Beginner’s Guide

Beginners guide to
circular dichroism
Alison Rodger Circular dichroism (CD) is used to give information about the chirality or handedness of molecular
(Macquarie University, systems. It is particularly widely applied to determine the secondary structure of proteins such as
Australia) biopharmaceutical products.

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Doug Marshall (Applied
Photophysics, UK) Introduction Challenges to acquiring a good CD
spectrum
CD is used to give information about the chirality or
handedness of molecular systems. CD is the difference CD spectra are now no more difficult to obtain than
in absorption of left and right circularly polarized light: the corresponding absorbance spectrum if one has
‍ CD = Al − Ar ‍ (1) access to a CD spectropolarimeter. CD signals can only
CD and the related phenomenon of optical rotation of be measured where there is absorbance, though the
linearly polarized light were studied in the 19th century relative magnitudes of different parts of a CD spectrum
by scientists including Biot, Fresnel, Pasteur, and Cotton. differ from those of the sample’s absorbance spectrum,
However, because CD signals are very small differences and some CD bands are positive and some negative
between two larger numbers, with the difference typi- (Figure  1). This article outlines what a CD spectrum
cally being four orders of magnitude smaller than absor- actually is, how to avoid at least some of the artefacts
bances, it is really only in the last 50 years that electronics that result in quite a lot of published CD data being
and instrumentation have enabled CD studies of solu- nonsense and what information can be extracted from a
tions of molecules. CD is used to give information about CD spectrum. The focus is on electronic CD, particularly
the chirality or handedness of molecular systems. of proteins, though the principles largely translate to any
electronic or vibrational CD experiment.
Why CD is relevant As with all analytical tools, care should be taken
to ensure that the CD spectropolarimeter is calibrated
Applications for CD range from the search for extra-­ (intensity and wavelength – preferably not just a single-­
terrestrial life to 3D display technology, but with most point calibration) and well maintained and that users
emphasis on chiral molecules. The spectra of two have sufficient training to collect good quality data.
enantiomeric (mirror image) molecules are equal in Most instruments are supplied with instructions, and
magnitude and opposite in sign (Figure  1). Within the manufacturers readily support new users because their
life sciences, CD is most used for rapid comparison and reputations depend on high-­quality data from their
characterization of protein secondary structures. It offers systems being published. Camphor sulphonic acid is
a way to determine the effect a mutation or a change in the primary standard for CD and its ammonium salt
the environment of a protein (temperature, pH, ionic is a less hygroscopic alternative. More recently the
strength) might have on the overall structure, often prior CoEDDS of Figure  1, for which both enantiomers
to more resource-­intensive characterization. This can are available, has been used as it has the advantage of
avoid resources going to waste in expensive analyses of remaining stable in solution for years. Although one
protein samples that, e.g., did not retain the native fold. concentration (e.g., 4 mM) can be used across the
In addition, CD is used to study protein-­folding, stability visible and UV regions of the spectrum, better data are
and ligand binding and for characterization studies in obtained if a higher concentration is used in the visible
the development of biotherapeutics. CD is also used in region.
the study of nucleic acids and small biomolecules. Although equation (1) is the definition of CD, it
is not actually what most instruments measure and
plot. CD machines measure the ratio of an AC current
corresponding to the difference in photon intensities, I,
of left (l) and right (r) circularly polarized light reaching
the detector (i.e., the light that is not absorbed) and a

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Figure 1.  (a) Absorbance (4 and 80 mM in 1-­cm path length cuvettes) and (b) CD spectra (0.072 mM in a 1-­cm path length
cuvette) of RR- and SS-­CoEDDS (EDDS: ethylene diamine disuccinic acid). (c) Structures of RR- and SS-­EDDS and the CoEDDS
complex, SS-­mer is illustrated.

