聚乙二醇（PEG）对Cys-93(β)的可逆保护改变了聚乙二醇化血红蛋白的结构和功能特性。

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

作者信息

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

机构信息

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.

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.

出版信息

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

DOI:10.1016/j.bbapap.2014.04.005

PMID:24747784

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.

摘要

作为一种潜在的基于血红蛋白（Hb）的氧载体（HBOC），聚乙二醇化血红蛋白因其无肾毒性而备受关注。然而，聚乙二醇化会对血红蛋白的结构和功能特性产生不利影响。聚乙二醇化的位点是决定聚乙二醇化血红蛋白结构和功能的重要因素。因此，保护血红蛋白的一些敏感残基不被聚乙二醇化对于将聚乙二醇化血红蛋白开发为HBOC具有重要意义。在此，血红蛋白的Cys-93(β)通过腙键和二硫键与20 kDa聚乙二醇（PEG20K）偶联。然后，使用酰化化学方法用聚乙二醇5000琥珀酰亚胺碳酸酯（PEG5K-SC）对该偶联物进行修饰，随后通过腙水解和二硫键还原去除PEG20K血红蛋白。PEG20K在Cys-93(β)处的可逆偶联可以保护血红蛋白的Lys-95(β)、Val-1(α)和Lys-16(α)不被PEG5K-SC聚乙二醇化。在用PEG20K保护后，聚乙二醇化血红蛋白的自氧化速率、氧亲和力、结构扰动和四聚体不稳定性显著降低。本研究有望提高聚乙二醇化血红蛋白作为氧治疗剂的疗效。