Use of immobilized light-harvesting chlorophyll a/b protein to study the stoichiometry of its self-association

Abstract

D.J. Davis and E.L. Gross (1976) previously observed that the light-harvesting chlorophyll a/b protein [spinach] or chlorophyll protein complex II self-associated as determined by ultracentrifugation. The stoichiometry of complex formation was determined by immobilizing the monomer on ethylenediamine Sepharose 4B and determining the ability of immobilized protein to bind the free protein. The amount of soluble protein bound to the immobilized protein increased as the concentration of soluble protein increased. The binding was maximal between pH 7 and 8. The maximum binding was 3 molecules bound per 1 molecule of protein immobilized. These results indicate that a tetramer is the intrinsic structural unit of the light-harvesting chlorophyll a/b protein in the chloroplast membrane. Upon complex formation, the chlorophyll fluorescence was decreased without any spectral change. The maximum binding was approximately doubled upon addition of 0.5 mM CaCl2 whereas 5 mM NaCl had no effect. Addition of CaCl2 had no effect on the fluorescence of the monomer. The light-harvesting chlorophyll a/b protein was isolated from a sodium lauryl sulfate extract of chloroplasts by affinity chromatography using the immobilized light-harvesting chlorophyll a/b protein.