saving in higher throughput situations since it does not involve
repeated phases of baseline re-equilibration. However, it is
essential that the binding kinetics (k obs ) is faster than the rate
of change in ligand concentration during the single injection
which limits the time economy of the method. In light of the
relatively low throughput of ITC in standard or SIM mode and
the modest gains in experimental time, it is perhaps not
surprising that SIM is not very commonly employed.
8. Buffers will have a characteristic heat (enthalpy) of ionization in
the equilibrium; BH $ B + H
+ , and any net proton flux
inherent to protein–ligand interactions will be provided or
absorbed in this associated buffering process [22]. The magnitude of the ionization enthalpy also determines the sensitivity
of the buffer system to changes in temperature; through mass
action effects, the ionization equilibrium is driven in the direction of endothermic heat absorption as temperature is
increased. Most buffers become more ionized and the pH
decreases, in the worst case for TRIS buffer, by ~0.3 pH units
for +10
C.
9. The ProtParam program uses protein sequence to sum the
molar extinction coefficients of aromatic amino acid content
along with minor contributions from cystine (cysteine residues
oxidized in disulfides). It thus reports values for the oxidized
and reduced forms of the protein. This approach is based on
two publications where extinction coefficients from solvent
exposed residues were used (denatured proteins or short
unstructured peptides) [23] or where average values were
obtained from a collection of folded proteins [24]. The values
are slightly different, reflecting the sensitivity of the aromatic
absorption to environment. ProtParam currently uses values
from the folded protein data set. However, it is clear that the
extinction coefficients obtained in this way are not without a
potential error of a few percent or so and other methods may be
considered depending on the sequence, oxidation state, etc.
(see Chapter 2).
10. Before measuring first check the quoted pathlength accuracy of
the cuvette or instrument device being used since this could be
another source of uncertainty in the absolute value measured.
The spectrometer should be blanked carefully using the exact
solvent that the protein is in. If the protein has been dialyzed
then the final dialysis buffer should be used. There can be
problems where DTT is present, often at mM levels, since its
oxidized state has absorbance with maxima 283 nm exactly
overlapping the aromatic absorbance region of proteins. This
can produce large errors where oxidation has proceeded at
differing rates in a protein sample compared to buffer stock
solutions as the buffer is no longer a suitable blank.
Isothermal Titration Calorimetry
155
repeated phases of baseline re-equilibration. However, it is
essential that the binding kinetics (k obs ) is faster than the rate
of change in ligand concentration during the single injection
which limits the time economy of the method. In light of the
relatively low throughput of ITC in standard or SIM mode and
the modest gains in experimental time, it is perhaps not
surprising that SIM is not very commonly employed.
8. Buffers will have a characteristic heat (enthalpy) of ionization in
the equilibrium; BH $ B + H
+ , and any net proton flux
inherent to protein–ligand interactions will be provided or
absorbed in this associated buffering process [22]. The magnitude of the ionization enthalpy also determines the sensitivity
of the buffer system to changes in temperature; through mass
action effects, the ionization equilibrium is driven in the direction of endothermic heat absorption as temperature is
increased. Most buffers become more ionized and the pH
decreases, in the worst case for TRIS buffer, by ~0.3 pH units
for +10
C.
9. The ProtParam program uses protein sequence to sum the
molar extinction coefficients of aromatic amino acid content
along with minor contributions from cystine (cysteine residues
oxidized in disulfides). It thus reports values for the oxidized
and reduced forms of the protein. This approach is based on
two publications where extinction coefficients from solvent
exposed residues were used (denatured proteins or short
unstructured peptides) [23] or where average values were
obtained from a collection of folded proteins [24]. The values
are slightly different, reflecting the sensitivity of the aromatic
absorption to environment. ProtParam currently uses values
from the folded protein data set. However, it is clear that the
extinction coefficients obtained in this way are not without a
potential error of a few percent or so and other methods may be
considered depending on the sequence, oxidation state, etc.
(see Chapter 2).
10. Before measuring first check the quoted pathlength accuracy of
the cuvette or instrument device being used since this could be
another source of uncertainty in the absolute value measured.
The spectrometer should be blanked carefully using the exact
solvent that the protein is in. If the protein has been dialyzed
then the final dialysis buffer should be used. There can be
problems where DTT is present, often at mM levels, since its
oxidized state has absorbance with maxima 283 nm exactly
overlapping the aromatic absorbance region of proteins. This
can produce large errors where oxidation has proceeded at
differing rates in a protein sample compared to buffer stock
solutions as the buffer is no longer a suitable blank.
Isothermal Titration Calorimetry
155