[12] Subchloroplast Particle Enriched in P700 and Iron-Sulfur Protein

Chloroplast membrane-bound iron–sulfur proteins are currently the focus of considerable attention because of their participation in the primary photochemistry of photosystem. Detailed characterization of the structure and function of these proteins in photosynthesis requires large quantities of highly purified reaction center particles enriched in iron–sulfur protein and P700. The procedure described in this chapter employs the nonionic detergent Triton X-100 to liberate an iron–sulfur and P700-containing reaction center particle from the chloroplast lamellae and to free a significant amount of light-harvesting chlorophyll from the P700-containing fragment. The use of gel filtration and ion-exchange chromatography leads to the isolation of a photoactive particle having a chlorophyll/P700 ratio of 25 and containing 10–12 moles of nonheme iron (NHI) and acid-labile sulfide (ALS) per mole of P700. The particle is nearly devoid of chlorophyll b, cytochromes f, b₆, and b-559 and has a greatly diminished content of β-carotene. The chapter describes the procedure for the preparation of the particle, fractionation scheme for spinach chloroplasts, and properties of the subchloroplast fragments.