pH-sensitive fluorescent dye as probe for proton uptake in photosynthetic reaction centers

Isolated and purified reaction centers (RC) from Rhodobacter sphaeroides R-26.1 were solubilised in detergent with excess quinone and external electron donors and illuminated in the presence of pyranine. The pH change accompanying the reaction center photocycle was monitored by recording the variation of the pyranine fluorescence intensity. Using QB-depleted reaction centers or blocking the photocycle with terbutryne strongly reduced the pH change. The usefulness and limits of this technique in monitoring the pH changes during the RC photocycle are also discussed.