Fourier‐transform Infrared Spectroscopic Investigation of the Secondary Structure of Aqueous and Dried Recombinant Human Deoxyribonuclease I

The secondary structure of aqueous and dried recombinant deoxyribonuclease I (rhDNase I) was studied by Fourier-transform infrared (FTIR) spectroscopy. The second derivative spectra for the amide I region, and also Gaussian curve-fitted original amide III spectra were examined. From the solution spectra, we discerned a mixed α/β-composition, consistent with the native secondary structure determined by X-ray crystallography. Upon freeze drying, the amide I second derivative showed an increased absorbance at about 1691 cm−1, probably due to increased intermolecular β-sheet formation. Gaussian curve-fitting of the amide III spectra also revealed an increase in β-sheets (exhibiting absorbance at 1220–1237cm−1), and some decrease in the α-helix content, a common occurrence for proteins upon drying. In addition, we tested several methodologies for preparation of solid rhDNase I samples (including transmission through the solid pressed directly between CaF2 windows) to confirm that the structural alteration was not a result of the conditions used in generating the spectra.