Improving real-time measurement of H/D exchange using a FTIR biospectroscopic probe

We describe the improvement of a novel approach to investigating hydrogen/deuterium (H/D) exchange kinetics in biomolecules using transmission infrared spectroscopy. The method makes use of a Fourier transform infrared spectrometer coupled with a microdialysis flow cell to determine exchange rates of labile hydrogens. With this cell system, the monitoring of exchange reactions has been studied here as a function of some cell characteristics such as: (a) dialysis membrane surface contacting both the H₂O and D₂O compartments; (b) molecular cutoff of dialysis membrane; and (c) distance between the cell-filling holes. The best improvement has been obtained by increasing the dialysis membrane surface followed by increase of molecular cutoff. However, not significant differences were found using various distances between filling holes. The fastest exchange rate which can be measured with the cell system used here is found to be k = 0.41 ± 0.02 min⁻¹, that is, about threefold greater than the one got in a previous work. This microdialysis flow cell has been used here for the study of H/D exchange in nucleic acids with subsequent structural analysis by 2D correlation spectroscopy.