Site-specific PEGylation of hemoglobin at cys-93(β):: Correlation between the colligative properties of the PEGylated protein and the length of the conjugated PEG chain
Site-specific PEGylation of hemoglobin at cys-93(β):: Correlation between the colligative properties of the PEGylated protein and the length of the conjugated PEG chain
复制标题
DOI：
10.1021/bc0200733
复制
发表时间：
2003-03-01
影响因子：
4.7
通讯作者：
Acharya, AS
中科院分区：
文献类型：
--
作者：
Manjula, BN;Tsai, S;Acharya, AS
已经提出增加无细胞血红蛋白（Hb）的分子大小作为减少其不良血管活性性质的方法。每个四聚体修饰有约10个拷贝的PEG 5 K的牛Hb表面具有血管活性的发现为这一概念提供了支持。PEG化的牛Hb具有比HbA大得多的分子半径（1）。聚乙二醇化牛血红蛋白的依数性不同于HbA，甚至聚合血红蛋白，这表明聚乙二醇化血红蛋白的依数性在中和非细胞血红蛋白的血管活性中的作用。为了将表面修饰的Hb的依数性与所连接的PEG的质量以及其血管活性相关联，我们开发了一种新的基于马来酰亚胺的方案，用于PEG与Hb的位点特异性缀合，利用氧基HbA的Cys-93（p）的异常高的反应性和马来酰亚胺与蛋白质硫醇的高反应性。5、10和20 kDa的PEG链已在其羟基之一处通过氨基甲酸酯键用马来苯基部分官能化，并用于在HbA的β-93 Cys处缀合PEG链以产生每个四聚体携带两个拷贝的PEG（不同链长）的PEG化Hb。通过离子交换色谱法分离了（SP-PEG 5 K）2-HbA、（SPPEG 10 K）2-HbA和（SP-PEG 20 K）2-HbA的均质制剂。聚乙二醇化后血红蛋白的氧亲和力略有增加，但聚乙二醇链的长度对氧-2亲和力的额外影响很小。血红蛋白的流体动力学体积和分子半径增加表面修饰与PEG链的质量呈线性相关。聚乙二醇化血红蛋白的粘度和胶体渗透压（COP）均随PEG链长的增加呈指数增加。与分子体积、粘度和COP相反，PEG化血红蛋白的血管活性与PEG链长没有直接相关性。PEG链长度似乎有一个阈值，超过该阈值，对血管活性的保护作用就会降低。这些结果表明，通过PEG的Hb的血管活性的调制可以是由PEG提供的表面屏蔽的函数，后者是PEG链在蛋白质表面上的布置的函数，这又是PEG链的长度的函数。因此，在本研究中描述的生物化学均匀的聚乙二醇化血红蛋白，表面修饰与适当大小的PEG链，可以作为潜在的候选人血红蛋白为基础的氧载体。
Increasing the molecular size of acellular hemoglobin (Hb) has been proposed as an approach to reduce its undesirable vasoactive properties. The finding that bovine Hb surface decorated with about 10 copies of PEG5K per tetramer is vasoactive provides support for this concept. The PEGylated bovine Hb has a strikingly larger molecular radius than HbA (1). The colligative properties of the PEGylated bovine Hb are distinct from those of HbA and even polymerized Hb, suggesting a role for the colligative properties of PEGylated Hb in neutralizing the vasoactivity of acellular Hb. To correlate the colligative properties of surface-decorated Hb with the mass of the PEG attached and also its vasoactivity, we have developed a new maleimide-based protocol for the site-specific conjugation of PEG to Hb, taking advantage of the unusually high reactivity of Cys-93(p) of oxy HbA and the high reactivity of the maleimide to protein thiols. PEG chains of 5, 10, and 20 kDa have been functionalized at one of their hydroxyl groups with a maleidophenyl moiety through a carbamate linkage and used to conjugate the PEG chains at the beta-93 Cys of HbA to generate PEGylated Hbs carrying two copies of PEG (of varying chain length) per tetramer. Homogeneous preparations of (SP-PEG5K)2-HbA, (SPPEG10K)2-HbA, and (SP-PEG20K)2-HbA have been isolated by ion exchange chromatography. The oxygen affinity of Hb is increased slightly on PEGylation, but the length of the PEG-chain had very little additional influence on the O-2 affinity. Both the hydrodynamic volume and the molecular radius of the Hb increased on surface decoration with PEG and exhibited a linear correlation with the mass of the PEG chain attached. On the other hand, both the viscosity and the colloidal osmotic pressure (COP) of the PEGylated Hbs exhibited an exponential increase with the increase in PEG chain length. In contrast to the molecular volume, viscosity, and COP, the vasoactivity of the PEGylated Hbs was not a direct correlate of the PEG chain length. There appeared to be a threshold for the PEG chain length beyond which the protection against vasoactivity is decreased. These results suggest that the modulation of the vasoactivity of Hb by PEG could be a function of the surface shielding afforded by the PEG, the latter being a function of the disposition of the PEG chain on the protein surface, which in turn is a function of the length of the PEG chain. Thus, the biochemically homogeneous PEGylated Hbs described in the present study, surface-decorated with PEG chains of appropriate size, could serve as potential candidates for Hb-based oxygen carriers.