摘要正常成人血红蛋白(hemoglobin,HbA0)与葡萄糖在非酶促反应下形成糖化血红蛋白(hemoglobin A1c,HbA1c) ,通过控制添加葡萄糖的浓度,糖化的酸碱度(pH) 、温度以及反应时间等参数对健康成人血红蛋白进行体外糖化。通过分析蛋白糖化过程中的紫外可见光谱图,免疫法测定其糖化率数据以及不变性-聚丙烯酰胺凝胶电泳(Native - PAGE)的结果获得最适的糖化条件。糖化后的血红蛋白,采用离子交换层析法和硼酸亲和层析法来分离纯化 HbA1c。 利用悬滴扩散法和微批量结晶法对纯化的HbA1c进行直接结晶, Native - PAGE 实验表明糖化后蛋白的性质发生改变,可能是导致难以直接结晶的方法得到HbA1c晶体的原因之一。同时通过与葡萄糖共结晶的血红蛋白得到菱形、片状晶体,浸泡血红蛋白晶体等制 HbA1c晶体,挑取长势较好的晶体冻存待数据收集。 27248
毕业论文关键词 糖化血红蛋白 葡萄糖 离子交换层析法 硼酸亲和层析法 结晶条件筛选
Title The glycosylation of human hemoglobin and its impact on the protein structure and function Abstract The glycosylation conditions of the pre-purified healthy adult hemoglobin in vitro were screened and optimized by varying concentration of glucose, reaction pH and temperature, etc. The glycated hemoglobin (HbA1c) was then isolated and purified by Bio-Rex 70 cation exchange chromatography followed by the boric acid affinity chromatography. The initial crystallization trials on the purified HbA1c were carried out by varying experimental parameters including pH, the precipitation agent concentration, and the concentration of salt ions. Native polyacrylamide gel electrophoresis (Native PAGE) results clearly showed that the properties of proteins had changed upon the glycosylation process, which may explain the difficulties in crystallization of the HbA lc. A large number of diamond shape, plate crystals were obtained successfully under co-crystallization of hemoglobin with glucose. The suitable sized crystals were harvested and flash frozen prior to the data collection. Keywords glycosylated hemoglobin glucose ion exchange chromatography agarose affinity chromatography crystallization screening
目次
1 绪论 . 1
1.1 糖化血红蛋白 . 1
1.2 糖基化对血红蛋白结构与功能的影响 . 2
1.3 糖化血红蛋白的分离、检测方法 . 2
2 糖化血红蛋白的制备 . 4
2.1 实验仪器和材料 . 4
2.2 实验方法 . 6
2.2.1 血红蛋白浓度的测定(UV-vis) . 6
2.2.2 糖化血红蛋白的制备 . 7
2.2.3 糖化率的测定 . 7
2.2.4 Native-PAGE 实验结果 8
2.3 结果与讨论 . 8
3 糖化血红蛋白的分离、提纯 13
3.1 实验仪器和材料 13
3.2 实验方法 15
3.2.1 糖化血红蛋白的分离 15
3.2.2 糖化血红蛋白的提纯 16
3.2.3 糖化血红蛋白的结晶条件的筛选 16
3.2.4 Native-PAGE 实验结果 . 18
3.3 结果与讨论 19
结 论 . 23
致 谢 . 24
参考文献 25
图2.1 不同葡萄糖浓度诱导的血红蛋白变化光谱图 . 9
图2.2 糖化血红蛋白的校准曲线图 11 人体血红蛋白糖基化过程对其结构影响的机理研究:http://www.751com.cn/shengwu/lunwen_21695.html