A new method for simultaneous quantification of uncoupling and inhibitory activity of organic pollutants in energy-transducing membranes

A new, mechanistically based approach is presented for the quantitative determination of the uncoupling and inhibitory activity of compounds interfering with energy transduction. Time-resolved spectroscopy of single-turnover events in the photosystem of the photosynthetic bacterium Rhodobacter sphaeroides can quantitatively distinguish uncoupling from inhibition. The decay kinetics of the membrane potential after a single turnover flash are used as a measure of uncoupling activity, and the redox kinetics of several components of the electron transfer chain are used as indicators of specific inhibition at various potential inhibitory sites. Results are presented for 21 nitrated and chlorinated phenols, some reference uncouplers, and some anisoles. Inhibition was exclusively detected at one specific quinone binding site, the quinone reductase site Qi. For most phenols, uncoupling was observed at lower concentrations than inhibition with the exception of alkylated 2,6-dinitrophenols and 2,4,6-trichlorophenol, where both effects occurred in the same concentration range. No direct correlation was observed between the uncoupling and inhibitory activity of a given compound. The data obtained with this new method correlate well with data from various bioassays on energy-transducing systems, indicating that this method may also be well suited as a screening tool for compounds suspected to interfere with energy transduction.