Huang Yubin, Komatsu Teruyuki, Wang Rong-Min, Nakagawa Akito, Tsuchida Eishun
Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555 Japan.
Bioconjug Chem. 2006 Mar-Apr;17(2):393-8. doi: 10.1021/bc050315+.
Artificial O2-carrying hemoprotein composed of human serum albumin including tetrakis(o-amidophenyl)porphinatoiron(II) (Fe4P or Fe3P) [HSA-FeXP] has been modified by maleimide- or succinimide-terminated poly(ethylene glycol) (PEG), and the formed PEG bioconjugates have been physicochemically characterized. 2-Iminothiolane (IMT) reacted with the amino groups of Lys to create active thiol groups, which bind to alpha-maleimide-omega-methoxy PEG [Mw: 2-kDa (PEG(M2)), 5-kDa (PEG(M5))]. On the other hand, alpha-succinimidyl-omega-methoxy PEG [Mw: 2-kDa (PEG(S2)), 5-kDa (PEG(S5))] directly binds to Lys residues. MALDI-TOF MS of the PEG-conjugated HSA-FeXP showed distinct molecular ion peaks, which provide an accurate number of the PEG chains. In the case of PEG(MY)(HSA-FeXP), the spectroscopic assay of the thiol groups also provided the mean of the binding numbers of the polymers, and the degree of the modification was controlled by the ratio of [IMT]/[HSA]. The viscosity and colloid osmotic pressures of the 2-kDa PEG conjugates (phosphate-buffered saline solution, [HSA] = 5 g dL(-1)) were almost the same as that of the nonmodified one, whereas the 5-kDa PEG binding increased the rheological parameters. The presence of flexible polymers on the HSA surface retarded the association reaction of O2 to FeXP and stabilized the oxygenated complex. Furthermore, PEG(MY)(HSA-FeXP) exhibited a long circulation lifetime of FeXP in rats (13-16 h). On the basis of these results, it can be concluded that the surface modification of HSA-FeXP by PEG has improved its comprehensive O2-transporting ability. In particular the PEG(MY)(HSA-FeXP) solution could be a promising material for entirely synthetic O2-carrying plasma expander as a red cell substitute.
由人血清白蛋白组成、包含四（邻氨基苯基）卟啉铁（II）（Fe4P或Fe3P）[HSA - FeXP]的人工携氧血红蛋白已通过马来酰亚胺或琥珀酰亚胺封端的聚乙二醇（PEG）进行了修饰，并且已对形成的PEG生物共轭物进行了物理化学表征。2 - 亚氨基硫杂环戊烷（IMT）与赖氨酸的氨基反应生成活性硫醇基团，该基团与α - 马来酰亚胺 - ω - 甲氧基PEG[分子量：2 kDa（PEG(M2)），5 kDa（PEG(M5)）]结合。另一方面，α - 琥珀酰亚胺基 - ω - 甲氧基PEG[分子量：2 kDa（PEG(S2)），5 kDa（PEG(S5)）]直接与赖氨酸残基结合。PEG共轭的HSA - FeXP的基质辅助激光解吸电离飞行时间质谱（MALDI - TOF MS）显示出明显的分子离子峰，这提供了准确的PEG链数量。在PEG(MY)(HSA - FeXP)的情况下，硫醇基团的光谱分析也提供了聚合物结合数的平均值，并且修饰程度由[IMT]/[HSA]的比例控制。2 kDa PEG共轭物（磷酸盐缓冲盐溶液，[HSA] = 5 g dL(-1)）的粘度和胶体渗透压与未修饰的几乎相同，而5 kDa PEG结合增加了流变学参数。HSA表面存在柔性聚合物阻碍了O2与FeXP的缔合反应并稳定了氧化复合物。此外，PEG(MY)(HSA - FeXP)在大鼠体内显示出FeXP的长循环寿命（13 - 16小时）。基于这些结果，可以得出结论，PEG对HSA - FeXP的表面修饰提高了其综合携氧能力。特别是PEG(MY)(HSA - FeXP)溶液可能是一种有前途的材料，可作为完全合成的携氧血浆扩容剂作为红细胞替代品。
