Isolation of pigment-binding early light-inducible proteins from pea

The early light-inducible proteins (ELLPs) in chloroplasts possess a high s equence homology with the chlorophyll a/b-binding proteins but differ from those proteins by their substoichiometric and transient appearance. In the present study ELIPs of pea were isolated by a two-step purification strateg y: perfusion chromatography in combination with preparative isoelectric foc ussing. Two heterogeneous populations of ELIPs were obtained after chromato graphic separation of solubilized thylakoid membranes using a weak anion ex change column. One of these populations contained ELIPs in a free form prov iding the first isolation of these proteins. To prove whether the isolated and pure forms of ELIP bind pigments, spectroscopic and chromatographic ana lysis were performed. Absorption spectra and TLC revealed the presence of c hlorophyll a and lutein. Measurements of steady-state fluorescence emission spectra at 77 K exhibited a major peak at 674 Mm typical for chlorophyll c t bound to the protein matrix. The action spectrum of the fluorescence emis sion measured at 674 nm showed several peaks originating mainly from chloro phyll a. It is proposed that ELIPs are transient chlorophyll-binding protei ns not involved in light-harvesting but functioning as scavengers for chlor ophyll molecules during turnover of pigment binding proteins.