awarding Institution
  • Pure and Applied Biochemistry
  • Bülow, Leif, Supervisor
Award date2009 May 19
ISBN (Print)978-91-628-7752-1
Publication statusPublished - 2009

Bibliographical note

Defence details

Date: 2009-05-19
Time: 09:15
Place: Lecture Hall B, Centre for Chemistry and Chemical Engineering, Getingevägen 60, Lund University Faculty of Engineering

External reviewer(s)

Name: Alayash, Abdu
Title: Dr.
Affiliation: Division of Hematology, Office of Blood Research and Review, Food and Drug Administration (FDA), USA


Subject classification (UKÄ)

  • Biochemistry and Molecular Biology


  • His-Gln tagging
  • protein purification
  • blood substitutes
  • radical scavenging
  • superoxide dismutase
  • E. coli
  • diglobin
  • Haemoglobin
  • protein engineering
  • aqueous two-phase systems
  • hydrophobic interaction chromatography


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