Biology Reference
In-Depth Information
18.2.2
Determination of protein concentrations
ITC is based on measuring the heat exchange during the interaction as a function of
the ratio of interacting molecules. This is why, just like any other quantitative anal-
ysis of interaction, knowing the concentration of tubulin and tau or stathmin at the
beginning of the experiment is also a critical point that should not be overlooked.
Tubulin concentration is usually determined spectrophotometrically at 275 nm using
an extinction coefficient of 109,000 M
1
cm
1
in 6 M guanidine hydrochloride
(
Andreu & Timasheff, 1982; Na & Timasheff, 1982
). This determination should
be done after tubulin full equilibration and as late as possible (just before the ITC
experiment). Indeed, in a buffer without glycerol or other tubulin stabilizer, tubulin
rapidly degrades, leading to significant errors in determination of thermodynamic
parameters of interaction (
Fig. 18.3
). Thus, prior to each subsequent ITC titration,
aggregated tubulin should be eliminated by centrifugation and concentration should
be measured again. As tau protein is unstructured and elongated, it induces some
scattering of light. Thus, to measure tau concentration, it is necessary to do a full
UV-spectrum of the sample and then correct it for light scattering to avoid overes-
timation of tau concentration (
Winder & Gent, 1971
). It should also be noted that
GTP, which has to be present in the final buffer in all binding experiments with tu-
bulin, strongly absorbs in the range used for measurement of tau concentration. Thus,
it is recommended to equilibrate tau in the absence of GTP, which should be added
just prior to the ITC titration.
FIGURE 18.3
Effect of tubulin degradation with time on binding isotherms. Three sequential ITC
experiments made with the same protein samples with a period of 1 h. The arrows indicate the
stoichiometry which decreases as time goes by, which indicates that less active tubulin is
available due to degradation over time.
