Rapid resolution by reversed-phase high performance liquid chromatography of the thylakoid membrane proteins of the photosystem II light-harvesting complex

This paper presents the results of a study performed to develop a rapid and straightforward HPLC method to resolve and identify the protein components of the photosystem II light-harvesting complex (LHC II), which is the major protein-chlorophyll a/b complex in higher plants. The method employs a reversed-phase Vydac Protein C4 column containing 5-?m porous butyl silica which is eluted by a 13.5-min linear water-acetonitrile gradient in the presence of 0.1% (v/v) trifluoroacetic acid. Peak identification was performed by denaturing SDS-PAGE analysis of the fractions of the eluate collected throughout the chromatographic run. This procedure allowed the identification of Lhcb2, Lhcb1 and Lhcb3 protein components of LHC II, which eluted as well-resolved peaks with highly repeatable retention times, in samples isolated from spinach leaves both by the same and by different preparations. The reversed-phase HPLC method has proven to be suitable for detecting dissimilarities in the protein components of LHC II isolated from different plants.