Using pH indicator dye techniques we have investigated the pH changes in dark-adapted chloro- plasts following excitation by short flashes. Two types of pH indicator, cresol red and neutral red, were used, to follow the pH changes either inside or outside the thylakoids, or the net change when the membrane was made permeable to protons by uncoupling agents. (1)With cresol red which showed the net pH changes inside and outside the thylakoids, an oscillation of the flash yield of H+ occurred with a periodicity of 4 (minima on the first and fifth flashes, the yield on the third being not significantly different from the yields on the second and fourth flashes). The pH changes did not occur in synchrony with O2-evolution. (2)The net flash yields without addition of electron acceptor were similar to those with benzyl- viologen. The results were comparable with those obtained with the glass electrode technique by Fowler and Kok (C. F. Fowler and B. Kok, Biochim. Biophys. Acta 357, 299 - 307 [1974]). (3)The net flash yields with ferricyanide as electron acceptor of photosystem I were higher than those in the absence of acceptor, or with benzylviologen. On the first and fifth flashes a net acidification was always observed. (4)In the presence of 3- (3,4-dichlorphenyl) -1,1-dimethylurea (DCMU) a rapid acidification also occured on the first flash, while the pH changes induced by subsequent flashes were inhibited. (5)The uncoupler methylamine did not inhibit the proton uptake outside the thylakoids. (6)With neutral red as indicator for the net pH change inside and outside the thylakoids, the same oscillation of the flash yield occured as with cresol red. (7)With neutral red in the precense of an external buffer, as a pH indicator for the internal aqueous phase alone, an oscillation of the flash yield with a periodicity of 4 also occured. The first and second flash yields were higher compared with the third than the equivalent yields of oxygen. (8)We discuss the results with respect to a model for the release of protons in the water- splitting enzyme reactions, in which protons are not released in synchrony with O2 , but in the transitions of all the states of the watersplitting enzyme with the exception of S1 → S2 . Our results are consistent with this model when account is taken of the release of protons inside the thylakoids with a periodicity of 2, associated with electron transfer from reduced plastoquinone.
Impact of impurities in bromocresol green indicator dye on
spectrophotometric total alkalinity measurements
