Cardiolipin containing liposomes are fully ionized at physiological pH. An FT-IR study of phosphate group ionization

Cardiolipin containing liposomes serve as model systems for probing cytochrome c binding to the inner membrane of mitochondria. In order to interpret binding isotherms properly it is important to identify the protonation state of the two phosphate groups of this phospholipid. Thus far, the literature proposed conflicting views based on experimentally and computationally support for both the fully protonated and semi-protonated state as the physiologically dominant species. In order to clarify this issue we measured the FT-IR spectra of 100% 1,1′2,2′-tetraoleyl cardiolipin (TOCL), 20% TOCL/80% 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) and 100% DOPC as a function of pH between 2 and 11. While the integrated intensities of all DOPC bands were found to be independent of pH within purely statistical variations, we identified specific spectral regions in the TOCL spectrum for which the intensities either decrease and or increase with decreasing pH below pH 4. It was obvious that these regions contain contributions from PO4− and PO stretching modes, respectively. We confirmed this with DFT-based normal mode calculations for a truncated TOCL head group, which revealed that the spectra regions between 1050 and 1100 cm−1 as well as between 1200 and 1300 cm−1 can be ascribed as a superpositions of bands mostly assignable to collective CH deformation modes and PO4 stretching modes. Our results strongly suggest that the protonation of the phosphate groups occur in the acidic region and that they exist in their fully ionized state at physiological pH.