77-K chlorophyll fluorescence spectra; Hordeum vulgare; photochemical efficiency; polyacrylamide gel electrophoresis; thylakoid membranes isolation

The present study was conducted to examine changes in photosynthetic pigment composition and functional state of the thylakoid membranes during the individual steps of preparation of samples that are intended for a separation of pigment-protein complexes by nondenaturing polyacrylamide gel electrophoresis. The thylakoid membranes were isolated from barley leaves (Hordeum vulgare L.) grown under low irradiance (50 ?mol m-2 s-1). Functional state of the thylakoid membrane preparations was evaluated by determination of the maximal photochemical efficiency of photosystem (PS) II (FV/FM) and by analysis of excitation and emission spectra of chlorophyll a (Chl a) fluorescence at 77 K. All measurements were done at three phases of preparation of the samples: (1) in the suspensions of osmotically-shocked broken chloroplasts, (2) thylakoid membranes in extraction buffer containing Tris, glycine, and glycerol and (3) thylakoid membranes solubilized with a detergent decyl-?-D-maltosid.