Determination of proton concentration at cardiolipin-containing membrane interfaces and its relation with the peroxidase activity of cytochrome c†

The activities of biomolecules are affected by the proton concentrations at biological membranes. Here, we succeeded in evaluating the interface proton concentration (−log[H+] defined as pH′) of cardiolipin (CL)-enriched membrane models of the inner mitochondrial membrane (IMM) using a spiro-rhodamine-glucose molecule (RHG). According to fluorescence microscopy and 1H-NMR studies, RHG interacted with the Stern layer of the membrane. The acid/base equilibrium of RHG between its protonated open form (o-RHG) and deprotonated closed spiro-form (c-RHG) at the membrane interface was monitored with UV-vis absorption and fluorescence spectra. The interface pH′ of 25% cardiolipin (CL)-containing large unilamellar vesicles (LUVs), which possess similar lipid properties to those of the IMM, was estimated to be ∼3.9, when the bulk pH was similar to the mitochondrial intermembrane space pH (6.8). However, for the membranes containing mono-anionic lipids, the interface pH′ was estimated to be ∼5.3 at bulk pH 6.8, indicating that the local negative charges of the lipid headgroups in the lipid membranes are responsible for the deviation of the interface pH′ from the bulk pH. The peroxidase activity of cyt c increased 5–7 fold upon lowering the pH to 3.9–4.3 or adding CL-containing (10–25% of total lipids) LUVs compared to that at bulk pH 6.8, indicating that the pH′ decrease at the IMM interface from the bulk pH enhances the peroxidase activity of cyt c. The peroxidase activity of cyt c at the membrane interface of tetraoleoyl CL (TOCL)-enriched (50% of total lipids) LUVs was higher than that estimated from the interface pH′, while the peroxidase activity was similar to that estimated from the interface pH′ for tetramyristoyl CL (TMCL)-enriched LUVs, supporting the hypothesis that when interacting with TOCL (not TMCL), cyt c opens the heme crevice to substrates. The present simple methodology allows us to estimate the interface proton concentrations of complex biological membranes

Detection of Curvature-Radius-Dependent Interfacial pH/Polarity for Amphiphilic Self-Assemblies: Positive versus Negative Curvature

It is possible that a defined curvature at the membrane interface controls its pH/polarity to exhibit specific bioactivity. By utilizing an interface-interacting spiro-rhodamine pH probe and the Schiff base polarity probe, we have shown that the pH deviation from the bulk phase to the interface (ΔpH)/interfacial dielectric constant (κ­(i)) for amphiphilic self-assemblies can be regulated by the curvature geometry (positive/negative) and its radius. According to ¹H NMR and fluorescence anisotropy investigations, the probes selectively interact with an anionic interfacial Stern layer. The ΔpH/κ­(i) values for the Stern layer are estimated by UV–vis absorption and fluorescence studies. For the anionic sodium bis-2-ethylhexyl-sulfosuccinate (AOT) inverted micellar (IM) negative interface, the highly restricted water and proton penetration into the Stern layer owing to tight surfactant packing or a reduced water-exposed headgroup area may be responsible for the much lower ΔpH ≈ −0.45 and κ­(i) ≈ 28 in comparison to ∼−2.35 and ∼44, respectively, for the anionic sodium dodecyl sulfate (SDS) micellar positive interface with a close similar Stern layer. With increasing AOT IM water-pool radius (1.7–9.5 nm) or [water]/[AOT] ratio (<i>w</i>₀) (8.0–43.0), the ΔpH and κ­(i) increase maximally up to ∼−1.22 and ∼45, respectively, due to a greater water-exposed headgroup area. However, the unchanged ΔpH ≈ −0.65 and κ­(i) ≈ 53.0 within radii ∼3.5–8.0 nm for the positive interface of a mixed Triton X-100 (TX-100)/SDS (4:1) micelle justify its packing flexibility. Interestingly, the continuously increasing ΔpH trend for IM up to its largest possible water-pool radius of ∼9.5 nm may rationalize the increase in ΔpH (∼−1.4 to −1.6) with the change in the curvature radii (∼15 to 50 nm) for sodium 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphorylglycerol (DMPG)/1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphocholine (DMPC) (2:1) large unilamellar vesicles (LUV) owing to its negative interface. Whereas, similar to the micellar positive interface, the unchanged ΔpH at the positive LUV interface was confirmed by fluorescence microscopic studies with giant unilamellar vesicles of identical lipids composition. The present study offers a unique and simple method of monitoring the curvature-radius-dependent interfacial pH/polarity for biologically related membranes