Many of the complex phenomena associated with food processing, including cooking, involve the interactions of biopolymers. These interactions are frequently triggered by changes in the three-dimensional conformation of the biopolymers, which may be caused by changes in temperature, pH, or ionic strength, or by modifications made by hydrolytic enzymes. The understanding of such processes, necessary for their control, requires knowledge of the dependence of the conformational changes and interactions on environmental conditions. Circular dichroism (CD) can provide a sensitive indicator of these changes at the molecular level (Li-Chan, 1998), provided the sample is in the form of an optically clear solution and, for most applications, contains purified components. This unit reflects the fact that CD has been used mainly to study proteins, but it can, in principle, be used for any molecules containing a chromophore that absorbs radiation in an accessible region of the spectrum, such as nucleic acids (Gray et al., 2002), carbohydrates (Johnson, 1987), or porphyrins (Huang et al., 2000).
There are two requirements for a molecule or group of atoms in a molecule to exhibit a circular dichroism (CD) spectrum. The first is the presence of a chromophore—i.e., a group that can absorb radiation by virtue of the electronic configuration of its resting or ground state at room temperature. The energy absorbed results in a transition to a higher-energy or excited state, which has a different distribution of electrons around the nucleus. It can therefore interact with its environment in a way that differs from the ground state. In proteins, tryptophan, tyrosine, and phenylalanine are the main chromophores in the near-UV (240- to 320-nm) region; the peptide bond is the main chromophore in the far-UV (180- to 240-nm) region. Disulfide bonds and histidine residues are two other chromophores whose contribution to CD are, in general, less marked. Most chromophores exhibit more than one transition (e.g., see Fig. B3.5.1, Fig. B3.5.2, and Table B3.5.2) and their spectra are a composite of several absorption bands. It is an important feature of CD that, unlike optical rotation, the wavelength region within which spectra are observed is limited strictly to the wavelength region of the individual absorption bands. This confers greater specificity on spectra and allows a certain degree of assignment to particular chromophores.
The second requirement for CD is that the chromophore be in, or be closely associated with, an optically asymmetric environment. The chromophores in proteins are themselves not chiral and exhibit no optical activity. The phenolic group of tyrosine, for example, exhibits a CD spectrum only because it is connected to an optically asymmetric carbon atom. However, when the same group is packed in a folded protein in an environment that is asymmetric with respect to polarity, or is interacting through the phenolic hydroxyl, it exhibits a different CD spectrum that is specific to the particular environmental influences acting on the chromophore. In practice, the latter spectrum is usually much more intense than that for free tyrosine and may also differ in shape. When the peptide-bond chromophore is part of a regularly folded structure, with particular backbone angles and hydrogen-bond interactions as in an a-helix or P-sheet, it becomes closely associated with a conformationally asymmetric structure—i.e., one that cannot be superimposed on its mirror image. A CD spectrum for the protein will be generated that is a composite of the individual spectra corresponding to each of the peptide-bond absorption transitions (Fig. B3.5.1). The intensity of the spectrum may be further affected by interaction between neighboring chromophores (Bayley, 1980).
Plane-polarized radiation comprises two circularly polarized vectors of equal intensity, one right-handed and the other left-handed (Fig. B3.5.3A), which are separately measured in the CD spectrometer by means of a photoelastic modulator. A chromophore situated
Characterization of Proteins
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