Acharya Seetharama A, Acharya Vivek N, Kanika Nirmala Devi, Tsai Amy G, Intaglietta Marcos, Manjula Belur N
Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA.
Biochem J. 2007 Aug 1;405(3):503-11. doi: 10.1042/BJ20070238.
TetraPEGylated canine Hb, [SP (succinimidophenyl)-PEG5K]4-canine-Hb, with PEGylation at its four reactive cysteine residues (a111 and b93) has been prepared and characterized. The hydrodynamic volume and the molecular radius of (SP-PEG5K)4-canine-Hb are intermediate to those of di- and hexaPEGylated human Hb as expected. However, the COP (colloidal osmotic pressure) of tetraPEGylated canine Hb is closer to that of hexaPEGylated human Hb than to that of diPEGylated human Hb. The O2 affinity of tetraPEGylated canine Hb is higher than that of canine Hb and comparable with that of hexaPEGylated Hb. The O2 affinity of tetraPEGylated canine Hb is not responsive to the presence of DPG (diphosphoglycerate) or chloride, but it retains almost full response to L-35, an allosteric effector that interacts at the aa-end of the central cavity. The tetraPEGylated canine Hb is vasoinactive in hamster in 10% top load infusion studies. It is also essentially non-hypertensive in an extreme exchange haemodilution protocol in hamster just as di- and hexaPEGylated human Hb. The O2 delivery by tetraPEGylated canine Hb is comparable with that of hexaPEGylated Hb but not as efficient as diPEGylated Hb. These results demonstrate that PEGylation-induced solution properties of PEG [poly(ethylene glycol)]-Hb conjugates are dictated by the level and chemistry of PEGylation and the interplay of these plays a critical role in tissue oxygenation. The studies imply the need to establish the right level (and/or pattern) of PEGylation and O2 affinity of Hb-PEG adducts in designing O2-carrying plasma volume expanders, and this remains the primary challenge in the design of PEGylated Hb as blood substitutes.
已制备并表征了四聚乙二醇化犬血红蛋白[SP（琥珀酰亚胺基苯基）-PEG5K]4-犬血红蛋白，其在四个反应性半胱氨酸残基（a111和b93）处进行了聚乙二醇化修饰。正如预期的那样，（SP-PEG5K）4-犬血红蛋白的流体力学体积和分子半径介于二聚乙二醇化和六聚乙二醇化人血红蛋白之间。然而，四聚乙二醇化犬血红蛋白的胶体渗透压（COP）更接近六聚乙二醇化人血红蛋白，而不是二聚乙二醇化人血红蛋白。四聚乙二醇化犬血红蛋白的氧亲和力高于犬血红蛋白，与六聚乙二醇化血红蛋白相当。四聚乙二醇化犬血红蛋白的氧亲和力对二磷酸甘油酸（DPG）或氯离子的存在无反应，但对L-35（一种在中央腔aa端相互作用的变构效应剂）仍保持几乎完全的反应。在仓鼠10%最大负荷输注研究中，四聚乙二醇化犬血红蛋白无血管活性。在仓鼠的极端交换性血液稀释方案中，它也基本不引起高血压，就像二聚乙二醇化和六聚乙二醇化人血红蛋白一样。四聚乙二醇化犬血红蛋白的氧输送与六聚乙二醇化血红蛋白相当，但不如二聚乙二醇化血红蛋白有效。这些结果表明，聚乙二醇（PEG）-血红蛋白缀合物的聚乙二醇化诱导的溶液性质由聚乙二醇化的水平和化学性质决定，并且它们之间的相互作用在组织氧合中起关键作用。这些研究表明，在设计携氧血浆容量扩充剂时，需要确定血红蛋白-PEG加合物的聚乙二醇化正确水平（和/或模式）和氧亲和力，而这仍然是设计聚乙二醇化血红蛋白作为血液替代品的主要挑战。
