224
B. Czarnik-Matusewicz and Y.M. Jung
tion could lead to an adsorption process. According to [127], this process may be
responsible for substantial structural distortions in the secondary structure and for
additional spectral variations that cannot be eliminated through any mathematical
pretreatment procedure. therefore, it was interesting to compare the adsorption-induced changes with the changes caused by concentration [47]. to answer this question, power spectra that present the overall extent of the intensity changes caused
by the given perturbation process have been used. A comparative analysis of these
power spectra has shown that the concentration changes were 10 times more intense
from those arising from adsorption for the example of β-lactoglobulin measured as
a function of concentration (from 1 to 5 % wt) and time for a 5 % wt system. this
result has convinced us that concentration was the dominant perturbation and that
2d results can be discussed while the minor contributions induced by adsorption
are neglected.
Similar experimental protocols have been used to study the thermal denaturation
of human serum albumin [92] and the molten globule-like state of ovalbumin at
acidic ph values [95]. In these analyses, the advantages of power and slice spectra
in monitoring the process of protein unfolding have been demonstrated. In [92],
a plot of the slice spectra at different temperatures extracted from concentrationperturbed asynchronous contour maps at 4,600 cm
−1
, the amide B–amide II combination transition, has clearly indicated a sudden change in the secondary structure
near 60 °C. A similar plot for the band near 7,000 cm
−1
, assigned to a combination
mode of water, has confirmed the active role of water in the process. the change
from near-IR to mid-IR radiation has allowed the study of the roles of different secondary structures and side chains in the transition [60]. the asynchronous spectrum
generated from the concentration-dependent spectral variations at a temperature
before the transition has shown that the exposed part of protein composed from
the side chains, random coil, and extended chains has been more sensitive to the
concentration changes than the α-helices and α-turns hidden in the protein core.
however, the identical analysis of the concentration-dependent spectra collected
at a temperature above the transition has revealed the opening of the compact core
because the changes assigned to β-turns were identified before those in the extended
chain, random coil structures, and α-helices.
Concentration has also been used as an external perturbation by Kamerzell et al.
[98], who have investigate the interactions that modulate the nonideal behavior of
two immunoglobulins (Igg) in highly concentrated solutions. With the same data
pretreatment as in [47], the nonspecific spectral changes were minimized, and the
remaining changes that deviated from linearity with concentration increase from 20
to 120 mg/mL were subjected to 2dCoS. the sequence of spectral events as a function of increased concentration of IgG has revealed that the variations in the β-sheet
and turn regions have occurred before the intensity variations in the disordered and
α-helical regions. Moreover, the 2DCoS results supported by PCA have shown that
glu and Asp residues triggered the processes that resulted in association of Igg. In
summary, increased intermolecular hydrogen bonding interactions and electrostatic
interaction modulate the intermolecular association and the nonideal behavior.
B. Czarnik-Matusewicz and Y.M. Jung
tion could lead to an adsorption process. According to [127], this process may be
responsible for substantial structural distortions in the secondary structure and for
additional spectral variations that cannot be eliminated through any mathematical
pretreatment procedure. therefore, it was interesting to compare the adsorption-induced changes with the changes caused by concentration [47]. to answer this question, power spectra that present the overall extent of the intensity changes caused
by the given perturbation process have been used. A comparative analysis of these
power spectra has shown that the concentration changes were 10 times more intense
from those arising from adsorption for the example of β-lactoglobulin measured as
a function of concentration (from 1 to 5 % wt) and time for a 5 % wt system. this
result has convinced us that concentration was the dominant perturbation and that
2d results can be discussed while the minor contributions induced by adsorption
are neglected.
Similar experimental protocols have been used to study the thermal denaturation
of human serum albumin [92] and the molten globule-like state of ovalbumin at
acidic ph values [95]. In these analyses, the advantages of power and slice spectra
in monitoring the process of protein unfolding have been demonstrated. In [92],
a plot of the slice spectra at different temperatures extracted from concentrationperturbed asynchronous contour maps at 4,600 cm
−1
, the amide B–amide II combination transition, has clearly indicated a sudden change in the secondary structure
near 60 °C. A similar plot for the band near 7,000 cm
−1
, assigned to a combination
mode of water, has confirmed the active role of water in the process. the change
from near-IR to mid-IR radiation has allowed the study of the roles of different secondary structures and side chains in the transition [60]. the asynchronous spectrum
generated from the concentration-dependent spectral variations at a temperature
before the transition has shown that the exposed part of protein composed from
the side chains, random coil, and extended chains has been more sensitive to the
concentration changes than the α-helices and α-turns hidden in the protein core.
however, the identical analysis of the concentration-dependent spectra collected
at a temperature above the transition has revealed the opening of the compact core
because the changes assigned to β-turns were identified before those in the extended
chain, random coil structures, and α-helices.
Concentration has also been used as an external perturbation by Kamerzell et al.
[98], who have investigate the interactions that modulate the nonideal behavior of
two immunoglobulins (Igg) in highly concentrated solutions. With the same data
pretreatment as in [47], the nonspecific spectral changes were minimized, and the
remaining changes that deviated from linearity with concentration increase from 20
to 120 mg/mL were subjected to 2dCoS. the sequence of spectral events as a function of increased concentration of IgG has revealed that the variations in the β-sheet
and turn regions have occurred before the intensity variations in the disordered and
α-helical regions. Moreover, the 2DCoS results supported by PCA have shown that
glu and Asp residues triggered the processes that resulted in association of Igg. In
summary, increased intermolecular hydrogen bonding interactions and electrostatic
interaction modulate the intermolecular association and the nonideal behavior.