Application of Fourier transform infrared spectroscopy to studies of aqueous protein solutions

Abstract Modern protein Fourier transform infrared (FT-IR) spectroscopy has proven to be a versatile and sensitive technique, applicable to many aspects of protein characterization. The major practical drawback for the FT-IR spectroscopy of proteins is the large absorbance band of water, which overlaps the amide I resonances. D 2 O is often substituted for H 2 O in infrared experiments. Removal of water from protein samples can be complicated and tedious and potentially lead to denaturation, aggregation, or sample loss. Solvent removal by dialysis is difficult for suspensions and sols. A new method called the D 2 O dilution technique (Ddt) is described which simplifies the sample preparation step and improves the solvent subtraction. The effect of the D 2 O concentration on the IR spectrum of aqueous solutions of several model proteins was studied. Dilution of aqueous samples with D 2 O yields good quality spectra. The Ddt has been evaluated for quantitative analysis using standard proteins and its applicability to solutions and suspensions of a genetically engineered malaria antigen is demonstrated. Use of resolution-enhancement techniques with spectra in mixed solvents has also been investigated.