Measuring local pH at interfaces from molecular tumbling: A concept for designing EPR-active pH-sensitive labels and...

Molecular probes and indicators are broadly employed for pH measurements in bulk media and at interfaces. The underlying physical principle of pH measurements of most of these probes is based on a change in the electronic structure that, for example, results in a shift of the emission peak of the fluorescence probes, changes in NMR chemical shifts due to the affected electronic shielding, or magnetic parameters of pH-sensitive nitroxides as measured by EPR. Here we explore another concept for measuring local protonation state of molecular tags based on changes in rotational dynamics of electron spin-bearing moieties that are readily detected by conventional continuous wave X-band EPR. Such changes are especially pronounced at biological interfaces, such as lipid bilayer membranes, due to the probe interactions with adjacent charges and polarizable dipoles. The concept was demonstrated by synthesizing a series of pH-sensitive nitroxides and spin-labelled phospholipids. EPR spectra of these newly synthesized nitroxides exhibit relatively small - about 0.5 G - changes in isotropic nitrogen hyperfine coupling constant upon reversible protonation. However, spin-labelled phospholipids incorporated into lipid bilayers demonstrated almost 6-fold change in rotational correlation time upon protonation, readily allowing for pK_a determination from large changes in EPR spectra. The demonstrated concept of EPR-based pH measurements leads to a broader range of potential nitroxide structures that can serve as molecular pH sensors at the desired pH range and, thus, facilitates further development of spin-labelling EPR methods to study electrostatic phenomena at chemical and biological interfaces.