Chromatographic Purification and Determination of the Carboxy-Terminal Sequences of Photosystem II Reaction Center Proteins, Dl and D2

The Dl and D2 subunits of the reaction center of photosystemII are intrinsic proteins, each with a molecular mass of about30 kDa. They exhibit considerable homology to each other interms of primary structure. A procedure was developed for theseparation and purification of these two proteins on a largescale from the photosystem II reaction center complex of spinachby high-performance liquid chromatography on a gel-permeationcolumn in the presence of sodium dodecyl sulfate. The purificationwas achieved by a combination of two gel-permeation chromatographicsteps performed with different concentrations of phosphate buffer,200 mM and 50 mM, as the mobile phase. The purified Dl and D2proteins were subjected to determination of their carboxy-terminalsequences by digestion of the proteins with carboxypeptidaseY. Comparison of the sequences deduced from the enzymatic analysiswith the sequences deduced from the psb A and psb D genes ofspinach indicates that the Dl protein ends at Ala-344 and theD2 protein at Leu-353. Thus, it appears that the Dl proteinloses 9 amino acid residues from the carboxy-terminus, fromAla-345 to Gly-353, during maturation, while the D2 proteindoes not lose any amino acid residues from the carboxy-terminus. (Received July 27, 1989; Accepted December 28, 1989)