人体血红蛋白糖基化过程对其结构影响的机理研究
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
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