Samples for measurement should have an absorbance
between 0.1 and 1.5 at the measurement pathlength (optimally
absorbance 0.87) or should be diluted. Pipettes used for any
dilutions should be reliable and properly calibrated. Alternatively, the dilution can be done with a balance assuming 1 g/
mL density. It is also important to measure the full spectrum of
the protein from 240 nm to at least 360 nm or longer wavelengths. Examining the spectrum gives important additional
information about the sample. A 280/260 nm ratio >1.6 is
characteristic of pure proteins while lower values can indicate
nucleic acid or nucleotide contributions as contaminants or
bound ligands. At longer wavelengths >320 nm, the protein
should not absorb, unless it contains an additional prosthetic
group. Thus, absorbance above zero in this range is indicating
scattering from aggregated material or other particles in the
sample. Scattering has a reciprocal dependence on wavelength
raised to power between 2 and 4 and so at lower wavelengths,
such as where the protein is being quantified, the apparent
absorbance will be even higher. If scattering absorbance is
observed >320 nm, it can be corrected for at 280 nm by
plotting the log of absorbance observed above 320 nm and
fitting this to a linear function. The extrapolated log of absorbance at 280 nm should be converted to absorbance and subtracted from the measured value. If this correction indicates
more than a few percent of measured absorbance, then it could
be a cause of some concern indicating that the sample contains
significant levels of aggregated and presumably inactive
materials.
11. The SI unit of heat is the Joule. Unsurprisingly, in the area of
calorimetry, there is some attachment to the older unit of
calories as has been used here. NB: 1 cal ¼ 4.184 J.
12. The background control heat associated with the ITC experiment can be determined in a separate experiment injecting
ligand into buffer. Solutions used should be identical to those
of the binding experiment. The diluted ligand can be recovered
from the ITC at the end of the experiment and recycled. The
background end point heat has contributions from the
mechanical and instrumental heat associated with an injection
(seen when injecting water into water) and from the heat of
dilution of the ligand associated with its ~100-fold drop in
concentration and any subtle solvent differences between the
protein and ligand solutions. Solvent effects can be minimized
by dialyzing extensively both components against buffer. Heats
of dilution will vary from ligand to ligand because they are
inherent to its solvation and chemistry. In addition, each injection of a particular ligand increases its concentration in the cell
so that the effective dilution factor decreases during the
156
Christopher M. Johnson
between 0.1 and 1.5 at the measurement pathlength (optimally
absorbance 0.87) or should be diluted. Pipettes used for any
dilutions should be reliable and properly calibrated. Alternatively, the dilution can be done with a balance assuming 1 g/
mL density. It is also important to measure the full spectrum of
the protein from 240 nm to at least 360 nm or longer wavelengths. Examining the spectrum gives important additional
information about the sample. A 280/260 nm ratio >1.6 is
characteristic of pure proteins while lower values can indicate
nucleic acid or nucleotide contributions as contaminants or
bound ligands. At longer wavelengths >320 nm, the protein
should not absorb, unless it contains an additional prosthetic
group. Thus, absorbance above zero in this range is indicating
scattering from aggregated material or other particles in the
sample. Scattering has a reciprocal dependence on wavelength
raised to power between 2 and 4 and so at lower wavelengths,
such as where the protein is being quantified, the apparent
absorbance will be even higher. If scattering absorbance is
observed >320 nm, it can be corrected for at 280 nm by
plotting the log of absorbance observed above 320 nm and
fitting this to a linear function. The extrapolated log of absorbance at 280 nm should be converted to absorbance and subtracted from the measured value. If this correction indicates
more than a few percent of measured absorbance, then it could
be a cause of some concern indicating that the sample contains
significant levels of aggregated and presumably inactive
materials.
11. The SI unit of heat is the Joule. Unsurprisingly, in the area of
calorimetry, there is some attachment to the older unit of
calories as has been used here. NB: 1 cal ¼ 4.184 J.
12. The background control heat associated with the ITC experiment can be determined in a separate experiment injecting
ligand into buffer. Solutions used should be identical to those
of the binding experiment. The diluted ligand can be recovered
from the ITC at the end of the experiment and recycled. The
background end point heat has contributions from the
mechanical and instrumental heat associated with an injection
(seen when injecting water into water) and from the heat of
dilution of the ligand associated with its ~100-fold drop in
concentration and any subtle solvent differences between the
protein and ligand solutions. Solvent effects can be minimized
by dialyzing extensively both components against buffer. Heats
of dilution will vary from ligand to ligand because they are
inherent to its solvation and chemistry. In addition, each injection of a particular ligand increases its concentration in the cell
so that the effective dilution factor decreases during the
156
Christopher M. Johnson