Biomedical Engineering Reference
In-Depth Information
Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) is a powerful tool used to monitor changes
in protein and polypeptide secondary structure during processing. After exposure of a
protein to infrared light, its secondary structure can be determined from the spectra obtained
from the absorption of different wavelengths corresponding to specific vibration frequencies
of the amide bonds (Jackson and Mantsch, 1995a).
Two kinds of the vibrations, known as amide I and amide II vibrations, are directly
correlated to the molecular structure of proteins and polypeptides. The stretching of the
carbonyl C = O double bonds is linked to the amide I vibration, and the deformation of the
N-H bonds causes the amide II vibration. FTIR determines the amount of light absorbed
corresponding to each of the vibrations over a wide range of frequencies (Kumosinski and
Farrell, 1993). Empirical correlations between the frequency of the amide I and amide II
absorptions of the protein and the secondary structure composition in helix, extended
β
-sheets, loops and unordered structures have been determined (Jackson and Mantsch,
1995 ).
The amide I absorption region (1700-1600 cm -1 ) in the FTIR is widely used for the
determination of the secondary structure of proteins (Álvarez et al ., 2008 ). Parallel and
antiparallel
β
-sheets are attributed to bands at 1622 and 1632 cm -1 , respectively, whereas,
α
-turns and unordered struc-
tures can be detected respectively at band positions of 1692 and 1645 cm -1 (Jackson and
Mantsch, 1995 ).
-helix structures correspond to the band at ~1655 cm -1 .
β
Circular dichroism
Circular dichroism (CD) is a powerful method for the study of protein structure and folding
in solution under various conditions. The technique has been widely utilized to monitor
structural transitions of food and bioproduct proteins resulting from changes in different
processing conditions (Martin and Schilstra, 2008). CD is a spectroscopic technique based
on the difference in protein interactions with left (L) and right (R) circularly polarized light
(Sreerama and Woody, 2004). Hence, CD analysis of proteins is based on the measurements
of the difference in absorbance between L and R circularly polarized components expressed
as ellipticity (
) in degrees (Kelly et al ., 2005). CD spectra thus result from the chirality of
some structures, such as the carbon atom bound to four different substituents, the C-S-S-C
structure and molecular asymmetric environments (Kelly et al ., 2005 ). Table 3.2 shows the
spectral region and contributing protein chromophores. The determination of the relative
proportions of
θ
-turns and random coil structures, as well as the overall
features of the secondary structure, could be determined by CD measurements in the far UV
region (Sreerama and Woody, 2004). All
α
-helix,
β
-sheets,
β
-lactalbumin and
lysozyme, are characterized by an intense negative band with two peaks at 208 and 222 nm,
and a strong positive band at 191-193 nm (Barbana et al ., 2006 ), whereas the
α
-helix rich proteins, such as
α
sheets rich
proteins are distinguished by a negative band at 210-225 nm and a stronger positive band at
β
Table 3.2 Contributing chromophores in CD spectra of proteins.
Chromophore
CD spectra region
Backbone amides
<250 nm (far UV)
Aromatic groups
250-300 nm (near UV)
Extrinsic groups
Above 300 nm (near UV-visible region)
 
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