DC current corresponding to the average of the two for the CD of normal solutions of, e.g., biomolecules.
intensities. More care is, however, required if one is using a CD
2(I −I )
AC/DC = (Ill+Ir )r ‍ (2) spectropolarimeter for linear dichroism spectroscopy or
‍
to study samples such as cholesteric liquid crystals.
The most common operating mode for CD machines is
When the HT voltage is above about 600 V (i.e., a
for a variable high-­tension (HT) voltage to be applied
highly absorbing sample), too few photons are reaching
to the photomultiplier so that the DC signal is kept
the detector for the DC to be effectively held constant.
constant. The software of the instrument then plots the
This issue becomes more and more significant below 200
approximation
( ) nm in most bench-­top CD machines because the photon
2+AC/DC AC
CD = log10 2−AC/DC ≈ DC log10 e (3) flux of the Xe arc lamp used as the light source only has 1%
‍ ‍
Consideration of these equations leads to some experi- of its 500-­nm flux. In older instruments, this is obvious
mental precautions needed to ensure real data, not arte- because of high levels of noise. In newer instruments,
facts, are being measured. Instruments sometimes but attempts to enhance the signal:noise sometimes obscure
not always provide an error flag for these issues. this problem. Any stray light, including from holes in the
(i) Equation (3) is an approximation, that requires sample compartment, of whatever frequency will simply
AC<<DC. be counted by the photomultiplier tube, thus artificially
(ii) The DC current must be accurately held constant depressing the HT. If an instrument has the option of
(assuming one is working in a fixed-­DC mode). setting the CD scale, it may be advisable to use a scale
In practice, as long as the CD signal is less than 20% of orders of magnitude larger than the CD signal – at least
the absorbance, the equation (3) approximation error is to check that the CD signal is not being damped by the
less than 1%. The approximation is therefore satisfactory electronics of the instrument.

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Beginner’s Guide
In this context it should be noted that achiral a number of models for calculating the CD spectrum
molecules absorb light – so, e.g., Cl– (including that in may be appropriate and the handedness of the molecule
phosphate-­buffered saline) and other buffer components may be able to be determined quite simply. For example,
reduce the photons available for the analytes of interest the coupling of electric dipole transition moments
to absorb. Proteins often need to have relatively high in the aromatic phenanthroline ligands of [Ru(1,10-­
concentrations of buffers or ions present to maintain phenanthroline)3]2+ gives rise to the sharp in-­ ligand
their stability. If the protein structure is concentration bands of Figure  2. The fact that the zero CD point
independent, an alternative to reducing buffer corresponds with the absorbance maximum confirms
concentrations is to increase the protein concentration that the CD is dominated by the coupled oscillator model.
and reduce the path length to ensure the absorbance The d-­d bands arise from a very different mechanism: the
does not exceed 2. For path lengths less than 1 mm it perturbation of magnetic dipole allowed d-­d transitions
is important to measure it for each cell and each user by in-­ligand transitions arranged in a helical geometry
as path lengths of demountable cells are affected by the about the metal centre.

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volume of sample used and how the cell is assembled. For biomacromolecules, such as proteins and DNAs,
Further artefacts can occur in any form of we usually know the chirality of the component amino
spectroscopy, including CD, due to heterogeneity acids or nucleosides and we are more interested in what
of the sample. Consider an inhomogeneous sample the CD spectrum tells us about how they are arranged
where all the absorbing molecules are put in the left-­ into secondary or even tertiary structures (Figure  3).
hand half of the cuvette with an absorbance of, say, The use one makes of a CD spectrum depends on what
( ) ( )
1 (‍A=log I0 /I = log 10/1 ‍). The 50% of photons question is being asked: is an absolute measure of helix
passing through the right-­hand side of the cuvette are and sheet content of a protein required? Or does one
all transmitted whereas those on the left are attenuated simply want to know whether a peptide folds in a lipidic
making the total transmission environment or a nucleic acid or protein unfolds when
I +I
0 heated? The latter questions can often be answered by
‍ 2I0 ‍ (4)
visual inspection of spectra (Figure  3). However, one
whose absorbance is
( ) ( ) does need to take care as to what information is being
A=log I2I 0
+I = log 20
11 = 0.26‍ (5) displayed. In Figure  3c, fluorescence tells us that the
‍ 0

