Bromothymol blue and bromocresol purple as indicators of pH changes in chromatophores of Rhodospirillum rubrum.

Abstract

Chromatophores in suspension were found to bind about 80% of bromothymol blue and about 20% of bromocresol purple in solution at low concentration. Binding of bromothymol blue was associated with a shift in pKa of the indicator from 7.1 to 7.9, and with a decrease in extinction coefficient. No change of pKa or extinction was found for bromocresol purple in the presence of chromatophores. On illumination of a suspension of chromatophores containing bromothymol blue, the indicator showed an absorbancy change opposite in sign to that expected from the pH change in the medium. The absorbancy change of bromocresol purple on illumination was indicative of the pH in the external medium. Increasing the buffering power of a medium containing chromatophores and bromothymol blue or bromocresol purple increased the response of bromothymol blue on illumination, but eliminated the response of bromocresol purple. It is suggested that a proportion of the bromothymol blue present was able to respond to external pH, while virtually all the bromocresol purple responded to changes in the external pH. Bromothymol blue was found to undergo a change in availability to external titration when the energetic state of the chromatophores was changed. Less bromothymol blue responded to addition of acid or alkali in coupled, illuminated chromatophores than in uncoupled chromatophores, or chromatophores in the dark. No similar change in availability of bromocresol purple was observed on changing the energetic state. The kinetics of the bromothymol blue absorbancy change in a highly buffered medium and of the change in availability of bromothymol blue to external titration on illumination were similar. The kinetics reflected the rate of response of bromothymol blue to the energetic state, and not the onset of the energetic state itself. Valinomycin and nigericin markedly changed the kinetics of the bromothymol blue response. The effects of the antibiotics are discussed in terms of their effects on H+ uptake and on the energetic state of the chromatophores and in terms of the dual response of bromothymol blue to external pH and energetic state. It is concluded that while proportions of the bromothymol blue present in a suspension of chromatophores responded both to external and internal pH changes, the major proportion of the bromothymol blue change was a slow response to the energetic state reflecting neither internal not external pH, nor kinetically, the onset of the energetic state. The response of bromocresol purple to changes in pH of the external medium has been used to follow the kinetics of H+ uptake, and these have been compared with rates of electron flow. In the absence of valinomycin, an initial burst of H+ uptake of about 1 H+/10 bacteriochlorophyll was observed. In the presence of valinomycin, the initial H+ uptake was more rapid, and more extensive (about 2 H+/bacteriochlorophyll). The initial rate of H+ uptake in the presence (1.28 H+/bacteriochlorophyll per sec) and absence of valinomycin (0.6 H+/bacteriochlorophyll per sec) were faster than rates of electron flow (0.5 electron/bacteriochlorophyll per sec) measured under similar conditions. The results are discussed in terms of current theories of energy conservation coupled to photoelectron flow.