Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria

The reponses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the 'light-minus-dark' difference spectrum of the chromatophores. The oxonols appear to respond to the light-induced 'energization' by shifting their absorption maxima. In the presence of K+, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes, respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift. The dye response has an apparent second-order rate constant of approx. 2 . 10(6) M-1 . s-1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not possess intrinsic probes of potential.