Cordeiro, M.M.; Filipe, H.A.L.; Santos, P.D.; Samelo, J.; Ramalho, J.P.P.; Loura, L.M.S.; Moreno, M.J. Interaction of Hoechst 33342 with POPC Membranes at Different pH Values. Molecules2023, 28, 5640.
Cordeiro, M.M.; Filipe, H.A.L.; Santos, P.D.; Samelo, J.; Ramalho, J.P.P.; Loura, L.M.S.; Moreno, M.J. Interaction of Hoechst 33342 with POPC Membranes at Different pH Values. Molecules 2023, 28, 5640.
Cordeiro, M.M.; Filipe, H.A.L.; Santos, P.D.; Samelo, J.; Ramalho, J.P.P.; Loura, L.M.S.; Moreno, M.J. Interaction of Hoechst 33342 with POPC Membranes at Different pH Values. Molecules2023, 28, 5640.
Cordeiro, M.M.; Filipe, H.A.L.; Santos, P.D.; Samelo, J.; Ramalho, J.P.P.; Loura, L.M.S.; Moreno, M.J. Interaction of Hoechst 33342 with POPC Membranes at Different pH Values. Molecules 2023, 28, 5640.
Abstract
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.
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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