whereas the absorbance of the sample after it has been tryptophans in α-lactalbumin unfold in two phases,
thoroughly mixed is expected to be 0.5. This absorbance the first being slow and the second from 55oC to 65°C.
flattening also affects CD and can have a significant effect In light of the fluorescence data, the aromatic CD
on, e.g., protein samples in membranes. (which is relatively noisy) is probably telling the same
A final artefact that must be considered is that any story, though it could be one broad transition. By way
light scattered by a sample is deemed by the spectrometer of contrast, the backbone protein spectrum measured
to have been absorbed – the instrument simply counts at 222 nm unfolds in a single concerted step from 55oC
the photons that are incident on the photomultiplier to 65°C, at the same time as the environments of some
tube and ‘assumes’ all others have been absorbed. of the tryptophans is exposed to water as shown by the
The above issues all mean that significant care must fluorescence.
be taken when using a bench-­top CD machine in the UV By far the most widespread use of CD spectroscopy
region of the spectrum – precisely where the CD of most is to identify protein secondary structure content.
biomolecules has useful data. The success of CD structure-­ fitting is predicated
on the observed spectrum being the sum of the
contributions from different motifs within the protein.
Interpreting a circular dichroism To get quantitative answers significantly better than
spectrum an experienced spectroscopist can provide just by eye,
it is essential to have a good reference set of spectra
Assuming that the above pitfalls have been avoided from proteins of known structure, preferably including
and one has measured a good CD spectrum, we ask proteins in some way related to the unknown one being
what can be deduced from it. A non-­zero spectrum studied. Various reference sets and algorithms have been
tells you that the system being studied is chiral, with collated on the Protein Circular Dichroism Data Bank
helical rearrangements of electrons during electronic and the Dichroweb site.
transitions. For small molecules, fairly high-­ quality It has long been accepted that the further down in
CD spectra can be calculated for known (or proposed) wavelength one measures, the more accurately more
geometries and compared with experiment. Alternatively, types of backbone structures can be extracted from CD
if a molecule can be divided into achiral chromophores spectra. However, some recent Bayesian analysis has
whose transition polarizations are known, then one of reinforced the value of collecting data to at least 190

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Figure 2.  (a) Molar absorbance and (b) CD spectrum of Λ-[Ru(1,10-­phenanthroline)3]2+ in a 1-­cm path length cuvette and (c)
schematic of the electric dipole transition moments that couple to give the dominant in-­ligand CD signal.

nm but made it clear that only three types of structure units include α-chymotrypsin and chymotrypsinogen,
(a collation of helical structures, a collation of sheet both of which Sreerama and Woody categorize as βII
structures and ‘other’) can be extracted from a normal proteins. The CD significance of this is that although
reference set even if it has data down to 175 nm. In the best matching proteins have βII structure, the
addition, the similarity of unfolded, polyproline II and spectrum of βIIs look like random coil spectra so the
βII spectra (Figure 3a) means that care must be taken 34% β-sheet assignment at 100oC is misleading and
to investigate the reference spectra selected by any should be 34% unfolded. This shuffling of assigned
algorithm for its final fit. content between β-sheet and random coil always needs
Our own self-­ organizing map structure-­ fitting to be considered.
approach (SOMSpec) has the advantage of enabling
the user to interrogate the output to identify which
reference proteins have been used to estimate the Some recent applications of CD
secondary structure of the unknown. This helps an spectroscopy
analysis of the quality of the secondary structure
estimate to be made and makes it possible to look for As a general rule CD is applied as one of a number
the source of any anomalies. For example, we know that of techniques to characterize complex molecules or
lysozyme is significantly unfolded at 100°C (Figure 4a) molecular assemblies with chiral components. This is
but a straightforward fit with the SOMSpec algorithms illustrated with three examples.
(and other) suggests it increases its β-sheet content
from 16% at room temperature to 34% at 100°C (with Biosimilars
no evidence of, e.g., amyloid fibre formation). The Biosimilar products are usually a different company’s
reason proves to be that the selected best matching attempts to produce a biopharmaceutical that is ‘highly

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Figure 3.  (a) Far UV (below 250 nm) CD spectra for a selection of proteins of different secondary structure content. (b)
CD spectra for some DNAs with differing GC content. The Z-­DNA was formed by adding spermine. (c) Melting curves
of α-lactalbumin measured by CD (at two wavelengths) and fluorescence (presented as the ratio of 350 nm to 330 nm
intensities).

