Towards label-free and site-specific probing of the local pH in proteins: pH-dependent deep UV Raman spectra of histidine and tyrosine

The site-specific pH is an experimental probe for assessing models of structural folding and function of a protein as well as protein–protein and protein–ligand interactions. It can be determined by various techniques such as NMR, FT-IR, fluorescence and EPR spectroscopy. The latter require the use of external labels, i.e., employ pH-dependent dyes and spin labels, respectively. In this contribution, we outline an approach to a label-free and site-specific method for determining the local pH using deep ultraviolet resonance Raman (UVRR) spectroscopic fingerprints of the aromatic amino acids histidine and tyrosine in combination with a robust algorithm that determines the pH value using three UVRR reference spectra and without prior knowledge of the p K a .