have mixed with the cell component by diffusion during the instrument’s equilibration phases after loading, but before measurement.
The data from this injection are not normally included during
analysis (see Note 7).
The injections are spaced at intervals of 2–4 min, depending on
the instrument, its settings, and the duration of the heat effect that
is determined by the kinetics of binding (see Eq. 2). Initially the
interval is better left longer so all the heat can be captured by the
instrument and a stable baseline reestablished before any
subsequent injections. Once initial binding is characterized, this
interval and the number of injections may be reduced if
appropriate.
2.2 A Realistic Test
Reaction: Lysozyme
Binding a Simple
Trisaccharide Ligand
1. Binding of tri-acetyl glucosamine (N,N
0 ,N
00 -Triacetylchitotriose)
to hen egg-white lysozyme is a useful test reaction to gain experience in performing ITC measurements. Both components are
available as lyophilized materials of high purity and at modest
cost. The binding K d is single μM level and the interaction has
been quantified by ITC and by a variety of orthogonal binding
assays with comparable results (see Chapter 2).
2. Both should be carefully weighed using an analytical balance
and dissolved in an experimental buffer at pH 5 or lower since
this avoids the possibility of lysozyme forming higher order
species such as dimers [1]. 0.1 M acetate buffer pH 5, is a
suitable choice as it has been widely used in the literature for
this interaction. It also has an advantage of a near zero enthalpy
of ionization and will thus maintain its pH at different temperatures if required (see Note 8). The buffer can be prepared
using an online recipe calculator (https://www.liverpool.ac.
uk/pfg/Research/Tools/BuffferCalc/Buffer.html) and the
appropriate molarities of basic and acidic components can simply be mixed in the calculated quantities (using weight or
volume). The pH should be 5 if checked with a calibrated pH
meter. The mixing approach is highly reproducible and avoids
the use of a pH meter that is normally a communal resource in a
lab and requires careful maintenance and calibration for reliable
performance.
3. Measuring the concentration of the materials used is key to
obtaining reliable and quantitative ITC data as discussed in
more detail below. Most proteins contain aromatic amino
acids that contribute absorbance at 280 nm giving a quick
and accurate method of determining concentration. Molar
extinction coefficients can be obtained from the literature or
using the protein sequence and the online program ProtParam
(https://web.expasy.org/protparam/) (see Note 9). For hen
egg-white lysozyme this value is 37,970 M
À1 cm
À1
. Unfortunately, there is not an easy way of checking the concentration of
tri-acetyl glucosamine stock solutions and so these must be
Isothermal Titration Calorimetry
139
The data from this injection are not normally included during
analysis (see Note 7).
The injections are spaced at intervals of 2–4 min, depending on
the instrument, its settings, and the duration of the heat effect that
is determined by the kinetics of binding (see Eq. 2). Initially the
interval is better left longer so all the heat can be captured by the
instrument and a stable baseline reestablished before any
subsequent injections. Once initial binding is characterized, this
interval and the number of injections may be reduced if
appropriate.
2.2 A Realistic Test
Reaction: Lysozyme
Binding a Simple
Trisaccharide Ligand
1. Binding of tri-acetyl glucosamine (N,N
0 ,N
00 -Triacetylchitotriose)
to hen egg-white lysozyme is a useful test reaction to gain experience in performing ITC measurements. Both components are
available as lyophilized materials of high purity and at modest
cost. The binding K d is single μM level and the interaction has
been quantified by ITC and by a variety of orthogonal binding
assays with comparable results (see Chapter 2).
2. Both should be carefully weighed using an analytical balance
and dissolved in an experimental buffer at pH 5 or lower since
this avoids the possibility of lysozyme forming higher order
species such as dimers [1]. 0.1 M acetate buffer pH 5, is a
suitable choice as it has been widely used in the literature for
this interaction. It also has an advantage of a near zero enthalpy
of ionization and will thus maintain its pH at different temperatures if required (see Note 8). The buffer can be prepared
using an online recipe calculator (https://www.liverpool.ac.
uk/pfg/Research/Tools/BuffferCalc/Buffer.html) and the
appropriate molarities of basic and acidic components can simply be mixed in the calculated quantities (using weight or
volume). The pH should be 5 if checked with a calibrated pH
meter. The mixing approach is highly reproducible and avoids
the use of a pH meter that is normally a communal resource in a
lab and requires careful maintenance and calibration for reliable
performance.
3. Measuring the concentration of the materials used is key to
obtaining reliable and quantitative ITC data as discussed in
more detail below. Most proteins contain aromatic amino
acids that contribute absorbance at 280 nm giving a quick
and accurate method of determining concentration. Molar
extinction coefficients can be obtained from the literature or
using the protein sequence and the online program ProtParam
(https://web.expasy.org/protparam/) (see Note 9). For hen
egg-white lysozyme this value is 37,970 M
À1 cm
À1
. Unfortunately, there is not an easy way of checking the concentration of
tri-acetyl glucosamine stock solutions and so these must be
Isothermal Titration Calorimetry
139