【作者】 施海；

【导师】 蔡谨；

【作者基本信息】 浙江大学 ， 生物化工， 2001， 硕士

【摘要】 从新鲜猪血出发，经过超速离心，微孔滤膜过滤等步骤分离纯化得到无基质猪血红蛋白。猪血红蛋白纯化的产物经SDS—PAGE方法分析显示只有一条条带，证明了无基质血红蛋白容易发生四聚体的解聚现象，测定了无基质猪血红蛋白的半饱和氧分压(P₅₀)和希尔系数（n）等有关猪血红蛋白（pHb）携氧能力的参数，并考察了别构效应调节剂对pHb携氧能力的影响。 用小分子试剂双（3，5—二溴水杨酸）延胡索酸酯（DBBF）对pHb进行修饰。与天然血红蛋白相比，DBBF修饰后的血红蛋白具有抗解聚的特性，经SDS—PAGE分析发现DBBF修饰产生的衍生物存在血红蛋白亚基之间的交联。比较了修饰时pHb所处的空间构象（脱氧状态的T构象和氧合状态的R构象）、修饰剂的用量、修饰时间等条件对DBBF修饰pHb的影响，并且DBBF对pHb的修饰还受到别构效应调节剂的影响。 PEG大分子化pHb是延长其在循环系统中的停留时间、降低免疫原性、减少肾脏毒性的有效办法，而PEG修饰一般都使pHb携氧能力下降。我们比较了4种不同方法活化的PEG衍生物对pHb修饰产物携氧能力的影响，还研究了修饰时pHb所处构象对修饰的影响，又用PEG修饰用DBBF内交联的pHb，考察了PEG用量，修饰时间，修饰温度，修饰缓冲液pH值等不同条件对修饰衍生物的携氧能力的影响。在有别构效应调节剂存在的条件下，用DBBF内交联处于脱氧状态的pHb（T态），再用PEG修饰，可以得到携氧能力好，分子量适宜，四聚体稳定的修饰产物。更多还原

【Abstract】 Stroma-free porcine hemoglobin is separated and purified from fresh pig blood by centrifugation and filtration with 0.45 um to 0.22um films. SDS-PAGE analysis of purified porcine hemoglobin shows only one band. It is proved that the dissociation of porcine hemoglobin from tetramer to dimer is taken place. We have determined the oxygen pressure corresponding to the half-saturation of stroma free hemoglobin (P50) and the Hill coefficient (n), and also investigated the dependence of allosteric cofactor(lHP) upon oxygen affinity of pHb.The pHb modified by DBBF shows the special character to resist the dissociation of tetramer in comparison with nature pHb. According to the analysis in SDS-PAGE, the DBBF modified derivatives are intramolecular crosslinked protein. DBBF is utilized to modify oxyhemoglobin and deoxyhemoglobin respectively. The derivatives show different results. We also investigated the affection of the mass of modifier and the modified time to pHb modified by DBBF. The oxygen affinity of pHb modified by DBBF is also affected by allosteric cofactor(IHP).Increasing the total molecular weight of hemoglobin through PEG coupling has been proved to be a good choice for prolonging vascular retention time. As a long, liner, hydrophilic molecule, PEG exerts significant influence on the oxygen-carrying properties of pHb conjugates. PEG-hemoglobin’s conjugates show a wide range of different properties that are attributed to the size of PEGs amounts of bound PEGs, and the presence of allosteric cofactors. Combination of three procedures , DBBF intramolecular crosslinking, allosteric cofactors treating and activated PEG conjugating, result in a stable tetramer pHb derivative with a large molecular weight and well oxygen-carrying capacity.更多还原