Hoechst 33342 (H33342) is a fluorescent probe that is commonly used to stain DNA of living cells. To do so, it needs to interact with and permeate through cell membranes. The pH of biological media may vary quite significantly and this influences H33342 ionization state, and may change its rate of permeation through biomembranes. In this work, we address the effect of pH in the association of H33342 with lipid bilayers using a combined experimental and computational approach. The partition of H33342 to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes was experimentally quantified using fluorescence spectroscopy and isothermal titration calorimetry (ITC) measurements. Quantum chemical calculations were performed to select the most stable isomer of H33342 for the overall charges 0, +1, and +2, expected to predominate across the 5 < pH < 10 range. The interaction of these isomers with POPC bilayers was then studied by both unrestrained and umbrella sampling molecular dynamics simulations. While probe insertion occurs for all ionization states, with similar overall equilibrium location, orientation was found to depend on the probe charge, with the neutral form lying mostly parallel and the doubly charged displaying a significant tilt relative to the membrane plane. Both experimental results and computational free energy profiles indicate that the partition coefficient of H33342 displays a small variation over a wide pH range, not exceeding one order of magnitude. The enthalpy of interaction is negative and is larger at lower pH values. This suggests efficient hydrogen bonding between the probe and the lipid, namely for the protonated +2 form. The relatively high lipophilicity obtained for the charged species contrasts with the decrease in their general hydrophobicity as estimated from water and octanol partition. This highlights the distinction between lipophilicity and hydrophobicity, and the importance of considering association with lipid bilayers when predicting the affinity for biomembranes.

The membrane dipole potential that arises from the interfacial water and constitutive dipolar groups of lipid molecules modulates the interaction of amphiphiles and proteins with membranes. Consequently, its determination for lipid mixtures resembling the existing diversity in biological membranes is very relevant. In this work the dipole potential of monolayers, formed at the air-water interface, from pure or mixed lipids (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-ol0eoyl-sn-glycero-3-phosphatidyserine (POPS), Sphingomyelin (SpM) and cholesterol) were measured and correlated with the mean area per lipid. The results showed that, as previously observed, cholesterol increases the dipole potential in correspondence with the decrease in the average area per lipid and that the replacement of phosphocholine lipids by sphingomyelin leads to a decrease in the dipole potential. We have also observed that 20 mol% of POPE induces a monolayer condensation which is not affecting the measured dipole potential, except when cholesterol is added to the mixture. Additionally, at the small mole fractions encountered in biomembranes, the presence of the negatively charged lipid POPS increases the dipole potential of monolayers even though an enhancement in the monolayer packing is not evident. This increase in the dipole potential due to POPS and cholesterol and the smaller dipole potential of monolayers containing large amounts of SpM generates a non-zero transbilayer dipole potential in the asymmetric plasma membrane of eukaryotic cells.