Isolation and characterization of chloroplast Photosystem II antenna of spinach by reversed-phase liquid chromatography

The protein components of the Photosystem II antenna system, isolated from spinach thylakoids, have been resolved by reversed-phase high performance l iquid chromatography (RP-HPLC) using a butyl-silica stationary phase packed either into analytical or semi-preparative columns. Peak identification ha s been accomplished by a combination of various SDS-PAGE systems employing either Comassie (or silver) staining or immunological detection using polyc lonal antibodies raised against LHC II and against CP29, CP26 and CP24 prot eins and by aminoacid microsequence. Moreover, peak identification is consi stent with the molecular masses determined by Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS). The developed RP-HPLC method allows the resolut ion of all the protein components of the Photosystem II major Light Harvest ing Complex (LHC II) and minor PS II antenna complex (CP24, CP26 and CP29) from grana membranes (BBY) and estimation of their relative stoichiometry i n natural and stressed conditions, avoiding the expensive and time consumin g separation procedure by sucrose-gradient ultracentrifugation and isoelect rofocusing.