葛仙米藻胆蛋白及其酶解产物活性构象和生理功能的研究

Nostoc sphaeroides kützing, belonged to Nostocaceae Cynophytn, is a kind of single-cell cyanobacterium with lower evolution status.Because Nostoc sphaeroides kützing could be used as both a wild precious medicine and food, its biological activity was investigated by a large number of domestic and foreign scientists. According to those researches, phycobiliprotein, the major proteins in Nostoc sphaeroides kützing, showed a strong biological activity. However, the relationship between phycobiliprotein active conformation, structure of its degradation products in vivo and physiological function has not been reported yet. The aims of the study were focused on several points as mentioned below: the first is to confirm the relationship between phycobiliprotein active conformation especially aggregation and its physiological function; The second is to study how external environmental conditions such as pH influence the phycobiliprotein active conformation and its physiological function,while provide the optimized methods for phycobiliproteins extraction and purification; The third is to establish metabolism dynamics model of phycobiliprotein; The forth is to confirm the relationship between physiological function and structure of active center, when the phycobiliprotein was enzymed by major gastrointestinal protease. This study will be helpful to explain how phycobiliprotein play its important role in vivo.

葛仙米为蓝藻门念珠藻科的一种低等的单细胞蓝藻，是一种珍贵野生的食药两用蓝藻，国内外大量实验证明藻胆蛋白具有很强的生理活性，而藻胆蛋白的活性构象（藻胆蛋白在溶液中具有生理功能活性的蛋白构象）及其在体内的降解产物的结构与其生理功能的关系还未见相关报道。因此本课题旨在进行以下研究：一是明确葛仙米藻胆蛋白的结构尤其是聚集态与其生理功能的关系；二是系统研究外界环境条件如pH如何影响藻胆蛋白活性构象与其生理功能的关系，为藻胆蛋白提取纯化方法的优化提供基础资料；三是建立葛仙米藻胆蛋白体内代谢动力学模型；四是明确藻胆蛋白经主要胃肠蛋白酶作用后，其活性部分的结构与其生理功能的关系。本研究将有助于深入理解藻胆蛋白在体内发挥其生理功能的作用机理。

葛仙米是一种珍贵野生的食药两用蓝藻，富含藻胆蛋白，国内外大量实验证明藻胆蛋白具有很强生理活性，但藻胆蛋白活性构象及其体内降解产物结构与功能的关系的研究还未见报道，因此本课题重点研究了（1）葛仙米藻胆蛋白的聚集态与其生理功能的关系；（2）外界环境条件如pH 对其活性构象与生理功能的影响；（3）建立葛仙米藻胆蛋白体外代谢动力学模型；（4）葛仙米藻胆蛋白经主要胃蛋白酶和胰蛋白酶作用后，其活性部位的结构与生理功能的关系。主要结果如下：藻胆蛋白聚合度对其生理功能尤其是抗氧化作用具有一定影响，但不同抗氧化体系其影响机理不同，对红细胞溶血和脂质过氧化抑制而言，聚合度越小抗氧化效果越好；浸提液pH通过影响藻胆蛋白聚合度进而影响其生理功能，中性pH时藻胆蛋白聚合度最大，中性略微偏酸会降低葛仙米藻胆蛋白的聚合度。体外模拟显示藻蓝蛋白胃液中半衰期小于1min，葛仙米藻胆蛋白胃液降解10min后可溶性蛋白含量基本趋于平衡，肠液持续至150min，胃液处理60min后游离氨基含量变化不显著，肠液持续到150min变化不显著。胃肠液降解产物的抗氧化作用随降解程度不同而不同，但模拟胃液和肠液消化产物的抗氧化活性具有明显差异性。葛仙米藻胆蛋白胃液消化产物对ABTS、•OH和FRAP体系的还原作用均强于肠液；均随消化时间延长而增强，但对•OH清除作用初期下降；另肠液消化产物对脂质过氧化的抑制作用强于胃液。偏相关分析表明模拟胃肠液消化产物的抗氧化指标除O2-•外，其他均与可溶性蛋白质含量呈负相关，模拟胃液产物对红细胞溶血抑制、FRAP、•OH和脂质过氧化与游离氨基含量显著正相关，模拟肠液消化产物对ABTS、•OH和O2-•、脂质过氧化抑制与游离氨基含量达到极显著水平。消化产物经过SEC-100层析分离，主要得到A和B两类物质，A相对分子质量约80-90ku，B相对分子质量约4-9ku，抗氧化物质主要集中在B部分物质，比较发现，模拟肠液消化产物对于ABTS+•、DPPH自由基的清除能力和铁离子还原能力均优于模拟胃液消化产物。