Biology Reference
In-Depth Information
Determination of stathmin concentration is even more challenging, as stathmin
bears no tyrosine or tryptophane residues. We tested several approaches, including
colorimetric methods (DC Protein Assay, Biorad) with BSA as standards. As none of
these techniques proved to be satisfactory enough for ITC, we often had to adjust
stathmin concentration after ITC experiments in order to reach the expected stath-
min:tubulin stoichiometry of 0.5. Ideally, the most precise method would be to con-
stitute a stock of stathmin of known concentration (e.g., previously determined by
amino acid composition) and aliquot them to use as standards for colorimetric
methods (instead of BSA) every time stathmin concentration is measured.
18.2.3
Temperature
As described above, the enthalpy of binding (
D
H
) depends on experimental temper-
ature. At a certain temperature, when
0, it is impossible to carry out ITC ex-
periments (
Fig. 18.4
). This is why it is necessary to collect ITC titration at least at two
different temperatures before considering that an interaction cannot be measured
using ITC. In case of stathmin-tubulin interaction, the absolute value of
D
H
ΒΌ
H
is min-
imal at a temperature close to 25
C, which is traditionally used as a standard tem-
perature for ITC experiments. This means that for temperatures lower than 25
C, the
stathmin-tubulin interaction can be monitored by the endothermic signal (
D
0),
whereas above 25
C the interaction will be monitored by the exothermic signal
(
D
H
>
D
H
<
0).
FIGURE 18.4
Temperature dependence of enthalpy of stathmin binding to tubulin. Plot is based on data
obtained by Honnappa with coauthors (
Honnappa, Cutting, Jahnke, Seelig, & Steinmetz,
2003
) (open squares) and our data (circles). The slope of temperature dependence of
enthalpy corresponds to molar heat capacity change of interaction (
DC
p
).
