Circular Dichroism in Protein Analysis
57
1
Chirality and Polarized Light
At a molecular level, much of matter is
asymmetrical, like our left and right hands,
which are mirror images. In the world
of organic chemistry, many compounds
have chiral centers, typically carbon atoms
bonded to four different atoms or groups.
Chiral compounds have two forms (left-
and right-handed), which cannot be su-
perimposed on each other by rotation,
although they have the same chemical
composition. The asymmetric character
of chiral compounds induces different in-
teractions with electromagnetic radiation,
which gives rise to optical activity.
A beam of natural light is a set of
electromagnetic waves propagating in a
single direction, but with all possible
orientations perpendicular to the direction
of propagation. When it passes through a
linear polarizer, only a single orientation
is selected and we get linearly polarized
light. It may be divided into two chiral
components of equal intensity, left- and
right-circularly polarized light (Fig. 1). The
electric vector of each circularly polarized
component rotates along the direction of
propagation. The overall effect of these two
vectors is a planar sinusoidal wave.
Circular dichroism (CD) spectroscopy
is the differential absorption of left- and
right-circularly polarized light by a chi-
ral
medium.
When
linearly
polarized
light passes through achiral molecules,
the absorption of left- and right-circularly
polarized light is equal and no CD spec-
trum results. However, when the medium
comprises chiral molecules, such as pep-
tides, proteins, or DNA, there is differential
absorption. The resultant of these two com-
ponents is an ellipse with a speci±c optical
rotational angle (Fig. 1). CD spectroscopy
is based on the observation of optical
rotation or circular birefringence, which
can reflect detailed information about
the three-dimensional conformation of
biomolecules. The differential absorption
arises because the left- and right-handed
circularly polarized components experi-
ence different refractive indices on passing
through chiral molecules.
The absorbance of light by a sample is
given by the Beer–Lambert law:
A
=
log
µ
I
0
I
=
ε
cl
(
1
)
where
A
is the absorption,
c
is the concen-
tration in mol dm
3
,
I
0
is the intensity of
the incident light,
l
is the pathlength in cm,
and
ε
is the molar absorption coef±cient in
units of dm
3
mol
1
cm
1
. The differential
absorption is then
1
A
=
A
L
A
R
=
L
ε
R
)
cl
=
cl
(
2
)
where
is the difference in the molar
absorption coef±cients for left- and right-
circularly polarized components. This may
also be described, removing the linear
dependence on pathlength
and
solute
concentration, by
=
ε
L
ε
R
=
θ
cl
(
3
)
where
θ
is the ellipticity in degrees. The
ellipticity is equal to the ratio of the minor
radius to the major radius of the now
elliptically polarized light, that is
A
/
B
,as
shown in Fig. 1, and it is related to the
differential absorption by
θ
=
32
.
98
1
A
(
4
)
For a peptide or protein, the mean residue
ellipticity in units of degree cm
2
dmol
1
is
de±ned as
[
θ
]
=
100
θ
cl
=
3298
=
θ
×
0
.
1
×
MRW
cl
(
5
)
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