title: Reversible protection of Cys-93(β) by PEG alters the structural and functional properties of the PEGylated hemoglobin.
abstract: As a potential hemoglobin (Hb)-based oxygen carrier (HBOC), the PEGylated Hb has received much attention for its non-nephrotoxicity. However, PEGylation can adversely alter the structural and functional properties of Hb. The site of PEGylation is an important factor to determine the structure and function of the PEGylated Hb. Thus, protection of some sensitive residues of Hb from PEGylation is of great significance to develop the PEGylated Hb as HBOC. Here, Cys-93(β) of Hb was conjugated with 20 kDa polyethylene glycol (PEG20K) through hydrazone and disulfide bonds. Then, the conjugate was modified with PEG5K succinimidyl carbonate (PEG5K-SC) using acylation chemistry, followed by removal of PEG20K Hb with hydrazone hydrolysis and disulfide reduction. Reversible conjugation of PEG20K at Cys-93(β) can protect Lys-95(β), Val-1(α) and Lys-16(α) of Hb from PEGylation with PEG5K-SC. The autoxidation rate, oxygen affinity, structural perturbation and tetramer instability of the PEGylated Hb were significantly decreased upon protection with PEG20K. The present study is expected to improve the efficacy of the PEGylated Hb as an oxygen therapeutic.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China; Graduate University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Wang
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Lijing
authors.initials: L
authors.lastname: Sun
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Shaoyang
authors.initials: S
authors.lastname: Ji
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China; Graduate University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Zhao
authors.affiliation: Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu 610052, Sichuan Province, China.
authors.forename: Jiaxin
authors.initials: J
authors.lastname: Liu
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, China. Electronic address: zgsu@home.ipe.ac.cn.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. Electronic address: thu@ipe.ac.cn.
authors.forename: Tao
authors.initials: T
authors.lastname: Hu
mesh_terms: D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D006441:Hemoglobin A / Q000737:chemistry* / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry*; D014461:Ultracentrifugation
chemical_list: D011092:Polyethylene Glycols; D006441:Hemoglobin A; D003545:Cysteine; D010100:Oxygen
keywords: Autoxidation; Hemoglobin; PEGylation; Tetramer stability