Extension arm facilitated pegylation of alphaalpha-hemoglobin with modifications targeted exclusively to amino groups: functional and structural advantages of free Cys-93(beta) in the PEG-Hb adduct.
Cys-93(beta) of hemoglobin (Hb) was reversibly protected as a mixed disulfide with thiopyridine during extension arm facilitated (EAF) PEGylation and its influence on the structural and functional properties of the EAF-PEG-Hb has been investigated. Avoiding PEGylation of Cys-93(beta) in the EAF-PEG-Hb lowers the level of perturbation of heme pocket, alpha1beta2 interface, autoxidation, heme loss, and the O(2) affinity, as compared to the EAF-PEG-Hb with PEGylation of Cys-93(beta).The structural and functional advantages of reversible protection of Cys-93(beta) during EAF PEGylation of oxy-Hb has been compared with Euro PEG-Hb generated by EAF PEGylation of deoxy Hb where Cys-93(beta) is free in the final product. The alphaalpha-fumaryl cross-linking and EAF PEGylation targeted exclusively to Lys residues has been combined together for generation of second-generation EAF-PEG-Hb with lower oxygen affinity. The PEG chains engineered on Lys as well as PEGylation of Cys-93(beta) independently contribute to the stabilization of oxy conformation of Hb and hence increase the oxygen affinity of Hb. However, oxygen affinity of the EAF-PEG-alphaalpha-Hb is more sensitive to the presence of PEGylation on Cys-93(beta) than that of the EAF-PEG-Hb. The present modified EAF PEGylation platform is expected to facilitate the design of novel versions of the EAF-PEG-Hbs that can now integrate the advantages of avoiding PEGylation of Cys-93(beta).
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Dongxia
D
Li
Tao
T
Hu
Belur N
BN
Manjula
Seetharama A
SA
Acharya
D003432:Cross-Linking Reagents; D003545:Cysteine; D015195:Drug Design; D006454:Hemoglobins / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D008239:Lysine / Q000737:chemistry; D015394:Molecular Structure; D010100:Oxygen / Q000737:chemistry*; D010108:Oxyhemoglobins; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000737:chemistry*; D015202:Protein Engineering
D003432:Cross-Linking Reagents; D006454:Hemoglobins; D010108:Oxyhemoglobins; C109960:PEG-hemoglobin; D011092:Polyethylene Glycols; D008239:Lysine; D003545:Cysteine; D010100:Oxygen