Infrared absorption bands are broad and often overlap with neighboring bands to produce a complex absorption profile. To elucidate the structural components of the protein, it is necessary to determine the component bands that overlap and generate the composite spectrum. The amide I band is most often subject to such an analysis of secondary structure. Two resolution enhancement techniques, differentiation and Fourier self-deconvolution, are commonly used to identify the component bands. Both methods do not increase instrumental resolution, but are mathematical procedures that yield narrower component bands. Although in many cases deconvolved amide I bands have been used to determine secondary structure by curve fitting, it should be recognized that resolution enhanced spectra, especially derivative spectra do not reproduce true band intensities and relative component fractions cannot be obtained directly from them. Finally, noise is enhanced in these spectra, which, depending on initial data quality, puts a limit on the extent to which resolution can be enhanced. Despite these shortcomings, both methods are extremely useful for identifying component frequencies in complex spectra and these band positions can then be used as fixed input parameters in component band-fitting routines on the original unprocessed spectra. Before these techniques are applied, peaks due to residual water vapor must be carefully subtracted from the raw spectra, because spurious spectral contaminants like those from water vapor will also be enhanced by these methods. The sharp peaks of water vapor can be reliably subtracted by inspecting the amide I region (~1650 cm–1) of the spectrum. When H2O (in contrast to D2O) buffers are used and an analysis of the amide I band is attempted, the spectral region around 2125 cm–1 is well suited to check for the correct subtraction of liquid water because this region is usually free from protein and lipid absorbance. In some cases a complete subtraction of the water bands is not possible because water bound to proteins can exhibit altered band shapes.
Infrared absorption bands are broad and often overlap with neighboring bands to produce a complex absorption profile. To elucidate the structural components of the protein, it is necessary to determine the component bands that overlap and generate the composite spectrum. The amide I band is most often subject to such an analysis of secondary structure. Two resolution enhancement techniques, differentiation and Fourier self-deconvolution, are commonly used to identify the component bands. Both methods do not increase instrumental resolution, but are mathematical procedures that yield narrower component bands. Although in many cases deconvolved amide I bands have been used to determine secondary structure by curve fitting, it should be recognized that resolution enhanced spectra, especially derivative spectra do not reproduce true band intensities and relative component fractions cannot be obtained directly from them. Finally, noise is enhanced in these spectra, which, depending on initial data quality, puts a limit on the extent to which resolution can be enhanced. Despite these shortcomings, both methods are extremely useful for identifying component frequencies in complex spectra and these band positions can then be used as fixed input parameters in component band-fitting routines on the original unprocessed spectra. Before these techniques are applied, peaks due to residual water vapor must be carefully subtracted from the raw spectra, because spurious spectral contaminants like those from water vapor will also be enhanced by these methods. The sharp peaks of water vapor can be reliably subtracted by inspecting the amide I region (~1650 cm–1) of the spectrum. When H2O (in contrast to D2O) buffers are used and an analysis of the amide I band is attempted, the spectral region around 2125 cm–1 is well suited to check for the correct subtraction of liquid water because this region is usually free from protein and lipid absorbance. In some cases a complete subtraction of the water bands is not possible because water bound to proteins can exhibit altered band shapes.
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