similar’ to another already approved biological medicine atomic level, especially with glycosylation patterns, may
whose patent has expired. Unlike small-­molecule generic be allowed. Comparisons of far UV CD (from 250 nm
drugs, biosimilars require not only the primary structure down to at least 190 nm) is generally deemed to be a good
(what is bonded to what), but also secondary (local indicator of whether the secondary structure content is
folds), tertiary and even quaternary structure similarity. changing. For many proteins, the near UV region (from
Also, unlike small molecules, some differences at the 300 nm to 250 nm) can be used to give an indication of

Figure 4.  (a) CD spectrum of lysozyme as a function of temperature. (b) CD spectrum of the 96-­residue protein G (PG) and of
PG with the addition of 1, 2, 3 or 4 copies of a 21-­residue linker peptide.

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Figure 5.  Baseline-­corrected and zeroed CD spectra of three batches of a biopharmaceutical protein product in the far UV (a)
and near UV (b).

tertiary structure – namely how the secondary structure of βII and unfolded structures. Close inspection of the
units assemble. The near UV region is often referred to data suggests there is a small difference in the secondary
as the aromatic region – though it includes disulphide-­ structure of three batches of the product. The aromatic
bond contributions and seldom shows any contribution CD suggests the tertiary structures of the different
from phenylalanines. So, the near UV is dominated batches also differ.
by tryptophan signals and shows contributions from
tyrosines. Identification of unfolded domains in proteins
Lysozyme CD as a function of temperature (Figure 4a)
An example of batch-­to-­batch CD comparison of a
illustrates a relatively sharp transition from the fully
biopharmaceutical product is given in Figure  5. Visual
folded protein (with 39% α-helix and 16% β-sheet)
inspection of the spectra suggests it is largely unfolded
to a largely unfolded protein (11% α-helix and 11%
(negative maximum at 200 nm), though CD structure-­
β-sheet). Similar profile differences are observed for
fitting suggests it is β-sheet. The resolution of this, as the CD spectra of a series of proteins composed of
discussed above, is that it is probably a combination a truncated form of a Streptococcus protein G linked
to increasing numbers of a silica-­ binding peptide
(Figure  4b). A more quantitative analysis indicates
that the added peptides are unfolded as are a few
N-­terminus residues of protein G.

Neutrophil granule myeloperoxidase

Myeloperoxidase (MPO) is an important glycoprotein
which plays a role in neutrophil-­mediated immunity.
The CD of neutrophil-­derived MPO (nMPO) before and
after treatment with Endoglycosidase H (EH), as judged
by comparing the nMPO spectrum and the difference
between EH-­ treated nMPO and EH, suggests there
may be a small difference between the two proteins,
but Figure  6 is not entirely convincing. However, the
simple experiment of measuring CD as a function of
temperature shows that the treated nMPO is much more
stable (beginning to melt at 72°C) than untreated nMPO
(beginning to melt at 60°C). Melting experiments can be
Figure 6.  CD of nMPO and EH-­treated nMPO, and done by measuring full wavelength scans at temperature
the theoretical spectrum of EH-­treated nMPO with EH intervals or by selecting a key wavelength (such as 208
subtracted. It should be noted that all spectra were baseline nm for α-helical proteins) to measure the signal as the
corrected by subtracting a buffer spectrum before EH was temperature is changed. Some CD instruments have an
subtracted from EH-­treated nMPO. option to measure both in one experiment. ■
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Beginner’s Guide
Further reading

• Bansal, R., Elgundi, Z., Goodchild, S.C., et al. (2020) The effect of oligomerization on a solid-­binding peptide binding to
silica-­based materials. Nanomaterials, 10, 1–15. DOI: 10.3390/nano10061070
• Berova, N., Nakanishi, K. and Woody, R.W. (eds.) (2000) Circular dichroism principles and applications. Wiley-­VCH, New York
• Berova, N., Polavarapu, P.L., Nakanishi, K. and R.W. Woody (eds.) (2014) Comprehensive Chiroptical Spectroscopy Wiley-­
VCH, New York
• Coggan, D.Z., Haworth, I.S., Bates, P.J., et al. (1999) DNA binding of ruthenium tris-(1,10-­phenanthroline): evidence
for the dependence of binding mode on metal complex concentration. Inorg. Chem., 38, 4486−97. DOI: 10.1021/
ic990654c
• Goodchild, S.C., Jayasundera, K. and Rodger, A. (2019) ‘Circular dichroism and related spectroscopic techniques.’ In
Biomolecular and bioanalytical techniques: theory, methodology and applications, Ramesh, V. (ed.) John Wiley & Sons,

