Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Absorption and CD Spectroscopy 47
3.3. Sample Preparation
1. Samples should, of course, be of he highest possible purity. Near-UV CD signals,
in particular, can be seriously distorted by the presence of relatively small
amounts of protein impurities (if they have intense signals) and by the presence
of nucleic acids, which have intense CD bands in this region.
2. Far-UV CD spectra of proteins (260–178 nm) are intense and small amounts of
material are required to record them. Because all peptide bonds contribute to the
spectrum the amount of material required is effectively the same for any protein.
Typical quantities are 200 µL of a 0.1–0.15 mg/mL solution with a 1-mm path
length cuvet or 30 µL of a 1.0–1.5 mg/mL solution with a 0.1 mm (demountable)
cuvet. The latter is preferable for good far-UV penetration (see Subheading 3.3
step 4) but the material is not generally recoverable.
3. Near-UV CD spectra (340–255 nm) are much less intense than far-UV spectra
and recording them requires more material. Spectra are usually recorded under
conditions similar to those used for measuring a conventional absorption spectrum,
e.g., use a 10-mm cuvet and aim for an absorbance at 280 nm in the range
0.7–1.0. Less-concentrated solutions may be used if the CD signals are intense.
4. CD signals will be seriously distorted if too little light reaches the photomultiplier.
In practical terms, this means that one cannot make reliable measurements
on samples with an absorbance (sample plus solvent) much greater than 1. The
absorption spectrum of the sample should always be checked to see if (and where,
see Subheading 3.4 step 3) this absorbance limit is exceeded. In far-UV measurements,
the absorbance of the protein itself is generally rather small and the
major problems arise from absorption by buffer components, almost all of which
will limit far-UV penetration to some extent (see Note 5).
3.4. Data Collection
1. Set the scan speed and time constant. The product of the time constant and the
scan speed should always be less than 0.5 nm. Higher values will give errors in
both band position and band intensity (see refs. 6,9–11 for further discussion of
errors in CD measurements). Typical parameters are a scan rate of 100 nm/min
and a time constant of 0.25 s. Collecting multiple scans will improve the signal to
noise (S/N) ratio to acceptable levels: the S/N ratio is proportional to the square
root of the number of scans and to the square root of the time constant.
2. Set the spectral bandwidth. Increasing the spectral bandwidth reduces noise by
increasing light throughput. The bandwidth should always be 2 nm or less to
avoid distorting the spectrum. It may be necessary to use lower values in order to
resolve fine structure in near-UV spectra.
3. Set the wavelength range. Far-UV spectra should generally be scanned from 260
to the lowest possible wavelength. This low-wavelength limit will depend largely
upon the buffer being used (see Subheading 3.3 step 4). Near-UV spectra are
routinely scanned over the range 340–255 nm.