The available procedures for isolation and purification of photosystem I (PSI) from Chlamydomonas reinhardtii are time consuming and usually require several hours of sucrose gradient ultracentrifugation steps. This may lead to structural and functional impairment, including release of pigments and/or dissociation of protein subunits. Moreover, it is difficult to isolate intact complexes from thylakoids containing mutated PSI that accumulate to lower levels. Hence, isolation of intact PSI core complex depends on the speed of the procedure and the mildness of the extraction and purification. We have, therefore, modified the procedure for PSI isolation to both increase the yield of PSI and to reduce contamination by other pigment protein complexes. The modified procedure involves dodecyl maltoside solubilization of crude-thylakoid membranes followed by single-step column chromatography using a weak anion-exchanger. PSI eluted from the column between 13 mM and 15 mM Mg S04. This new rapid purification procedure yielded pure PSI preparations with a Chl/P700 ratio of approx 90 and showing typical absorption difference spectra with a maximum bleaching occurring at 696 nm. Femtosecond transient absorption spectroscopy of purified PSI complex revealed a high degree of similarity in terms of excitation energy transfer within the PSI core to observations in cyanobacterial PSI.
|Original language||English (US)|
|Number of pages||10|
|Journal||Methods in molecular biology (Clifton, N.J.)|
|State||Published - 2004|
ASJC Scopus subject areas
- Molecular Biology