THE SELECTIVE ENHANCEMENT AND SUBSEQUENT SUBTRACTION OF ATMOSPHERIC WATER-VAPOR CONTRIBUTIONS FROM FOURIER-TRANSFORM INFRARED-SPECTRA OF PROTEINS

The contributions from atmospheric water vapour distort resolution enh anced FTIR spectra of proteins and interfere with the analysis of the secondary structure sensitive amide I band. In this paper, we present a simple, yet very sensitive method for detecting extremely weak water vapour contributions and examine the effects of these weak bands on r esolution enhanced FTIR spectra. The spectra are mildly deconvolved us ing both a narrow bandwidth parameter and a little smoothing. Under th ese conditions, the intensities and shapes of the broad protein bands remain essentially unchanged, while the narrow water vapour contributi ons are enhanced in intensity by roughly 30 fold and their band shapes distorted. Both factors lead to spectra dominated by very narrow vapo ur bands with a sine function-like line shape indicative of over decon volution. The characteristic shape of the over deconvolved vapour band s is easily distinguished from both protein vibrations and the backgro und noise, and can be subtracted from the spectra leading to an accura te determination of the correct subtraction coefficient. It is shown t hat artifacts can occur in resolution enhanced FTIR spectra of protein s, even if the vapour contributions are too weak to be observed in the corresponding non-deconvolved absorbance spectra. The results demonst rate a clear requirement for the careful subtraction of vapour bands f rom all FTIR spectra before analysis of the amide I contour.