Preparation of highly enriched photosystem II membrane vesicles by a non-detergent method

An improved, non-detergent, method for preparative isolation of PS II membrane vesicles from spinach chloroplasts is presented. Thylakoids (chlorophyll (Chl) a/b ratio 2.8, Chl/P700 435) were fractionated by Yeda press treatment and aqueous two-phase partition to yield inside-out vesicles (1) (chl a/b 2.2, chl/P700 700). These vesicles were subjected a sonication - phase partitioning procedure; steps of sonication of inside-out vesicles, while still present in a dextran-polyethylene glycol two-phase system were alternated by phase partition. These steps selectively removed P700-containing membrane fragments from the inside-out vesicles and yielded a membrane fraction with improved PS II purity (Chl a/b ratio 1.9, Chl/P700 1500) and retained oxygen evolving capacity (295 μmol O2 mg Chl(-1) h(-1)).