Pigment Content of D1-D2-Cytochrome b559 Reaction Center Preparations after Removal of CP47 Contamination: An Immunological Study

Isolated D1-D2-cytochrome b₅₅₉ photosystem II reaction center preparations with pigment stoichiometry higher than 4 chlorophylls per 2 pheophytins can be contaminated with CP47 proximal antenna complex. Reaction centers prepared by a modification of the Nanba-Satoh procedure and containing about 6 chlorophylls per 2 pheophytins showed immuno-cross-reactivity when probed with a monoclonal antibody raised against the CP47 polypeptide. Furthermore, they could be fractionated successfully by Superose-12 sieve chromatography into two different populations. The first few fractions off the column contained a more definitive 435 nm shoulder corresponding to increased chlorophyll content, and showed strong immuno-cross-reactivity with the CP47 antibody. The peak fractions off the column displayed a less prominent 435 nm shoulder, and did not cross-react with the antibody. Moreover, when a 6-chlorophyll preparation was mixed with Sepharose beads coupled to CP47 antibody, the eluted material corresponded to a preparation of about 4 chlorophylls per 2 pheophytins and did not show any crossreaction with the antibody against CP47. The amount of CP47 protein in the 6-chlorophyll preparation as quantitated using Coomassie Blue staining or from gel blots was sufficient to account for most of the extra 2 chlorophylls. We conclude that D1-D2-cytochrome 7559 preparations containing more than 4 chlorophylls per 2 pheophytins can be contaminated with small amounts of CP47-Dl-D2-Cyt b₅₅₉ complex and that native photosystem II reaction centers contain 4 core chlorophylls per 2 pheophytins.