【作者】 张锐；

【导师】 陈旭伟；

【作者基本信息】 东北大学 ， 化学工程， 2014， 硕士

【摘要】 蛋白质是重要的生命物质,在生命活动中扮演着至关重要的角色。快速高效的将蛋白质从复杂基体中分离出来是蛋白质组学研究中的关键环节。本论文将石墨型氮化碳材料首次应用于蛋白质的分离纯化,建立了固相萃取分离纯化血红蛋白的新方法,拓展了石墨型氮化碳材料在生命科学领域中的应用范围。第一章简要综述了蛋白质分离纯化的意义、基本原则和方法,石墨型氮化碳材料的合成方法及其在各领域的应用进展。第二章制备并利用石墨型氮化碳材料(g-C3N4),建立了固相萃取选择性分离纯化全血中血红蛋白的新方法。以具有三嗪结构的三聚氰胺为原料,550℃保持4h,热聚合得到淡黄色的石墨型氮化碳。采用红外光谱、扫描电镜、X射线衍射、紫外-可见吸收光谱、Zeta电位对石墨型氮化碳材料进行了表征。考察了石墨型氮化碳材料在血红蛋白分离纯化中的性能,在pH 8.0的条件下,5 mg g-C3N4材料对1.0mL50mg·L-1血红蛋白的吸附效率为98%,吸附行为符合Freundlich模型及二级反应动力学方程。吸附的血红蛋白可用B-R (pH4.0)进行洗脱,洗脱率为60%。圆二色光谱表明吸附洗脱过程对血红蛋白的构象没有明显影响,说明g-C3N4材料具有良好的生物相容性。将所建立的方法用于人全血中血红蛋白的选择性分离,纯化的样品采用SDS-PAGE凝胶电泳进行纯度验证,结果表明该方法分离得到的血红蛋白纯度较高。更多还原

【Abstract】 As a kind of important biomacromolecules, protein plays crucial role in various life activities. Rapid and efficient separation of proteins from complex matrices is of great importance for proteomics. In the present work, graphite carbon nitride is first used for protein separation and a solid-phase extraction procedure is developed for the separation/purification of hemoglobin. The results further expanded the application potentials of Graphite carbon nitride in the field of life sciences.In chapter 1, brief reviews are given to the significance, principles and methodologies of protein separation/purification In addition, the properties of graphite carbon nitride as well as its applications in various fields have been discussed.In chapter 2, a solid-phase extraction protocol for the selective separation/purification of hemoglobin (Hb) from human whole blood is developed by using graphite carbon nitride as the sorbent. Melamine of Triazine structure is used as the original material in the synthesis of carbon nitride. After thermal polymerization of Melamine at 550℃ for 4 h, yellow carbon nitride is obtained. The obtained carbon nitride is characterized by means of SEM, FTIR, XRD, UV/Vis spectroscopy and Zeta potential. The performances of carbon nitride in the adsorption/isolation of hemoglobin are thus investigated. At pH 8.0, an adsorption efficiency of 98% for 50 mg·L-1 hemoglobin in a 1.0 mL solution is achieved by using 5.0 mg of Graphite carbon nitride as sorbent. The adsorption of hemoglobin onto graphite carbon nitride fits freundlich adsorption model and the secondary reaction kinetics equation. The retained Hb could be readily recovered by using a B-R buffer of pH 4.0, giving rise to a recovery of 60%. Circular dichroism spectra indicated that the adsorption/desorption procedures pose no obvious effect on the conformation of Hb, showing favorable biocompatibility of the Graphite carbon nitride. The developed extraction protocol is thus used for the selective isolation of Hb from human whole blood. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicate that high purity of hemoglobin is obtained.更多还原