Theory, design, and characterization of a microdialysis flow cell for Raman spectroscopy

The theory, design, and application of a dialysis flow cell for Raman spectroscopy are described. The flow cell permits rapid collection of Raman spectra concurrent with the efflux of small solute molecules or ions into a solution of macromolecules and is well suited to acquisition of data during hydrogen-isotope exchange reactions of biological molecules. Kinetic parameters of the device are described by a diffusion model, which accounts satisfactorily for the observed rates of efflux of deuterium oxide (K2H = 0.30 min-1), calcium ions (KCa = 0.10 min-1) and EGTA (KEGTA = 0.07 min-1). Application to the kinetics of glutamate protonation in a peptide copolymer [poly(Glu, Lys, Tyr)] shows that pH-titration rates as high as 3.3 pH units/min can be monitored. It is also shown that one can extract first-order hydrogen-isotope exchange rate constants from measured second-order exchanges by taking into account the rate of entry of 2H2O effluent into the bulk H2O solution. Deuterium exchanges of the single-stranded polyribonucleotides poly(rA) and poly(rU) and of the double-stranded RNA genome from bacteriophage phi 6 have been investigated. The measured nucleotide base exchange rates are comparable with those determined previously by other methods. The results indicate that base exchanges as fast as approximately 2 min-1 can be determined reliably with the present design. Application of the Raman flow cell to hydrogen-isotope exchange of the basic pancreatic trypsin inhibitor confirms consistency with results obtained previously on this protein by tritiation and NMR techniques.