Manjula Belur N, Tsai Amy G, Intaglietta Marcos, Tsai Ching-Hsuan, Ho Chien, Smith Paul K, Perumalsamy Krishnaveni, Kanika Nirmala Devi, Friedman Joel M, Acharya Seetharama A
Departments of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Protein J. 2005 Apr;24(3):133-46. doi: 10.1007/s10930-005-7837-2.
PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)(6)-Hb, that carries approximately six PEG5K chains/Hb - HexaPEGylated Hb. PEGylation increased the O(2) affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)(6)-Hb, its molecular volume, O(2) affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)(2)-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)(6)-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)(2)-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.
聚乙二醇化诱导的血红蛋白A（HbA）分子体积和溶液性质的变化被认为是其血管收缩活性的潜在调节因子。然而，我们最近对携带两条聚乙二醇链/血红蛋白的聚乙二醇化血红蛋白的研究表明，血红蛋白血管收缩活性的调节与聚乙二醇化血红蛋白的分子体积和溶液性质没有直接关联，而是暗示血红蛋白的表面电荷和/或聚乙二醇对血红蛋白的表面修饰模式起到了作用。现在，HbA已通过基于硫醇化介导的马来酰亚胺化学的聚乙二醇化进行修饰，这种修饰不会改变其表面电荷，并结合了多个聚乙二醇5000链拷贝。该方案已得到优化，以生成一种聚乙二醇化血红蛋白，即（SP-PEG5K）(6)-Hb，其携带约六条聚乙二醇5000链/血红蛋白——六聚乙二醇化血红蛋白。聚乙二醇化增加了血红蛋白的氧亲和力，并使该分子对离子强度、pH值和变构效应剂的影响不敏感，这可能是蛋白质周围形成水合聚乙二醇壳的结果。（SP-PEG5K）(6)-Hb中的聚乙二醇总质量、其分子体积、氧亲和力和溶液性质与另一种携带两条聚乙二醇20000链/血红蛋白的聚乙二醇化血红蛋白（SP-PEG20K）(2)-Hb相似。然而，（SP-PEG5K）(6)-Hb表现出比（SP-PEG20K）(2)-Hb显著降低的血管收缩介导反应。这些结果表明，通过将多个小聚乙二醇链拷贝与血红蛋白结合实现的分子大小和溶液性质增强，在降低其血管收缩活性方面比使用少量大聚乙二醇链结合相当质量的聚乙二醇更有效。
