Circular dichroism (CD) spectroscopy is a form of
light absorption spectroscopy that measures the
difference in absorbance of right- and left-circularly
polarized light (rather than the commonly used
absorbance of isotropic light) by a substance.
Jasco J-810 Circular Dichroism System
Important Basic concepts
• Definitions of asymmetry
• What is optical Activity
• What is polarized light
Circular Dichroism (CD) phenomena that result when
asymmetrical molecules interact with plane polarized light.
• The function of purging the CD instrument with
nitrogen is to remove oxygen from the lamp
housing, monochromator, and the sample
• The presence of oxygen is detrimental for two
reasons. When deep ultraviolet light strikes
oxygen, ozone is produced. Ozone causes
degradation of optics and can cause respiratory
problems. The second reason for removing
oxygen is that oxygen absorbs deep UV light, thus
reducing the light available for the measurement.
Sample preparation and measurement
• Additives, buffers and stabilizing compounds: Any
compound which absorbs in the region of interest
(250 –190 nm) should be avoided (see below).
• Protein solution: The protein solution should
those chemicals necessary to maintain protein
at the lowest concentrations possible. Any additional
protein or peptide will contribute to the CD signal.
• Data collection: Initial experiments are useful to
establish the best conditions for the "real"
experiment. Cells of 0.5 mm path length offer a good
Protein Concentration: 0.5 mg/ml
Cell Path Length: 0.5 mm
Stabilizers (Metal ions, etc.): minimum
Buffer Concentration : 5 mM or as low as
possible while maintaining protein stability
Cutoff Wavelengths For Common Solvents and
• The molar ellipticity [ ] is related to the difference
in extinction coefficients
[ ] = 3298 Δε.
• Here [ has the standard units of degrees cm2
• The molar ellipticity has the units degrees
deciliters mol-1 decimeter-1.
CD spectra of calf thymus DNA titrated with BPSQ; at (a) r 0 ¼ 0; (b) 0.08;
(c) 0.16; (d) 0.24; (e) 0.40; and (g) 0.72.
Applications of CD spectroscopy
A. Estimation of protein and nucleic acid
B. Determination of conformational changes
due to the interactions of asymmetric
1. Protein-protein interactions
2. Protein-DNA interactions
3. Protein-Ligand interactions
4. DNA-Ligand interactions
C. Determination of the thermodynamics of
folding and unfolding of proteins and nucleic
D. Determination of binding constants by:
1. direct titrations
2. serial dilutions of complexes
3. changes in stability to thermal or chemical
E. Kinetics of
• It has been shown that CD spectra between 260
and approximately 180 nm can be analyzed for
the different secondary structural types: alpha
helix, parallel and anti-parallel beta sheets, turns,
• A number of excellent review articles are
available describing the technique and its
application (Woody, 1985 and Johnson, 1990).
• Modern secondary structure determination by
CD are reported to achieve accuracies of 0.97
for helices, 0.75 for beta sheet, 0.50 for turns,
and 0.89 for other structure types (Manavalan &
• For proteins we will be mainly concerned with
absorption in the ultraviolet region of the spectrum
from the peptide bonds (symmetric chromophores)
and amino acid sidechains in proteins.
• Protein chromophores can be divided into three
classes: the peptide bond, the amino acid
sidechains, and any prosthetic groups.
• The lowest energy transition in the peptide
chromophore is an n → * transition observed at
210 - 220 nm with very weak intensity ( max~100).
• In a first approximation, a CD spectrum of a protein
or polypeptide can be treated as a sum of three
components: -helical, -sheet, and random coil
contributions to the spectrum.
• At each wavelength, the ellipticity (θ) of the
spectrum will contain a linear combination of these
• θT is the total measured ellipticity, θh the
contribution from helix, θs for sheet, θc for coil, and
the corresponding χ the fraction of this contribution.
• As we have three unknowns in this equation, a
measurement at 3 points (different wavelengths)
would suffice to solve the problem for χ, the
fraction of each contribution to the total measured
• We usually have many more data points available
from our measurement (e.g., a whole CD
spectrum, sampled at 1 nm intervals from 190 to
250 nm). In this case, we can try to minimize the
total deviation between all data points and
calculated model values. This is done by a
minimization of the sum of residuals squared
(s.r.s.), which looks as follows :
• CD bands in the near UV region (260 – 350 nm)
are observed in a folded protein where aromatic
side chains are immobilized in an asymmetric
• The CD of aromatic residues is very small in the
absence of ordered structure (e.g. short
• The signs, magnitudes, and wavelengths of
aromatic CD bands cannot be calculated; they
depend on the immediate structural and
electronic environment of the immobilized
• The near-UV CD spectrum has very high
sensitivity for the native state of a protein. It can
CD spectra before and after mixing SCM and Mj HSP16.5.
• Circular dichroism spectroscopy is used to gain
information about the secondary structure and
folded state of proteins and polypeptides in solution.
• Uses very little sample (200 ul of 0.5 mg/ml solution
in standard cells)
• Relative changes due to influence of environment
on sample (pH, denaturants, temperature, etc.) can
be monitored accurately.
• Interference with solvent absorption in the UV
• Only very dilute, non-absorbing buffers allow
measurements below 200 nm
• Absolute measurements subject to a number of
• Average accuracy of fits about +/- 10%
• CD spectropolarimeter is relatively expensive