蓝隐藻PC645的存在状态及其聚集态复合物的分离

Existent Status of PC645 in Chroomonas placoidea and Isolation of Its Aggregated Complex

以蓝隐藻(Chroomonas placoidea)为材料,探索细胞中PC645的天然存在状态,以及聚合态PC645存在条件与细胞破碎和离心分离时盐浓度及pH变化的关系。吸收和荧光光谱分析显示,除了小分子的PC645异二聚体之外,在高密度蔗糖梯度区还发现有PC-叶绿素-蛋白复合物与大分子藻蓝蛋白复合物的混合条带,说明隐藻藻胆蛋白的天然状态包括游离异二聚体、聚合形式以及与膜结合的多种形式,且结合态的PC645与叶绿素之间保持着活性的能量传递状态。通过检测和比较PC645的660 nm特征荧光强度,发现该条带中大分子藻蓝蛋白复合物在破藻缓冲液磷酸盐浓度为50 mmol/L、pH 4.92,蔗糖密度梯度离心时磷酸盐缓冲液浓度为300 mmol/L、pH 4.92时含量最高。一定浓度的磷酸盐和pH小于6.5的酸性条件有利于藻蓝蛋白复合物的形成和稳定。实验结果将为深入了解隐藻藻胆蛋白的结构、组装和功能提供重要依据。

The native existent status of PC645 isolated from Chroomonas placoidea and the effects of salt concentration and pH value on the formation of aggregated PC645 during cell breaking and centrifugation separation are investigated.Absorption and fluorescence spectra show that in addition to the small molecular of PC645 heterodimer,the PC-chlorophyll-protein complex mixed with macromolecular phycocyanin complex are also found in the high-density sucrose gradient region.The results indicate that cryptophytic phycobiliproteins may exist natively as free heterodimers,aggregated forms and membrane binding forms.The binding form of PC645 keeps efficient energy transfer to chlorophyll.By monitoring and comparing the intensity of 660nm typical fluorescence emitted by PC645,it is found that the aggregated PC645 in Band II is most abundant when the concentration of phosphate buffer is 50 mmol/L(pH 4.92)during cell broken,and 300 mmol/L(pH 4.92)during sucrose density gradient centrifugation.Accordingly,we conclude that a certain concentration of phosphate and acidic conditions with pH value below 6.5 are conducive to the formation and stabilization of PC645 complex in BandⅡ.The results will provide theoretical basis for further study on the structure,assembly and energy transfer of cryptophytic phycobiliprotein.