临床输血是多种疾病治疗的重要手段。然而，天然血液具有来源匮乏、应用前需要进行血型匹配以防止交叉感染等弊端，使得诸多学者开始进行红细胞代用品的研究。本文旨在借助化学修饰方法调控成人血红蛋白（Human adult hemoglobin，HbA）的氧亲和力以获得一种低氧亲和力、高四聚体稳定性和高自氧化速率的三重修饰血红蛋白氧载体。本课题以HbA作为研究对象，利用分子内交联、还原烷基化以及琥珀酰亚胺化学反应对其进行化学修饰，并研究修饰前后HbA及其衍生物相关的结构和功能特征。研究内容主要分为两个部分：首先，利用双（3，5-二溴水杨酸基）延胡索酸酯（Bis (3,5-dibromosalicylate) fumurate，DBBF）对血红蛋白分子进行分子内交联，提高游离血红蛋白的四聚体稳定性，使之具备类似于红细胞内的四聚体结构形式从而降低氧亲和力；在此基础上再利用乙醛酸钠对其进行末端羧甲基化反应，凭借静电排斥作用维持其低氧亲和力的T态，得到三种小分子修饰产物（αα-Hb、Glx-Hb以及Glx-αα-Hb）并探究了修饰前后血红蛋白分子的结构和功能性质等变化情况。随后，利用带有八臂琥珀酰亚胺基团的聚乙二醇（Polyethylene glycol，PEG）对HbA及其衍生物进行共价修饰得到四种PEG化血红蛋白分子（PEG-Hb、PEG-αα-Hb、PEG-Glx-Hb以及PEG-Glx-αα-Hb），以进一步提高其四聚体的稳定性，并增加其流体力学体积，以降低因血管外溢而引发的肾毒性同时也可以降低免疫原性。根据研究结果发现，双重修饰产物（Glx-αα-Hb）的P50值为34.6 mmHg，约为原蛋白的3倍，氧转运效率（Oxygen transfer efficient，OTE）则由原蛋白的9.1%增加到33.1%；三重修饰产物（PEG-Glx-αα-Hb）的P50和OTE较Glx-αα-Hb有轻微的降低，其中P50为27.8 mmHg且OTE为30.5%。同时伴随着自氧化速率的增加和高铁血红蛋白（metHb）含量的增大，值得指出的是化学修饰程度越高对应的自氧化速率和metHb含量越大。其中，Glx-αα-Hb的自氧化速率和metHb含量分别为0.036 h-1和12%（4小时），而PEG-Glx-αα-Hb则为0.044 h-1和15%。圆二色光谱远紫外区域（260~190 nm）的结果表明化学修饰并未对HbA二级结构没有产生影响，但近紫外区域（480~260 nm）的结果表明双重修饰和三重修饰均影响了对血红蛋白的四级结构和血红素的微环境。SDS-PAGE凝胶电泳结果显示，发生分子内交联反应后的修饰产物在32 kDa附近具有明显条带，这是由于分子内交联反应使得HbA的两个Lys-99(α)残基之间形成了富马酰亚胺桥的共价连接；经八臂PEG修饰后HbA的相对分子质量主要集中在90 kDa及以上区域，可见PEG化共价修饰能明显增加血红蛋白的相对分子质量。分子排阻层析法分析结果表明，经PEG修饰后血红蛋白的出峰位置较其所对应的原蛋白向左移动其流体力学体积明显增大，此结果也在一定程度上说明其与稳定性增强有关；拉曼光谱学图谱结果表明，双修饰产物在1340~1390 cm-1范围内拉曼强度明显增加，这与其四级结构的改变存在着联系。借助胰蛋白酶肽图谱和高效液相色谱确定双重化学修饰反应发生的位点是Val-1(α)和Lys-99(α)。根据紫外分光光度法动力学可知包括原蛋白在内的八种化学修饰产物中巯基数量并未发生改变，但不同的修饰方法对巯基反应活性的影响存在一定差异，主要是由于化学修饰对HbA的α1β2界面造成了扰动。基于分子内交联、还原烷基化以及琥珀酰亚胺化学反应修饰得到的三重修饰血红蛋白氧载体具有较低的氧亲和力能够有效地向缺氧组织释放氧气同时伴随着自氧化速率的增加和metHb的产生；由于富马酰亚胺桥的形成以及八臂PEG的修饰增强了亚基间的相互作用使得HbA的四聚体稳定性提高。;Blood transfusion is a major clinical therapy of multiple diseases. However, natural blood has many disadvantages, including insufficient sources and matching before application to prevent cross infection. Thus, many international researchers turned their interest on red cell substitutes. The article aims is regulating oxygen affinity of human adult hemoglobin by chemical modification to acquire triply-modified HbA with low oxygen affinity, high auto-oxidation rate and high tetramer stability.In the thesis, HbA as the research object, was modified by intramolecular cross-linking, reductive alkylation and succinimide chemistry. The functional and structural properties of the triply modified HbA were investigated. The project on chemical modification of HbA can be divided into two sections. At first, HbA was intramolecular cross-linked by using bis (3,5-dibromosalicylate) fumarate, which can improve tetramer stability and oxygen affinity similar with HbA in red blood cells. Subsequently, sodium glyoxylate monohydrate was used to modify N-terminus of crosslinked HbA by reductive alkylation. This method can maintain lower oxygen affinity state (T state) for the static repulsion. Three HbA samples (αα-Hb, Glx-Hb and Glx-αα-Hb) were acquired by these methods. Furthermore, the structural and functional properties of the modified HbA were investigated. Subsequently, four HbA samples were covalently