AHA- heterodimer of a class-2 uncoupler: pentachlorophenol

AHA- heterodimers formed by association of neutral molecules of weak acid (HA) with its conjugate anion (A-) have been proposed to be the charged membrane-permeable species of class-2 uncouplers. Past attempts to extract and identify AHA- heterodimers failed. We have measured optical spectra of HA+A- (1:1) solutions of pentachlorophenol (PCP) in various solvents and in the presence of PC liposomes. Optical studies were supplemented by nuclear magnetic resonance measurements of HA+A- (1:1) solutions of PCP in dichloroethane to gain insight into the formation of AHA- species in lipid membranes. From these experiments, we found evidence for AHA- formation in non-hydrogen-bonding solvents, then reported the AHA- formation constant Kf and the molar absorptivity epsilon AHA-(lambda). Kf decreases with increasing dielectric constant, kappa, from 1210 +/- 130 M-1 for dichloroethane (kappa 10.7), to 340 +/- 34 M-1 for acetonitrile (kappa 37.5); Kf also decreases with increasing concentration of water. In hydrogen-bonding solvents, octanol (kappa 10.3) and methanol (kappa 33.5) and in liposomes, AHA- heterodimers are not observed optically. We estimate Kf for PCP in lipid bilayers from a combination of data on membrane electrical conductivity and surface density of adsorbed PCP. Our estimate for lipid bilayer, 0.005 < Kf < 0.5 M-1, is consistent with our inability to detect the AHA- species optically in liposome suspensions. We propose that penetration of water into the membrane inhibits formation of AHA- in lipid bilayers.