Circular Dichroism in Protein Analysis
59
to the oscillator strength in electronic
absorption spectroscopy. It is related to
the product of the electric and mag-
netic transition moments and corresponds
to
the
area
under
the
band
in
the
CD spectrum.
Electronic CD spectra may be divided
into the far UV region (180–250 nm),
the near UV region (250–300 nm), and
the UV–vis region (300–700 nm). Absorp-
tion in the far UV region is dominated
by the excitation of electrons on amide
chromophores. It furnishes information
on secondary structure (Fig. 2). In the
near UV region, the CD arises from
side-chain chromophores, such as aro-
matic groups and disul±de bonds. In
this region, CD spectra can provide in-
formation on protein tertiary structure.
The UV–vis region reflects the confor-
mation and environment of the protein.
Transitionsinthisregionarisefromchro-
mophoric prosthetic groups, such as the
heme group.
2.1
Far Ultraviolet
Peptide bonds are the key chromophores
in proteins. To appreciate the origin of the
CD of proteins, one needs to understand
the electronic structure of the peptide
bond. The valence shell of the peptide
bond comprises four
π
electrons in three
π
orbitals (
π
+
0
, and the unoccupied
π
) and two electron pairs in two lone-
pair orbitals (n and n
0
). The n
π
and
π
0
π
electronic transitions of the peptide
chromophore occur in the far UV region.
The lowest energy transition is the n
π
transition, which is polarized along the
carbonyl bond. It is an excitation of an
electron from a lone pair on the oxygen
atom to an antibonding
π
orbital and
occurs at 210 to 230 nm. It is magnetically
allowed and electrically forbidden (Fig. 3).
The
transition
energy
is
sensitive
to
the extent of hydrogen-bonding to the
oxygen atom. In the lower symmetry of
40000
20000
20000
190
200
210
Wavelength
[nm]
Mean residue ellipticity
[deg cm
2
dmol
1
]
220
230
240
180
0
60000
Myoglobin
Concanavalin A
Elastase
Fig. 2
The standard CD spectra for
α
-helix,
β
-sheet, and random coil, as
typiFed by the proteins myoglobin, concanavalin A, and elastase
respectively; the spectra are plotted from the freely available
experimental data.
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