Downloaded from http://portlandpress.com/biochemist/article-pdf/43/2/58/908045/bio_2020_105.pdf by UK user on 07 May 2021

Hoboken
• Hall, V., Sklepari, M. and Rodger, A. (2014) Protein secondary structure prediction from circular dichroism spectra using
a self-­organizing map with concentration correction. Chirality, 26, 471–82. DOI: 10.1002/chir.22338
• Hall, V., Nash, A. and Rodger, A. (2014) SSNN, a method for neural network protein secondary structure fitting using
circular dichroism data. Anal. Methods, 6, 6721–6726. DOI: 10.1039/C3AY41831F
• Hiort, C., Nordén, B. and Rodger, A. (1990) Enantioselective DNA binding of [RuII(1,10-­phenanthroline)3]2+ studied with
linear dichroism. J. Am. Chem. Soc., 112, 1971–1982. DOI: 10.1021/ja00161a050
• Nordén, B., Rodger, A. and Dafforn, T.R.. (2010) Linear dichroism and circular dichroism: a textbook on polarized
spectroscopy. Royal Society of Chemistry, Cambridge.
• Ravi, J., Schiffmann, D., Tantra, R. et al. (2010) International comparability in spectroscopic measurements of protein
structure by circular dichroism: CCQM-­P59. Metrologia, 47, 08022. DOI: 10.1088/0026-1394/47/1A/08022
• Schipper, P.E., and Rodger, A. (1986) Generalized selection rules for circular dichroism: A symmetry
adapted perturbation model for magnetic dipole allowed transitions. Chem. Phys. 109, 173–193. DOI:
10.1016/0301-0104(86)87050-1
• Spencer, S.E.F., and Rodger, A. (2020) Bayesian inference assessment of protein secondary structure analysis using
circular dichroism data – how much structural information is contained in protein circular dichroism spectra? Anal.
Methods. 13, 359–368. DOI: 10.1039/d0ay01645d
• Sreerama, N., and Woody, R.W. (2003) Structural composition of βI- and βII-­proteins. Protein Sci. 12, 384–388. DOI:
10.1110/ps.0235003
• Sutherland, J.C. (2002) Simultaneous measurement of circular dichroism and fluorescence polarization anisotropy.
Proc. SPIE 4625, Clinical Diagnostic Systems: Technologies and Instrumentation. 126, 126–136. DOI: 10.1117/12.469782
• Tjondro, H.C., Ugonotti, J., Kawahara, R. et al. (2020) Hyper-­truncated Asn355- and Asn391-­glycans modulate the
activity of neutrophil granule myeloperoxidase. J. Biol. Chem. DOI: 10.1074/jbc.RA120.016342
• Whitmore, L., and Wallace, B.A. (2004) DICHROWEB: an online server for protein secondary structure analyses from
circular dichroism specroscopic data. Nucleic Acids Res., 32, W668–673. DOI: 10.1093/nar/gkh371
• Whitmore, L., Woollett, B., Miles, A.J. et al. (2011) PCDDB: the protein circular dichroism data bank, a repository for
circular dichroism spectral and metadata. Nucleic Acids Res. 39, D480–D486. DOI: 10.1093/nar/gkq1026

Alison Rodger began her career at the University of Sydney and after nearly 30 years in the UK at the
Universities of Cambridge, Oxford and Warwick moved to Macquarie University in 2017. Her research
focuses on understanding the structure and function of biomacromolecules and their assemblies and often
requires new techniques. Email: [email protected]

Doug Marshall began his research career as a postdoc at UCL in 2007 before moving on to University of
Pennsylvania and University of Essex. He specialized in understanding the structure and function of
respiratory chain proteins. Doug now works as product manager for CD technology at Applied Photophysics.

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