Combined in-silico and in-vitro experiments support acid-base equilibrium as a tool to estimate the localization...

The interaction and location of 4-nitrophenol (PNP) in biomembranes are relevant in the bioaccumulation and potentiation of the intensive toxic effects of this persistent organic pollutant. In this work, in-silico analyses predicted that, in a fluid phospholipid bilayer, the minimum energy of the protonated (PNPH) and deprotonated (PNP^-) species is located within the glycerol and choline region, respectively. This was experimentally confirmed by acid-base equilibrium experiments and theory, allowing the estimation of the mean location of PNP within a bilayer region with a dielectric constant D = 50.6 compatible with the phosphate/choline moiety of egg-yolk phosphatidylcholine unilamellar (EPC) vesicles. The comparison with the D = 43.2 value obtained in Triton X-100 micelles allow predicting a mean surface potential of ψ = 25.37 mV for the EPC-water interface. Changes in the chemical shifts and longitudinal relaxation times of EPC hydrogens by ¹H NMR confirm the deeper location of the PNPH within the glycerol region and at the choline region (PNP^-) at higher pH. Intermolecular PNP^-EPC dipolar interactions within the choline region was also demonstrated at pH 10.2 using ROESY experiments. Additional information was obtained trough ³¹P NMR, that detected an increase in the anisotropy at the membrane interface after insertion of PNP which probably act as a bridge between choline moieties rigidizing the crystalline structure at that spot. Concluding, here we provide experimental support to the "pH-piston hypothesis" proposed some decades ago in the pharmaceutical field, and that reinforce the importance of the environmental conditions (e.g. pH) to modulate the bioavailability of this highly toxic pollutant.