Reversible protection of Cys-93(β) by PEG alters the structural and functional properties of the PEGylated hemoglobin

Qingqing Wang; Lijing Sun; Shaoyang Ji; Dawei Zhao; Jiaxin Liu; Zhiguo Su; Tao Hu

doi:10.1016/j.bbapap.2014.04.005

Reversible protection of Cys-93(β) by PEG alters the structural and functional properties of the PEGylated hemoglobin

Biochim Biophys Acta. 2014 Jul;1844(7):1201-7. doi: 10.1016/j.bbapap.2014.04.005. Epub 2014 Apr 15.

Authors

Qingqing Wang¹, Lijing Sun², Shaoyang Ji², Dawei Zhao¹, Jiaxin Liu³, Zhiguo Su⁴, Tao Hu⁵

Affiliations

¹ 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.
² National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
³ Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu 610052, Sichuan Province, China.
⁴ 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.
⁵ National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. Electronic address: thu@ipe.ac.cn.

PMID: 24747784
DOI: 10.1016/j.bbapap.2014.04.005

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.

Keywords: Autoxidation; Hemoglobin; PEGylation; Tetramer stability.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Chromatography, Gel
Chromatography, High Pressure Liquid
Circular Dichroism
Cysteine / chemistry*
Hemoglobin A / chemistry*
Hemoglobin A / metabolism*
Oxygen / metabolism*
Polyethylene Glycols / chemistry*
Ultracentrifugation

Substances

Polyethylene Glycols
Hemoglobin A
Cysteine
Oxygen