Practicalities of CD
Nicholas C. Price
IBLS
University of Glasgow
Main sections of talk
Preparation of protein solutions
Suitable solvents/buffers
Determination of protein concentration
Protein concentration and cell pathlength
Calibration of CD instrument
Cleaning and care of cuvettes
Spectropolarimeter settings
Units for CD data
Preparation of protein solutions
Dialysis of glycerol stock solutions
Dialysis from (NH4)2SO4 suspensions
Dissolution of a freeze-dried powder
Requirements of sample
• Proteins should be >95% purity on SDS-PAGE
and free from nucleic acids.
• Solutions should be clear with no aggregates or
precipitated protein. If necessary, use a 0.2 m
Millipore filter or centrifugation (bench top
centrifuge).
Requirements of sample (contd)
• What amount and/or concentration of protein is
required and/or available?
• How stable is the protein over the time period of
the experiment?
• Is the sample subject to photo-damage?
What solvents/buffers to use?
• Need sufficient buffering capacity (especially for
highly charged ligands, e.g. ATP4-)
• Need to ensure that buffer does not give
excessive absorbance
Choice of buffers
• Suitable buffers for far UV (240 nm to 180 nm)
include phosphate, borate and Tris (at low
molarity).
• Avoid high concentrations of chloride ions,
carboxylate ions and Good’s buffers such as
HEPES and MOPS.
• Control of pH in the 4-6 range is difficult (buffers
usually based on carboxylate ions).
Choice of buffers
Effect of carboxylate on CD quality
100mM Na Phosphate buffer pH 7.0
50mM Tris Acetate buffer pH 7.0
100mM Tris Acetate buffer pH7.0
Lysozyme 0.18mg/ml ; Scan speed 100nm/min ; Time constant 0.25s; No. of scans 8;
Cell pathlength 0.02cm
Effect of chloride ions on CD quality
100mM Na Phosphate buffer pH7.0
100mM Na Phosphate, 0.2M NaCl pH7.0
Lysozyme 0.18mg/ml ; Scan speed 100nm/min ; Time constant 0.25s; No. of scans 8;
Cell pathlength 0.02cm
Maintaining ionic strength
• If it is necessary to maintain the ionic strength for
protein stability, avoid adding high [NaCl]
(chloride ions absorb strongly at 195 nm)
• Fluoride or sulphate salts are much better for far
UV CD
Detergents and solvents
• Detergents – avoid Triton, use alkyl glucosides,
e.g. lauryl maltoside or octyl glucoside instead.
SDS does not give problems with absorption.
• Organic solvents – avoid chlorinated solvents.
Dioxane absorbs above 200 nm. Good solvents
(down to 190 nm) include alcohols (MeOH, EtOH)
and acetonitrile – HPLC grade.
• Always run a solvent/buffer blank to check for
high absorbance, or any chiral components.
Subtract this from the sample spectrum.
Determination of protein concentration
• Accurate determination of [protein] is important
for reliable analysis of secondary structure.
• Many methods are available, e.g. dye binding,
Lowry, BCA reagent etc.
Protein concentration determination
Determination of protein concentration
(cont’d)
• A convenient method is based on A280, using the
calculated values of extinction coefficients.
• A280 (1 mg/ml) = (nW (5690) + nY(1280) + nC(60))
mol. mass (Da)
• Equation is valid provided:  no contribution from light scattering
  no other chromophore (e.g. cofactor) in the
protein
  no other absorbing contaminant, e.g. nucleic
acids
Determination of protein concentration
(cont’d)
• Record absorption spectrum over the range 400240 nm. If there is no absorption above 310 nm,
scattering is not a problem.
• Check the A260 and A280 values. For proteins
A280/A260 should be about 1.6; for nucleic acids,
this ratio is about 0.62.
Absorbance spectrum of Lysozyme
Absorbance and DNA contamination
Absorbance spectrum of SNARE protein syntaxin 4 contaminated with DNA and after
treatment with DNase
0.3
Absorbance
1 (no DNase)
0.2
0.1
2 (+ DNase)
0.0
240
260
280
300
Wavelength (nm)
320
340
Determination of protein concentration
(cont’d)
• The calculated extinction coefficient refers to
exposed W and Y side chains in proteins, i.e.
denatured protein.
