Protein Processing in Food and Bioproduct Manufacturing and Techniques for Analysis - Food and Industrial Bioproducts and Bioprocessing - page 104

0.0001

0

250 270 290 310 330 350

-0.0001

-0.0002

Wavelength (nm)

-0.0003

Figure 3.6 Circular dichroism spectra in the near UV region of bovine holo- (solid line) and apo- (dashed
line) -lactalbumin. Circular dichroism spectra were recorded at a protein concentration of 70 mM in 5 mM

Tris buffer, pH 7.0, at 20 °C. (Reproduced from Barbana et al ., copyright 2006, with permission from Elsevier.)

α

190-200 nm. Unordered proteins are generally characterized by a negative band at 195-200 nm,
and a much weaker signal between 215 and 230 nm (Martin and Schilstra, 2008).
CD spectra analysis at the near UV region provides important information about protein
tertiary structure. Spectra at the near UV region arise from the aromatic amino acids, and
depend on their number, mobility, spatial disposition and their environment (Kelly et al .,
2005). CD signals could also be useful for the study of the conformational changes in
proteins caused by ligand binding (Barbana et al ., 2006 , 2008 ). Figure 3.6 shows the
near UV CD spectra of bovine

α -lactalbumin in its holo and apo forms. As observed, apo-

α -lactalbumin has very low signal in the near UV region of the CD spectrum, indicative of

loss of the native tertiary structure with rapidly rotating aromatic side chains after the
release of Ca 2+ .

Fluorescence spectroscopy
Fluorescence is the light emitted subsequent to absorption of UV or visible light due to
electronic state transitions from excited singlet to several vibrational levels (Lakowicz,
1999 ; Hof et al ., 2005). The emission spectrum provides information for both qualitative
and quantitative analysis.
Fluorescence spectroscopy is a widely used biophysical tool for the study of the protein
structure based mainly on tryptophan fluorescence, which can only be excited by light of
specific wavelengths. The fluorophore that is able to emit fluorescence in the trypotophan
structure is the indole group. Generally, the fluorescence spectra recorded between 300 and
400 nm after excitation of the tryptophan fluorescence at 298 nm could be very useful for
understanding the effect of processing on protein structure. A shift in the maximum of tryp-
tophan fluorescence emission suggests an exposition of the tryptophanyl residues to a polar
environment. However, an increase in fluorescence intensity corresponds to an increase in
solvent polarity and reduced quenching of some tryptophanyl residues (Barbana et al ., 2006 ,
2008 ). Figure 3.7 shows the fluorescence spectra of

-lactalbumin before (holo) and after
(apo) the removal of Ca 2+ by chelating agent. The fluorescence intensity of the spectrum of

α