摘要
以纯化的蓝隐藻(Chroomonas plaoidea)藻蓝蛋白PC645为材料,经尿素变性、分子筛层析以及SDS-PAGE与IEF电泳技术分离、纯化其2类不同亚基,并测定了亚基的吸收光谱、荧光谱、等电点以及pH耐受性等.结果表明,隐藻PC645异二聚体的亚基结构稳定,8 mol/L尿素处理48 h方可令A 645=10的PC645样品变性完全.SDS-PAGE与IEF电泳结果表明,经Sephacryl S-100层析得到的α与β亚基组分达到了完全分离的效果,并证实PC645存在分子质量和等电点均不相同的2种β亚基.光谱分析显示,分离后的α和β亚基依然保持光谱活性,其中β亚基是PC645的660 nm特征荧光发射位点,并且在pH值为4.92的磷酸缓冲液中可保持45 d内光谱形态稳定;而α亚基所含的MBV色基不产生荧光,亚基的pH耐受性以pH值为7左右为最佳.实验结果将为研究藻蓝蛋白的亚基结构、能量传递途径等提供相应理论依据,并为进一步的亚基序列分析奠定了基础.
Two different subunits of phycocyanin PC645 from Chroomonas placoidea are separated and purified using molecular sieve chromatography,SDS-PAGE and IEF electrophoresis after urea denaturation.The absorption spectrum,fluorescence properties,isoelectric points,pH tolerance,and some other characteristics of the isolated subunits are determined.The results indicates that the structure of PC645 dimer is stable.PC645 with A 645=10 is completely denatured only after 48 hours of treatment with 8 mol/L of urea.SDS-PAGE and IEF electrophoresis profiles show that theαandβsubunits after Sephacryl S-100 chromatography are completely separated,and that the PC645 may contain two types ofβsubunits with different molecular weight and isoelectric point.Spectral analysis show that the spectral activities are still maintained in both isolatedαandβsubunits.Theβsubunit is the terminal emission site,which produces the characteristic fluorescence of PC645 at 660 nm.Furthermore,the isolatedβsubunit maintains their spectral morphology in PBS buffer during 45 days of storage if preserved at pH 4.92.MBV chromophore inαsubunit displays no fluorescence emission.And the isolatedαsubunit shows the best pH tolerance around pH 7.These results can provide useful theoretical and technical information for studying the subunit structure and energy transfer pathway of phycocyanin,and for further subunit sequence analysis.
作者
任慧慧
王玉璇
李雪薇
陈敏
REN Hui-hui;WANG Yu-xuan;LI Xue-wei;CHEN Min(School of Life Sciences,Yantai University,Yantai 264005,China)
出处
《烟台大学学报（自然科学与工程版）》
CAS
2020年第1期27-35,共9页
Journal of Yantai University(Natural Science and Engineering Edition)
基金
山东省自然科学基金资助项目(ZR2018LD009)
关键词
蓝隐藻
藻蓝蛋白
亚基分离
光谱特性
pH耐受性
Chroomonas placoidea
phycocyanin
subunit separation
spectral property
pH tolerance