• Perform parallel dilutions (1/4) into buffer and 8 M
GdmCl and compare the values of A280. Typical
ratio is 0.9 - 1.1, use this to correct the calculated
A280 value.
Protein concentration and cell pathlength
• CD can be used to study samples over a very
wide range of concentrations (100-fold or greater
in the far UV).
• The aim is to keep the total absorbance below
about 0.7; vary the [protein] and/or cell
pathlength (d) to achieve this.
• Cells of pathlength 0.01 cm to 1 cm are routinely
used; shorter pathlengths can be achieved by
using demountable cells.
• For cells of 0.01 cm to 0.05 cm pathlength,
volumes of 0.3-0.4 ml are required; most of this
can be recovered.
Typical absorbance values for proteins
Wavelength (nm) Absorbance (1 cm cell) of
1mg/ml protein
190
65
200
45
205
32
210
21
215
15
220
11
225
8
260
0.6
280
1
310
<0.05
Absorbance and concentration
• In order to keep the total A190 below 1.0,
for c = 1 mg/ml, d should be about 0.01 cm.
for c = 10 g/ml, d should be about 1 cm
Otherwise the absorbance (and noise) would be too
high.
• In the near UV, the range of concentrations is
more limited, but we can still study
concentrations in the range 1 mg/ml (d = 1 cm) to
10 mg/ml (d = 0.1 cm).
Effect of protein concentration on CD
spectra
Far UV CD of Lysozyme at 0.5mg/ml (pink) 1mg/ml (blue) and 5mg/ml (red).
Effect of protein concentration on CD
spectra
Far UV CD of Lysozyme at 0.5mg/ml (pink) 1mg/ml (blue) and 5mg/ml (red).
Calibration of CD instrument
• CSA ((1S-(+)-10-camphorsulphonic acid) is the
calibrating substance most commonly used (ideal
for near and far UV).
• CSA (mol. mass 232.3) can be purchased from
Sigma-Aldrich; it is made up in distilled H2O at
0.06% (w/v) (0.6 mg/ml).
• Note CSA (free acid) is hygroscopic, so in many
cases, the NH4+ salt is preferred (mol. mass
249.3).
Properties of CSA solutions
• Extinction coefficient of CSA (285 nm) is 34.5 M -1
cm-1.
• A 0.6 mg/ml solution of the free acid should have
an A285 of 0.089 (1 cm pathlength cuvette).
• The ellipticity of 0.6 mg/ml CSA solution (free
acid) should be +202 mdeg at 290.5 nm, and -420
mdeg at 192.5 nm (1 cm pathlength cuvette).
• A 0.6 mg/ml solution of the ammonium salt of
CSA should have an A285 of 0.083, and ellipticities
of +188 mdeg (290.5 nm) and -391 mdeg (192.5
nm).
• Ellipticity (mdeg) at 290.5 nm = A285 x 2270
CD spectra of 0.06% Ammonium-dCamphor- Sulphonate
CSA 1cm pathlength
CSA 0.5cm pathlength
CSA 0.1cm pathlength
CSA 0.1cm pathlength cell. Ratio of 192.5 :
290.5 is approximately 2:1
Calibration of CD instrument
• CSA solutions can be kept at 4oC for 1 week.
• CSA solutions are useful for checking the
pathlengths of cuvettes.
• The spectropolarimeter should be calibrated at
least monthly: for strict quality control purposes
this should be done before each set of
experiments.
• The key to successful work is regular
maintenance and servicing of the instrument.
Purchasing CD cuvettes
• Cuvettes are expensive (cylindrical cuvettes are
typically about £150 to 250 each – they are
obtained from Hellma and can take 2 months to
be delivered.
• Each cuvette should be ordered as "strain free for
polarimetry", and will have its own description of
its optical properties.
Cleaning and care of CD cuvettes
• Cuvettes are fragile – avoid mechanical damage
and severe thermal shock when handling.
