The efficiency in selective extraction of photosystem (PS) 2 oxygen evolving complexes was compared among seven detergents. These were applied to thylakoid membranes of the thermophilic cyanobacterium Synechococcus elongatus. Used were five non-ionic detergents with one ionic and one zwitterionic for comparison. To compare the suitability and efficiency of the detergents the following properties of the extracts were examined: maximum rate of oxygen evolution with various electron acceptors, the relative variable fluorescence (FV/FM), the contamination of the extract with photosystem (PS) 1, and the status of the electron acceptor side of PS2 reaction centre. None of the detergents yielded a highly selective extraction of the PS2 complexes (negligible contamination with PS1) which would simultaneously display a high photochemical activity and high structural intactness. Heptylthioglucoside and dodecylmaltoside yielded the nearest approximation to the optimum result. Kinetic fluorometry was applied here for the first time to characterize the functional and structural properties of PS2 particles from cyanobacteria. and E. Šetlíková ... [et al.].
a1_Photosystem (PS) II particles retaining a high rate of O2 evolution were isolated from the mesophilic filamentous cyanobacterium, Spirulina platensis. To achieve high production of PSII complexes in the cells, irradiance from halogen incandescent lamps was used. Disruption of cells by vibration of glass beads proved to be the most suitable procedure for isolation of thylakoid membranes. The selectivity of detergents for PSII particle preparation rose in the order of Triton X-100 < decyl-β-D-glucopyranoside < dodecyldimethyl-aminooxide < n-heptyl-β-D-thioglucoside < N-dodecyl-N,N-dimethylammonio-3-propane sulphonate < n-octyl-β-thioglycoside < octylglucoside < n-dodecyl-β-D-maltoside. The last four detergents yielded extracts, from which pure PSII particles not contaminated by PSI complexes could be obtained by sucrose-gradient centrifugation (20-45%) at the 43% sucrose level. We assumed both the acceptor and donor sides of the isolated n-dodecyl-β-D-maltoside (DM) particles to be intact due to high oxygen production by DM particles [1,500 meq(e-) mol-1 (Chl) s-1] achieved in the presence of all artificial acceptors tested. The PSII particle fraction from the sucrose gradient was used with immobilized metal (Cu2+) affinity chromatography (IMAC) for the preparation of the PSII core complex. By washing the column with a MES buffer containing MgCl2 and CaCl2, the phycobiliproteins were stripped off. The PSII core complex was eluted in a buffer containing 1% DM, mannitol, MgCl2, NaCl, CaCl2, and ɛ-aminocaproic acid. SDS-PAGE of the core complex provided pure bands of D1 and D2 proteins and PsbO protein from thylakoid membrane, which were used to raise polyclonal antibodies in rabbits. These antibodies recognized D1 and D2 not only as monomers of 31 and 32 kDa proteins, but also as heterodimers of D1, D2 corresponding to the band of 66 kDa on SDS-PAGE. This was in contrast to antibodies of, a2_synthetic determinants, which reacted only with the monomers of D1 and D2 proteins. These negative reactions against heterodimers of D1, D2 supported the hypothesis that dimeric forms of PSII reaction centre proteins have a C-terminal sequence sterically protected against a reaction with specific antibodies., and E. Šetlíková ... [et al.].