pKa Determination of a Histidine Residue in a Short Peptide Using Raman Spectroscopy

Research output: Contribution to journalArticle


Determining the pKa of key functional groups is critical to understanding the pH-dependent behavior of biological proteins and peptide-based biomaterials. Traditionally, ¹H NMR spectroscopy has been used to determine the pKa of amino acids; however, for larger molecules and aggregating systems, this method can be practically impossible. Previous studies concluded that the C-D stretches in Raman are a useful alternative for determining the pKa of histidine residues. In this study, we report on the Raman application of the C2-D probe on histidine's imidazole side chain to determining the pKa of histidine in a short peptide sequence. The pKa of the tripeptide was found via difference Raman spectroscopy to be 6.82, and this value was independently confirmed via ¹H NMR spectroscopy on the same peptide. The C2-D probe was also compared to other Raman reporters of the protonation state of histidine and was determined to be more sensitive and reliable than other protonation-dependent signals. The C2-D Raman probe expands the tool box available to chemists interested in directly interrogating the pKa's of histidine-containing peptide and protein systems.


External organisations
  • Haverford College
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biochemistry and Molecular Biology


  • 1H NMR spectroscopy, acid dissociation constant, deuterium replacement, histidine, isotopic labeling, peptides, proteins, Raman spectroscopy, vibrational probes
Original languageEnglish
Article number405
JournalMolecules (Basel, Switzerland)
Issue number3
Publication statusPublished - 2019 Jan 23
Publication categoryResearch