紫球藻藻红蛋白和藻蓝蛋白共价交联物的构建及其与天然藻胆体特性的比较研究

IOCAS-IR > 海洋环流与波动重点实验室

	紫球藻藻红蛋白和藻蓝蛋白共价交联物的构建及其与天然藻胆体特性的比较研究
	马圣媛
学位类型	博士
	2001
学位授予单位	中国科学院海洋研究所
学位授予地点	中国科学院海洋研究所
学位专业	海洋生物学
摘要	用蔗糖密度梯度超速离心的方法从单细胞红藻---紫球藻（Porphyridium cruentum）中分离纯化了完整的藻胆体。光谱分析表明，藻胆体在可见光区域有四个吸收峰（545nm, 565nm, 618nm 和 650nm）和一个吸收肩（498nm）；荧光发射峰分别在680nm和595nm，二者的比值（λ_(680nm)~(em)/λ_(595nm)~(em)）可高达10以上，说明分离的藻胆体非常完整。非变性电泳结果显示，分离的藻胆体至少有三种颜色的条带，分别为：B－PE，R－PC和b－PE，APC可能含量很少而未见到。SDS－PAGE电泳鉴定至少有三条明显的带，为PE的α和β的重叠带，γ亚基和RPC的亚基。用羟基磷灰石层析和Sephadex G-200层析的方法分离到了高纯度的B－藻红蛋白、b-藻红蛋白和较纯的R－藻蓝蛋白。光谱检验发现，分离得到的藻胆蛋白均为天然态，其中藻红蛋白的荧光发射峰在580nm左右，藻蓝蛋白的荧光发射峰在640nm左右。SDS－PAGE电泳分析，B-藻红蛋白有两条带，分子量大约在20kD，31kD。小分子量的条带极宽，是因为α和β亚基的分子量相近，所以有重叠；大分子量的条带是γ亚基。相对应的是b-藻红蛋白只有20kD的条带，无γ亚基的条带。藻蓝蛋白有两条明显的条带，大约为16kD和20kD。用戊二醛共价交连的方法得了紫球藻B-藻红蛋白和R－藻蓝蛋白和共价结合体。吸收光谱表明，交联体和对照最明显的区别是紫外光区278nm的峰高增加并且蓝移到268nm；交联体的荧光发射光谱与对照相比，498nm激发产生的峰值在588nm（游离的PE的发射峰）和650nm（交联体中PC的发射峰），而对照只有590nm的发射峰，并且交联体在588nm的峰高明显低于对照。非变性电泳鉴定，发现有与对照不同的新的条带出现。这一切证明藻胆蛋白能量传递模型共价交联成功。人工构建的交联物和天然的藻胆体相比，对低离子强度和低温（4 ℃）更为稳定，但能量传递效率相对较低。
其他摘要	Intact phycobilisomes were isolated and purified from Porphyridium cruentum, a unicell red algae by sucrose density gradient supercentrifugence. Spectra analysis indicates that it has four absorption peaks(545nm, 565nm, 618nm and 650nm) and a absorption shoulder(498nm) in visible light region. Its fluorescence emission peak in room tempretrue is 680nm and 595nm respectively, and the ratio of 680nm and 595nm can be up to more than 10, which indicated we have got very intact phycobilisome. Isolated phycobilisome contains at least three bands(B-PE, b-PE and R-PC) on non-denature PAGE electrophoresis, APC is not found accounting for its mall content. At least three clear bands were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis, α subunit and β subunit of phycoerythrin (overlapping), γ subunit of phycoerythrin and one subunit of R-phycocyanin. B-PE, b-PE and R-PC with high purity were obtained by the Sephadex G-200 gel chromatography. B-PE had tow absorption maxima (545nm, 565nm) and one absorption shoulder (498nm), with the A_(545nm)/A_(498nm) radio about 2.45. b-PE had its principal absorption mixima at 543nm and 563nm, but it did not have the absorption shoulder at 498nm, which is the main difference between B-PE and b-PE. The principal maxima of PC lied in 555nm and 618nm. Fluorescence emission peaks of PE and PC were at 580nm and 640nm respectively. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed two bands of B-PE, standing for molecular weights of 20KD and 31KD. The former band was rather wide because overlapping due to molecular weight similarity of subunit α and subunit β. The latter band was subunit γ. b-PE only showed one 20 KD band, while not possess γ subunit. Two clear bands of PC were also shown with molecular weights of 16KD and 20 KD. B-PE-R-PC conjugate was obtained using glutaraldehyde. Congujate had an absorption maximum at 268nm while the control showed a peak at 278nm which was chiefly attributed to aromatic amino acid residues. Conjugate showed two fluorescence emission peaks, 588nm and 640nm, attributed to free B-PE and R-PC in the conjugate respectively, while the control had only a fluorescence emmsion of 590nm originating from B-PE Furthermore, the 588nm peak of conjugate is far lower than that of the control. New bands appeared on non-denatured PAGE electrophoresis except for the B-PE and R-PC bands. All indicated that the phycobiliproteins energy transfer model is successfully covalently coupled. Artificial conjugate is more stable than natural phycobilisome in low ion strength and low temperature(4 ℃), but efficiency of energy transfer is comparatively lower.
页数	47
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/799
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	马圣媛. 紫球藻藻红蛋白和藻蓝蛋白共价交联物的构建及其与天然藻胆体特性的比较研究[D]. 中国科学院海洋研究所. 中国科学院海洋研究所,2001.

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