modified using polyethylene glycol with eight succinimide motifs (Eight-arm PEG-NHS 10 kDa) and acquired PEGylated HbA samples (PEG-Hb、PEG-αα-Hb、PEG-Glx-Hb and PEG-Glx-αα-Hb) to further improve the tetramer stability and increase the hydrodynamic volume for reduction of renal toxicity caused by vascular overflow and decrease immunogenicity.Results show that P50 value of Glx-αα-Hb is 34.6 mmHg, about three times than HbA. The dual modification significantly increased oxygen transfer efficient of HbA from 9.1% to 33.1%. Eight-arm PEG-based polymerization slightly decreased the P50 of the Hb derivatives to 27.8 mmHg and OTE to 30.5%, followed by increase in the auto-oxidation rate and the metHb concentrate. It is worth noting that the higher the degree of chemical modification, the greater auto-oxidation rate and the greater content of metHb. Glx-αα-Hb has the highest auto-oxidation rate of 0.036 h-1 and metHb concentration of 12% (after four hours), while PEG-Glx-αα-Hb was 0.044 h-1 and 15%. The far-UV region of circular spectrum (260~190 nm) results showed that chemical modification did not change the secondary structure of HbA. However, the near-UV region (480~260 nm) showed that dual modification and triple modification have some impacts on quaternary structure of hemoglobin and microenvironment of heme. SDS-PAGE analysis shows that the intramolecular cross-linking hemoglobin has a band at 32 kDa, due to that the fumarimide bridge is constructed at Lys-99(α) between two α subunits. The mass of PEGylated HbA is mainly in the region of 90 kDa. It can be seen that PEGylation can significantly increase the molecular mass of HbA. Size exclusion chromatography analysis showed that the peak position of PEGylated HbA move to the left compared with its corresponding original protein and hydrodynamic volume of PEGylated hemoglobin increased significantly, which was related to enhanced stability. The Raman spectrum shows that Raman intensity of dual modification significantly increases in the region of 1340~1390 cm-1, which has relation with the change of quaternary structure. The result of tryptic peptide mapping and reserved-phase high performance liquid chromatography shows that dual modified sites are Val-1(α) and Lys-99(α). According to the kinetics of UV spectrophotometry, the number of thiol number in eight chemical modified samples including the original protein has not changed, while the different chemical modified methods have the different effect on the reaction activity of thiol. The main reason is that chemical modification disturbs the α1β2 interface.The triple modified hemoglobin-based oxygen carriers based on intramolecular cross-linking, reductive alkylation and succinimide chemical reaction has low oxygen affinity and can effectively release oxygen to hypoxic tissues along with an increase in auto-oxidation rate and the content of metHb. Due to the formation of fumarimide bridges and the modification of eight-arm PEG-NHS, the interaction between subunits is enhanced and the stability of tetramer is improved.