Hu Tao, Li Dongxia, Wang Jun, Wang Qingqing, Liang Yangui, Su Yanlei, Ma Guanghui, Su Zhiguo, Wang Shihua
Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China.
Biochim Biophys Acta. 2012 Dec;1820(12):2044-51. doi: 10.1016/j.bbagen.2012.09.013. Epub 2012 Sep 26.
Hemoglobin (Hb)-based oxygen carriers (HBOCs) are potential pharmaceutical agents that can be used in surgery or emergency medicine. PEGylation can modulate the vasoactivity of Hb and is a widely used approach to develop HBOCs. However, PEGylation can significantly enhance the tetramer-dimer dissociation of Hb, which may perturb the structure of Hb and increase its observed adverse effect. Thus, it is necessary to increase the tetramer stability of the PEGylated Hb.
Propylbenzmethylation at Val-1(α) of HbA was carried out to stabilize the Hb tetramer. The propylbenzmethylated Hb at Val-1(α) (PrB-Hb) was used as the starting material for site-specific PEGylation at Cys-93(β) of Hb using maleimide PEG. Structural and functional properties, autoxidation rate and thermal stability of the resultant product (PEG-PrB-Hb) were measured.
Propylbenzmethylation at Val-1(α) led to 25-fold and 24-fold decreases in the tetramer-dimer dissociation constant of HbA and PEG-Hb, respectively. The increased tetramer stability is due to the enhanced hydrophobicity of the area around Val-1(α) and the increased polar interaction of Hb upon propylbenzmethylation. Thus, the structural and functional properties of PEG-Hb were improved, and its autoxidation rate and thermal denaturation were decreased.
Propylbenzmethylation at Val-1(α) showed higher ability than propylation at Val-1(α) to improve the structural and functional properties and decrease the side effect of PEG-Hb.
Our study can facilitate the biotechnological development of stable PEGylated Hb as more advanced HBOC. Our study is also expected to improve the stability of the tetrameric or dimeric proteins (e.g., uric oxidase) by propylbenzmethylation at their N-terminus.
基于血红蛋白（Hb）的氧载体（HBOCs）是可用于外科手术或急诊医学的潜在药物。聚乙二醇化可调节Hb的血管活性，是开发HBOCs的一种广泛使用的方法。然而，聚乙二醇化可显著增强Hb的四聚体 - 二聚体解离，这可能扰乱Hb的结构并增加其观察到的不良反应。因此，有必要提高聚乙二醇化Hb的四聚体稳定性。
对HbA的Val-1(α)进行丙基苄基化以稳定Hb四聚体。以Val-1(α)处丙基苄基化的Hb（PrB-Hb）为原料，使用马来酰亚胺聚乙二醇对Hb的Cys-93(β)进行位点特异性聚乙二醇化。测量所得产物（PEG-PrB-Hb）的结构和功能特性、自氧化速率和热稳定性。
Val-1(α)处的丙基苄基化分别使HbA和PEG-Hb的四聚体 - 二聚体解离常数降低了25倍和24倍。四聚体稳定性的提高是由于Val-1(α)周围区域疏水性增强以及丙基苄基化后Hb极性相互作用增加。因此，PEG-Hb的结构和功能特性得到改善，其自氧化速率和热变性降低。
Val-1(α)处的丙基苄基化在改善PEG-Hb的结构和功能特性以及降低其副作用方面比Val-1(α)处的丙基化表现出更高的能力。
我们的研究可促进稳定的聚乙二醇化Hb作为更先进的HBOC的生物技术开发。我们的研究还有望通过在其N端进行丙基苄基化来提高四聚体或二聚体蛋白质（如尿酸氧化酶）的稳定性。
