Determination of divalent metal ion-regulated proton concentration and polarity at the interface of anionic...

We studied the influence of trace quantities of divalent metal ions (M²⁺: Ca²⁺, Mg²⁺, and Zn²⁺) on proton concentration (-log[H⁺], designated as pH') and polarity at the interface of anionic PG-phospholipid membranes comprising saturated and unsaturated acrylic chains. A spiro-rhodamine-6G-gallic acid (RGG) pH-probe was synthesized to monitor the interfacial pH' of large unilamellar vesicles (LUVs) at a physiologically appropriate bulk pH (6.0-7.5). ¹H-NMR spectroscopy and fluorescence microscopy showed that RGG interacted with the LUV interface. The pH-dependent equilibrium between the spiro-closed and spiro-open forms of RGG at the interface from the bulk phase was compared using fluorescence spectra to obtain interfacial pH'. Interfacial dielectric constant (κ) was estimated using a porphyrin-based polarity-probe (GPP) that exhibits a κ-induced equilibrium between monomeric and oligomeric forms. M²⁺ interaction decreased LUV interfacial κ from ∼67 to 61, regardless of lipid/M²⁺ types. Fluorescence spectral and microscopic analysis revealed that low Ca²⁺ and Mg²⁺ amounts (M²⁺/lipid = 1 : 20 for unsaturated DOPG and POPG and ∼1 : 10 for saturated DMPG lipids), but not Zn²⁺, decreased LUV interfacial acidity from pH' ∼3.8 to 4.4 at bulk pH 7.0. Although membrane surface charges are normally responsible for pH' deviation from the bulk to the interface, they cannot explain M²⁺-mediated interfacial pH' increase since there is little change in surface charges up to a low M²⁺/lipid ratio of <1/10. M²⁺-induced tight lipid headgroup packing and the resulting increased surface rigidity inhibit interfacial H⁺/H₂O penetration, reducing interfacial acidity and polarity. Our findings revealed that in certain cases, essential M²⁺ ion-induced bio-membrane reactivity can be attributed to the influence of interfacial pH'/polarity.