Li Dongxia, Manjula Belur N, Ho Nancy T, Simplaceanu Virgil, Ho Chien, Acharya A Seetharama
Department of Physiology, Albert Einstein College of Medicine, Bronx, NY, USA.
Artif Cells Blood Substit Immobil Biotechnol. 2007;35(1):19-29. doi: 10.1080/10731190600974376.
The development of hexaPEGylated Hb, (SP-PEG5K)(6)-Hb, using the newly designed thiolation-mediated maleimide chemistry based PEGylation, has validated the concept that engineering 'plasma volume expander' -like properties to Hb neutralizes its vasoactivity. The high O(2) affinity of hexaPEGylated Hb has been attributed to the two PEG-5K chains on its two Cys-93(beta) residues. In an attempt to map the influence of the additional four PEG-5K chains of HexaPEGylated Hb on the O(2) affinity, we have now investigated the influence of PEGylation of the surface amino groups alone on the subunit interface interactions and O(2) affinity of Hb using rHb(betaC93A). The molecular radius of PEGylated rHb(betaC93A) was slightly smaller than that of (SP-PEG5K)(6)-Hb, and the overall site-selectivity of PEGylation in the PEGylated rHb(betaC93A) at Lys-residues was comparable to that of (SP-PEG5K)(6)-Hb. Proton NMR studies have shown that the conjugation of the protein with PEG-5K does not have any significant influence on its subunit interface interactions. Surprisingly, the influence of PEGylation on the O(2) affinity and Bohr effect of HbA and rHb(betaC93A) is also nearly the same. Apparently, conjugation of PEG-chains to Lys residues of Hb by the thiolation mediated PEGylation induces unique changes in the structure of the hydration shell of Hb (layer of tightly bound water molecules), which, in turn, induces constraints in its R to T conformational transition to favor the more hydrated R-state.
利用新设计的基于硫醇化介导的马来酰亚胺化学的聚乙二醇化方法开发的六聚聚乙二醇化血红蛋白（SP-PEG5K）(6)-Hb，验证了这样一个概念，即对血红蛋白进行工程改造使其具有“血浆容量扩充剂”样特性可中和其血管活性。六聚聚乙二醇化血红蛋白的高氧亲和力归因于其两个Cys-93(β)残基上的两条PEG-5K链。为了确定六聚聚乙二醇化血红蛋白额外的四条PEG-5K链对氧亲和力的影响，我们现在使用rHb(βC93A)研究了仅对表面氨基进行聚乙二醇化对血红蛋白亚基界面相互作用和氧亲和力的影响。聚乙二醇化rHb(βC93A)的分子半径略小于(SP-PEG5K)(6)-Hb，聚乙二醇化rHb(βC93A)中赖氨酸残基处聚乙二醇化的整体位点选择性与(SP-PEG5K)(6)-Hb相当。质子核磁共振研究表明，蛋白质与PEG-5K的结合对其亚基界面相互作用没有任何显著影响。令人惊讶的是，聚乙二醇化对HbA和rHb(βC93A)的氧亲和力和波尔效应的影响也几乎相同。显然，通过硫醇化介导的聚乙二醇化将PEG链与血红蛋白的赖氨酸残基结合，会导致血红蛋白水合壳层（紧密结合的水分子层）结构发生独特变化，进而在其从R态到T态的构象转变中产生限制，有利于更水化的R态。
