Hu Tao, Manjula Belur N, Li Dongxia, Brenowitz Michael, Acharya Seetharama A
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Biochem J. 2007 Feb 15;402(1):143-51. doi: 10.1042/BJ20061434.
The influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated {PEG [poly(ethylene glycol)]-conjugated} haemoglobin has been investigated. The sites and the extent of PEGylation of haemoglobin by reductive alkylation are not influenced by the presence of an alphaalpha-fumaryl cross-link at Lys-99(alpha). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)(6)-alphaalpha-Hb, exhibits a larger molecular radius and lower colloidal osmotic pressure than propylated hexaPEGylated non-cross-linked haemoglobin, (propyl-PEG5K)(6)-Hb. Perturbation of the haem microenvironment and the alpha1beta2 interface by PEGylation of haemoglobin is reduced by intramolecular cross-linking. Sedimentation velocity analysis established that PEGylation destabilizes the tetrameric structure of haemoglobin. (Propyl-PEG5K)(6)-Hb and (propyl-PEG5K)(6)-alphaalpha-Hb sediment as stable dimeric and tetrameric molecules, respectively. The betabeta-succinimidophenyl PEG-2000 cross-link at Cys-93(beta) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the alphaalpha-fumaryl cross-link within the central cavity. However, the influence of the two cross-links on the oxygen affinity of PEGylated haemoglobin are very distinct, indicating that the high oxygen affinity of PEGylated haemoglobin is not a direct consequence of the dissociation of the haemoglobin tetramers into dimers. alphaalpha-Fumaryl cross-linking is preferred to modulate both oxygen affinity and molecular properties of PEGylated haemoglobin, and cross-linking outside the central cavity could only modulate molecular properties of PEGylated haemoglobin. It is suggested that PEGylation induces a hydrodynamic drag on haemoglobin and this plays a role in the microcirculatory properties of PEGylated haemoglobin.
研究了分子内交联对聚乙二醇化（聚乙二醇共轭）血红蛋白的分子、结构和功能特性的影响。通过还原烷基化对血红蛋白进行聚乙二醇化的位点和程度不受 Lys-99(α) 处 αα-富马酰交联的影响。丙基化的六聚乙二醇化交联血红蛋白 (propyl-PEG5K)(6)-αα-Hb 比丙基化的六聚乙二醇化非交联血红蛋白 (propyl-PEG5K)(6)-Hb 具有更大的分子半径和更低的胶体渗透压。分子内交联减少了聚乙二醇化对血红素微环境和 α1β2 界面的扰动。沉降速度分析表明，聚乙二醇化使血红蛋白的四聚体结构不稳定。(propyl-PEG5K)(6)-Hb 和 (propyl-PEG5K)(6)-αα-Hb 分别以稳定的二聚体和四聚体分子形式沉降。中心腔外 Cys-93(β) 处的 ββ-琥珀酰亚胺苯基聚乙二醇-2000 交联也影响血红蛋白的分子特性，与中心腔内的 αα-富马酰交联相当。然而，这两种交联对聚乙二醇化血红蛋白氧亲和力的影响非常不同，表明聚乙二醇化血红蛋白的高氧亲和力不是血红蛋白四聚体解离成二聚体的直接结果。αα-富马酰交联更适合调节聚乙二醇化血红蛋白的氧亲和力和分子特性，而中心腔外的交联只能调节聚乙二醇化血红蛋白的分子特性。有人认为，聚乙二醇化会对血红蛋白产生流体动力学阻力，这在聚乙二醇化血红蛋白的微循环特性中起作用。
