Influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated haemoglobin

Tao Hu, Belur N. Manjula, Dongxia Li, Michael Brenowitz, Seetharama A. Acharya

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

The influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated {PEG [poly (ethylene glycol)]-conjugated} haemoglobin has been investigated. The sites and the extent of PEGylation of haemoglobin by reductive alkylation are not influenced by the presence of an αα-fumaryl cross-link at Lys-99(α). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)6- αα-Hb, exhibits a larger molecular radius and lower colloidal osmotic pressure than propylated hexaPEGylated non-cross-linked haemoglobin, (propyl-PEG5K)6-Hb. Perturbation of the haem microenvironment and the α1β2 interface by PEGylation of haemoglobin is reduced by intramolecular cross-linking. Sedimentation velocity analysis established that PEGylation destabilizes the tetrameric structure of haemoglobin. (Propyl-PEG5K)6-Hb and (propyl-PEG5K)6-αα-Hb sediment as stable dimeric and tetrameric molecules, respectively. The ββ-succinimidophenyl PEG-2000 cross-link at Cys-93(β) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the αα-fumaryl cross-link within the central cavity. However, the influence of the two cross-links on the oxygen affinity of PEGylated haemoglobin are very distinct, indicating that the high oxygen affinity of PEGylated haemoglobin is not a direct consequence of the dissociation of the haemoglobin tetramers into dimers. αα-Fumaryl cross-linking is preferred to modulate both oxygen affinity and molecular properties of PEGylated haemoglobin, and cross-linking outside the central cavity could only modulate molecular properties of PEGylated haemoglobin. It is suggested that PEGylation induces a hydrodynamic drag on haemoglobin and this plays a role in the microcirculatory properties of PEGylated haemoglobin.

Original languageEnglish (US)
Pages (from-to)143-151
Number of pages9
JournalBiochemical Journal
Volume402
Issue number1
DOIs
StatePublished - Feb 15 2007

Keywords

  • Cross-link
  • Haemoglobin
  • PEGylation
  • Reductive alkylation
  • Subunit dissociation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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