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A Size-Exclusion Chromatography/FT-IR (SEC/FT-IR) Technique for Improved FTIR Spectroscopy of Proteins in D2O Solution

Volume 45, Number 5 (June 1991) Page 868-873

Remsen, Edward E.; Freeman, John J.


A size-exclusion chromatography/Fourier transform infrared spectroscopic (SEC/FT-IR) method for improving the FT-IR spectroscopy of proteins in D2O solution is described. The technique employs low-volume SEC columns to exchange protein solutions into buffered D2O and effectively removes spectral interference from H2O and other low-molecular-weight compounds in the region of the protein amide carbonyl stretching band (1600-1700 cm−1). Application of the method with protein solutions prepared in deuterated, denaturing buffers completely exchanges labile protein hydrogens with deuterium and refolds the proteins into their native conformations for subsequent spectral characterization. Potential applications of the SEC/FT-IR method for kinetic monitoring of dynamic processes in proteins (e.g., secondary structure formation during protein folding) are also discussed.