Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography
Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography
复制标题
DOI：
10.1007/bf00029933
复制
发表时间：
1995-03
影响因子：
3.7
通讯作者：
E. Šetlíková;S. Ritter;R. Hienerwadel;J. Kopecký;J. Komenda;W. Welte;I. Šetlík
中科院分区：
文献类型：
--
作者：
E. Šetlíková;S. Ritter;R. Hienerwadel;J. Kopecký;J. Komenda;W. Welte;I. Šetlík
Oxygen-evolving PS II particles from the thermophilic cyanobacteriumSynechococcus elongatusare partially purified by centrifugation on a sucrose gradient and are bound to a Chelating Sepharose column loaded with Cu2+ions. Bound particles are then transformed into PS II RC complexes by two washing steps. First, washing with a phosphate buffer (pH=6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH=6.2) containing 0.2 M LiClO4and 0.05% of DM removes CP 47 and CP 43 and leaves bare PS II RC complexes on the column. These are then eluted with a phosphate buffer containing 1% of dodecylmaltoside (DM). The molar ratio of pigments in the eluate changes with the progress of elution but around the middle of the elution period a nearly stable ratio is maintained of Chla: Pheoa: Car: Cytb559 equal to 2.9: 1: 0.9: 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduced pheophytin (ΔA of 430–440 nm) and by the flash induced formation of P680+(ΔA at 820 nm). The relatively slow relaxation kinetics of the latter signal (t1/2≈ 1 ms) may suggest that in a substantial fraction of the RCs QAremains bound to the complex.
