The interaction of highly active uncouplers with mitochondria

A simple carrier model describes adequately the transport of protons across lipid bilayer membranes by the weak acid S-13. We determined the adsorption coefficients of the anionic, A−, and neutral, HA, forms of the weak acid and the rate constants for the movement of A− and HA across the membrane by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. These measurements agree with the results of voltage clamp and charge pulse kinetic experiments. We considered three mechanisms by which protons can cross the membranesolution interface. An anion adsorbed to the interface can be protonated by (i) a H+ ion in the aqueous phase (protolysis), (ii) a buffer molecule in the aqueous phase or (iii) water molecules (hydrolysis). We demonstrated that the first reaction cannot provide the required flux of protons: the rate at which H+ must combine with the adsorbed anions is greater than the rate at which diffusion-limited reactions occur in the bulk aqueous phase. We also ruled out the possibility that the buffer is the main source of protons: the rate at which buffers must combine with the adsorbed anions is greater than the diffusion-limited rate when we reduced the concentration of polyanionic buffer adjacent to the membrane-solution interface by using membranes with a negative surface charge. A simple analysis demonstrates that a hydrolysis reaction can account for the kinetic data. Experiments at acid pH demonstrate that the transfer of H+ from the membrane to the aqueous phase is limited by the rate at which OH combines with adsorbed HA and that the diffusion coefficient of OH− in the water adjacent to the bilayer has a value characteristic of bulk water. Our experimental results demonstrate that protons are capable of moving rapidly across the membrane-solution interface, which argues against some mechanisms of local chemiosmosis.

How do protons cross the membrane-solution interface? Kinetic studies on bilayer membranes exposed to the protonophore S-13 (5-chloro-3-tert-butyl-2'-chloro-4' nitrosalicylanilide).

Theory and applications of ion-selective electrodes part II

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 Q{sub i}. 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.

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

Several reports have suggested that people living in industrialized societies will generally be contaminated with pentachlorophenol (PCP) at concentrations of 1 ppb and higher. The most likely source for this contamination appears to be food chain exposure to PCP treated wood products. Another possible source of exposure is metabolism of hexachlorobenzene to pentachlorophenol. Pentachlorophenol has been shown to be a mutagen and commercial preparations of the compound are often contaminated with dioxins. Both of these observations may be significant to the toxicological evaluation of the present hazard from pentachlorophenol exposure.

W.H. Perman

Papers

Electrical conductivity in lipid bilayer membranes induced by pentachlorophenol.