• Avoid scratching the surface of the cuvette when
using the microsyringes to fill and empty.
• Quartz cuvettes can be washed and dried
between runs (H2O, EtOH; vacuum pump).
• Sticky protein deposits can be removed by
treatment with conc. HNO 3 (CARE – check local
safety rules) followed by thorough washing out
before new data are acquired.
Spectropolarimeter settings
Various machine settings can be adjusted to
improve the results:• time constant
• scan rate
• bandwidth
• number of scans.
Spectropolarimeter settings (contd)
• The product of time constant and scan rate
should not exceed 0.5 nm. (Typical conditions 2
sec and 10 nm/min).
• Bandwidth should be less than or equal to 1 nm.
• Increased number of scans will improve S/N ratio
(proportional to square root of number of scans).
• Typically 3-4 scans can be accumulated; depends
on time available and stability of protein sample.
Effect of instrument parameters on quality
of CD spectra
Lysozyme 0.18mg/ml; Scan rate 100nm/min; Time constant 0.25sec;
No. of scans 10; cell pathlength 0.02cm.
Lysozyme 0.18mg/ml; Scan rate 100nm/min; Time constant 0.25sec;
No. of scans 1; cell pathlength 0.02cm.
Lysozyme 0.18mg/ml; Scan rate 100nm/min; Time constant 2.0sec;
No. of scans 1; cell pathlength 0.02cm.
Units for CD data
• CD data presented in terms of either ellipticity
(degrees) or absorbance.
• In order to normalise data, they are referred to
molar concentrations of chromophore or
repeating unit.
• For far UV CD, the repeating unit is the peptide
bond.
• The mean residue weight (MRW) is calculated
from the molecular mass/(N – 1); N = number of
amino acids.
• For most proteins, MRW is around 110.
Units for CD data (contd)
To calculate mean residue ellipticity ([]mrw )
[]mrw = (MRW x obs) deg. cm2.dmol-1
10 x d x c
where
d
c
obs = observed ellipticity in degrees
= pathlength in cm
= concentration in g/ml
Units for CD data (contd)
So if in a cuvette of pathlength 0.05 cm, the
observed ellipticity at 225 nm of a 0.1 mg/ml
solution of protein (MRW = 110) was 8 mdeg, then
[]mrw
= (110 x 8 x 10-3)
(10 x 0.05 x 0.1 x 10-3)
= 17600 deg cm2 dmol-1
Units for CD data (contd)
If we know the molar concentration (m) of a
solution:[]molar = (100 x obs) deg.cm2.dmol-1
(d x m)
where d is the pathlength in cm.
Units for CD data (contd)
For data in absorption units, it is usual to calculate
the molar extinction coefficient
= 
L– 
R
in units of M-1.cm-1
If a solution of molar concentration m in a cuvette of
pathlength d cm gives a difference in absorbance
(AL - AR) of Aobs, the value of is given by:= Aobs
M-1.cm-1
dxm
Units for CD data (contd)
• Note the relationship
[]mrw = 3298 
• There is some debate about the correct choice of
molar units for near UV and visible CD – does it
refer to the MRW or to the intact protein?
• Always check to see what the paper says.
References
• Gill, S.C. and von Hippel, P. (1989) Analyt.
Biochem. 182, 319-326.
• Johnson, W.C. jr. (1988) Ann. Rev. Biophys.
Biophys. Chem. 17, 145-166.
• Johnson, W.C. jr. (1990) Proteins: Str. Func. Gen.
7, 205-214.
• Kelly, S.M. and Price, N.C. (1997) Biochim.
Biophys. Acta 1338, 161-185.
• Kelly, S.M. and Price, N.C. (2000) Curr. Prot. Pep.
Sci. 1, 349-384.
• Kelly, S.M., Jess, T.J. and Price N.C. (2005)
Biochim. Biophys. Acta 1751, 119-139.
• Price, N.C. (1996) in Enzymology Labfax (Engel,
P.C. ed.) Bios Scientific Publishers, Oxford, pp.
34-41.