Influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated haemoglobin.
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 alphaalpha-fumaryl cross-link at Lys-99(alpha). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)(6)-alphaalpha-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 alpha1beta2 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)-alphaalpha-Hb sediment as stable dimeric and tetrameric molecules, respectively. The betabeta-succinimidophenyl PEG-2000 cross-link at Cys-93(beta) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the alphaalpha-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. alphaalpha-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.
Hu
Tao
T
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Manjula
Belur N
BN
Li
Dongxia
D
Brenowitz
Michael
M
Acharya
Seetharama A
SA
D000478:Alkylation; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D006441:Hemoglobin A / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D011092:Polyethylene Glycols; D006441:Hemoglobin A