Abstract
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.
Highlights
There is currently a great deal of published data from infrared (IR) probes [1,2,3]; Raman scattering spectroscopy is gaining traction as a technique for collecting signals from vibrational probe groups
This study demonstrates clearly that Raman spectroscopy provides a reliable method to determine
Thisof study demonstrates clearly that Raman spectroscopy a reliableofmethod to the pKa histidine
Summary
There is currently a great deal of published data from infrared (IR) probes [1,2,3]; Raman scattering spectroscopy is gaining traction as a technique for collecting signals from vibrational probe groups. In comparison to IR absorption, complementary selection rules of Raman scattering mean that probe signals can sometimes be more observed in Raman than Fourier transform infrared (FTIR) spectroscopy [4]. Carbon-deuterium (C-D) stretches can be employed as a vibrational probe in both FTIR and. One of the first analyses of C-D bands was in the FTIR and Raman spectra of CD3 OH and CD3 OD [7], where exchanging a protium for a deuterium (C-H to C-D) caused a shift of ~800 cm−1 for the anti-symmetric and symmetric stretches from 2850–3000 cm−1 to 2050–2350 cm−1 , respectively. The anti-symmetric stretch around 2300 cm−1 is significantly more intense in the Raman spectrum compared to in the FTIR spectrum
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