Protein-protein interactions of the light-harvesting chlorophyll a/b protein. II. Evidence for two stages of cation independent association.

In a previous paper, we observed a two-stage cation-independent association of the light-harvesting chlorophyll a/b protein from spinach chloroplasts based on concentration-dependent changes in the sedimentation coefficient. The two stages of association occurred between (2-4) and (4-7) mug/ml chlorophyll. In this paper, we provide further evidence for this association. This includes: (1) A decrease in the number of divalent cation binding sites in the second stage of association. (2) A corresponding decrease in the extent of the cation-dependent association. (3) A positive deviation from Beer's law for chlorophyll b for both stages of the cation-independent association and a positive deviation for chlorophyll a for the second stage of association only. (4) A change in the fluorescence emission of both chlorophyll a and b. The change for chlorophyll b was observed for both steps of association whereas that for chlorophyll a was observed for the second step of association only. Therefore, the first stage of association affects only chlorophyll b whereas the second stage alters the environment of both chlorophyll a and b. (5) In addition, divalent cations quenched chlorophyll fluorescence. However, the quenching which required 200-300 muM divalent cation for half-maximal effects was related neither to divalent cation binding nor to the divalent cation-induced association of the protein.