1. Use the following procedure to calculate the area of the
H 2 O-libration and H 2 O-bending combination band of each
spectrum (each labeled with its own #):
(a) Open spectrum and select the spectral region between
2690 and 1965 cm
À1 .
(b) Calculate the baseline-corrected area from 2690 to
1965 cm
À1 (the region may need to be adjusted dependent on the sample), and save the result.
(c) Steps (a) and (b) need to be applied to all the spectra that
have been recorded during the run.
2. In a spreadsheet program, enter column listing: (1) spectrum
number, (2) time point of collection and/or sample temperature during collection, and (3) area of the H 2 O-libration and
H 2 O-bending combination band (AνH 2 O). Create a plot in
which AνH 2 O is plotted as a function of the sample
temperature.
3.8 Spectral
Analysis: Protein
Secondary Structure
and Heat Denaturation
Protein secondary structure can be evaluated by analyzing the
amide-I band in FTIR spectra (1700–1600 cm
À1 wave number
range). This region contains the C¼O stretching vibration band
around 1655 cm
À1 , arising from the protein backbone. Different
bands in the amide-I region represent different types of secondary
structure: α-helical structures and turn/β-sheet structures can be
found at ~1655 and ~1635 cm
À1 , respectively. The H 2 O band
interferes with the amide-I band, which complicates analysis. Difference spectra analysis can be used to resolve this issue. Alternatively, specimens can be measured in D 2 O. Protein denaturation
coincides with an abrupt change in the amide-I band profile, which
is reflected in the band area of second derivative difference spectra
(see Fig. 8):
1. Use the following procedure/macro to calculate the area of the
amide-I band of each spectrum (each labeled with its own #):
(a) Open spectrum (recorded at a particular time point or
temperature) and subtract the first recorded spectrum
(reference spectrum, typically recorded at 0 or 20
C).
(b) Calculate the second derivative of the difference spectrum
obtained in step (a), using a 13-point smoothing factor.
(c) Select the spectral region between 1700 and 1600 cm
À1 .
(d) Invert the spectrum, by multiplication by À1.
(e) Calculate the baseline-corrected area from 1640 to
1605 cm
À1 (the region may need to be adjusted dependent on the sample), and save the result.
(f) Steps (a) through (e) need to be applied to all the spectra
that have been recorded during the run.
344
Willem F. Wolkers and Harrie ¨ tte Oldenhof
H 2 O-libration and H 2 O-bending combination band of each
spectrum (each labeled with its own #):
(a) Open spectrum and select the spectral region between
2690 and 1965 cm
À1 .
(b) Calculate the baseline-corrected area from 2690 to
1965 cm
À1 (the region may need to be adjusted dependent on the sample), and save the result.
(c) Steps (a) and (b) need to be applied to all the spectra that
have been recorded during the run.
2. In a spreadsheet program, enter column listing: (1) spectrum
number, (2) time point of collection and/or sample temperature during collection, and (3) area of the H 2 O-libration and
H 2 O-bending combination band (AνH 2 O). Create a plot in
which AνH 2 O is plotted as a function of the sample
temperature.
3.8 Spectral
Analysis: Protein
Secondary Structure
and Heat Denaturation
Protein secondary structure can be evaluated by analyzing the
amide-I band in FTIR spectra (1700–1600 cm
À1 wave number
range). This region contains the C¼O stretching vibration band
around 1655 cm
À1 , arising from the protein backbone. Different
bands in the amide-I region represent different types of secondary
structure: α-helical structures and turn/β-sheet structures can be
found at ~1655 and ~1635 cm
À1 , respectively. The H 2 O band
interferes with the amide-I band, which complicates analysis. Difference spectra analysis can be used to resolve this issue. Alternatively, specimens can be measured in D 2 O. Protein denaturation
coincides with an abrupt change in the amide-I band profile, which
is reflected in the band area of second derivative difference spectra
(see Fig. 8):
1. Use the following procedure/macro to calculate the area of the
amide-I band of each spectrum (each labeled with its own #):
(a) Open spectrum (recorded at a particular time point or
temperature) and subtract the first recorded spectrum
(reference spectrum, typically recorded at 0 or 20
C).
(b) Calculate the second derivative of the difference spectrum
obtained in step (a), using a 13-point smoothing factor.
(c) Select the spectral region between 1700 and 1600 cm
À1 .
(d) Invert the spectrum, by multiplication by À1.
(e) Calculate the baseline-corrected area from 1640 to
1605 cm
À1 (the region may need to be adjusted dependent on the sample), and save the result.
(f) Steps (a) through (e) need to be applied to all the spectra
that have been recorded during the run.
344
Willem F. Wolkers and Harrie ¨ tte Oldenhof